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Horizontal Gene Transfer 2nd Edition Michael Syvanen 2024 Scribd Download
Horizontal Gene Transfer 2nd Edition Michael Syvanen Digital Instant Download Author(s): Michael Syvanen, Clarence I. Kado ISBN(s): 9780126801262, 0126801266 Edition: 2 File Details: PDF, 5.67 MB Year: 2002 Language: english
Foreword The era of high-speed sequencing and computer- based annotating systems have generated enor- mous data bases from which many novel discov- eries are being made. Comparative genomic nucleotide and amino acid sequence analyses have revealed many sequence cassettes that appear highly conserved, thus raising the ques- tion whether these sequences were introduced by horizontal gene transfer mechanisms, or whether they were fortuitous occurrences. The prior recombinant DNA era provided valuable insights on the extent of conserved genetic mechanisms and brought to realization that genes can be moved across species barriers, a finding that was supported 20 years earlier than the recombinant DNA era by bacterial genetic ex- periments. In fact, a number of elegant bacterial genetic studies had implicated genetic transmis- sion of foreign DNA into bacteria and into plants. The natural transfer of plasmid DNA from Agrobacterium tumefaciens to plant cells, resulting in the integration of the foreign plasmid DNA into the chromosome of the plant followed by its expression to generate phenotypic change is the best case of horizontal gene transfer that occurs in nature. Certainly, bacteriophages were well known to mediate horizontal gene transfer long before the Agrobacterium story. Although this was a dramatic discovery, horizontal gene transfer among microbes did not have the impact of that mediated between a microbe and a eukaryote. The genomics era and its ever growing data bases provide vast opportunities to explore po- tential horizontal gene transfer systems that may exist between microbes and eukaryotes and be- tween lower eukaryotes and higher eukaryotes. The direction of horizontal gene transfer may be dominant in one direction, but may occur in the opposite direction (retrotransfer). In reviewing the chapters of this book, I have come to the realization that the discovery of hor- izontal gene transfer among distinct organisms has increased substantially in this new millen- nium and a “new” scientific vocabulary has been introduced. The terms “cross-species gene transfer”, “lateral gene transfer” and “horizontal gene transfer” have been used interchangeably, with the last two terms used most frequently. To avoid future confusion, the term “horizontal gene transfer” should represent the transfer of genes across distinct species, especially when interkingdom gene transfer takes place. The term “lateral gene transfer” could be retained to accommodate gene transfer between distinct species within a kingdom, viz., between pro- karyotes, or between eukaryotes. Clarence I. Kado 9
Preface The seminal experiment that illustrated the ability of genetic information to flow between species slipped by largely unnoticed. In 1959, Tomoichiro Akiba and Kunitaro Ochia discov- ered antibiotic resistance plasmids. The most sur- prising attribute of this new class of plasmids was that they carried resistance genes to multiple anti- biotics and that they moved among different bac- terial species, spreading resistance genes, and thereby demonstrating that genetic information can flow from one species to another (Akiba et al., 1960; Ochia et al., 1959). The implications of this finding would have profound effects ranging from the applied field of genetic engineering to the very theory of evolution. Early papers prob- ing the deeper theoretical implications of hori- zontal gene transfer began to appear in the 1970s, though they were not widely acknowledged or accepted. Fritz Went, in 1971, wrote a review on similar traits that are shared by unrelated flower- ing plants thereby illustrating many examples of parallel evolution. In addition, he noted that the traits are shared among plants that occupied the same ecosystems. In this context he proposed that these unrelated plants were exchanging genes. He cited bacterial plasmid transfer as a precedent for such events. Krassilov in 1977 ar- rived at a similar model for flowering plant evo- lution based on his paleontological studies of the emergence of angiosperms in the fossil record. Anderson in 1970 and Reanney in 1976 suggested that horizontal gene transfer could affect evolu- tion in the animal kingdom, and Hartman, in 1976, suggested that horizontal gene transfer might effect speciation. There were a series of theoretical papers that cited horizontal gene transfer as an explanation for the widespread occurrence of parallelisms in the fossil record (Krassilov, 1977; Erwin and Valentine, 1984; Reanney, 1976; Jeppsson, 1984; Syvanen, 1985). Meanwhile, genetic engineering experiments began to produce startling results. In 1976, Struhl et al. placed DNA from yeast into a histidine defi- cient mutant of Escherichia coli that resulted in the restoration of histidine biosynthesis. This DNA contained a histidine biosynthesis gene from the yeast genome. What seems commonplace today was difficult to comprehend back in 1976 – genes from a eukaryotic organism artificially intro- duced into a bacterium could actually function. Davies and Jimenez in 1980 showed that a bacte- rial neomycin phosphotransferase gene would express aminoglycoside resistance in yeast, showing that a bacterial gene could be expressed in a eukaryote. As a bacteriologist, I had personally incorpo- rated the findings of Akiba and Ochia into my sci- entific world-view. I was intrigued by the implication of Struhl’s experiment. In the course of a discussion of a review of Crick’s book about the unity of the genetic code entitled Life Itself (Crick, 1981), it occurred to me that horizontal movement of genes could shed light on this ques- tion provided such gene transfer was a factor in major evolutionary transitions. If this conjecture was correct, it could provide an alternative expla- nation for not only the unity the genetic code, but many other biological unities as well. At this point, I was unaware of the works of Went, Reanney, Krassilov, Hartman and Anderson. I wrote up my ideas in 1982, and they were finally published in 1985. During this period, the field of 11
genetic engineering was exploding. Palmiter et al. in 1983 produced the first transgenic mouse that expressed a foreign gene, the human growth hormone gene. Result after result confirmed that it was possible for genes to cross species bound- aries and to express their phenotype. These ex- periments all demonstrated that genes could be made to cross species boundaries in the labora- tory. The fundamental question that remained was whether these events occurred in nature, and whether they occurred at a frequency high enough to effect evolution. Hopefully, this collec- tion of articles will be but one of many which will begin to explore this question. By the mid-1980s, numerous mechanisms for horizontal gene transfer were firmly established, not only for bacteria but also for metazoans and, in addition, many heretofore difficult to explain biological phenomena were easily handled by a horizontal gene transfer theory. However, there was a paucity of observations giving direct sup- port to these speculations. With the rapid in- crease in the nucleic acid database over the past decade, the situation has changed. This book covers some of these more recent developments. Today, researchers in many unrelated areas are making observations related to horizontal gene transfer, which has resulted in the unusual breadth of topics included in this volume. This book does not attempt a comprehensive survey of horizontal gene transfer, but rather attempts to sample various areas with a primary focus on material from active research areas. The chap- ters in this book deal with three questions. First, can genes, or more specifically DNA move from one species to an unrelated one? Thus, a section of this book is devoted to the subject of transfer mechanisms, a phenomenon well documented in bacteria but also found in plants and animals. Obviously transfer mecha- nisms exist, the subsidiary questions are: how widespread are the mechanisms? And, do they operate in natural environments? Second, what is the evidence that horizontal gene transfer contributes to existing genotypes of species? The primary evidence supporting evolutionary significant horizontal transfers in- volves phylogenetic reasoning. This is an area where the evidence is accumulating in the gene and protein sequence databases. Two problems are repeatedly encountered – defining the to- pology of a gene tree and estimating divergence times following molecular clock assumptions. There are a number of contributions discussing results obtained from phylogenetic analysis and problems associated with this approach. The third question raised by the central hypothesis is that if the mechanisms exist and events can be documented, does horizontal gene transfer actually play any significant evolu- tionary role? Or, does a theory that incorporates migrant DNA have utility in explaining more general biological phenomena. To this end, more conjectural papers that directly address macro- evolutionary patterns and trends are presented. REFERENCES Akiba, T., Koyama, K., Ishiki, Y., Kimura, S. and Fukushima, T. (1960) The mechanism of the development of multiple- drug-resistant clones of Shigella. Jpn J. Microbiol. 4: 219. Anderson, N.G. (1970) Evolutionary significance of virus infection. Nature 227: 1346–1347. Crick, F. (1981) Life Itself: Its Origin and Nature, Simon and Schuster, New York. Davies, J. and Jimenez, A. (1980) A new selective agent for eukaryotic cloning vectors. Am. J. Tropical Med. Hygiene 29(5 Suppl): 1089–1092. Erwin, D.H. and Valentine, J.W. (1984) Hopeful monsters, transposons and metazoan radiation. Proc. Natl Acad. Sci. USA 81: 5482–5483. Hartman, H. (1976) Speculation on viruses, cells and evolut- ion. Evolution Theory 3: 159–163. Jeppsson, L. (1986) A possible mechanism in convergent evolution. Paleobiology 12: 37–44. Krassilov, V.A. (1977) The origin of angiosperms. Bot. Rev. 43: 143–176. Ochia, K., Yamanaka, T., Kimura, K. and Sawada, O. (1959) Inheritance of drug resistance (and its transfer) between Shigella strains and between Shigella and E. coli strains. Nihon Iji Shimpo 1861: 34 [in Japanese]. Palmiter, R.D., Norstedt, G., Gelinas, R.E. et al. (1983) Metallothionein–human GH fusion genes stimulate growth of mice. Science 222(4625): 809–814. Reanney, D. (1976) Extrachromosomal elements as possible agents of adaptation and development. Bacteriol. Rev. 40: 552–590. Struhl, K., Cameron, J.R. and Davis, R.W. (1976) Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc. Natl Acad. Sci. USA 73(5): 1471–1475. Syvanen, M. (1985) Cross-species gene transfer; impli- cations for a new theory of evolution. J. Theor. Biol. 112: 333–343. Went, F.W. (1971) Parallel evolution. Taxon 20: 197–226. Michael Syvanen xii PREFACE 12
Contributors Ronald M. Adkins Biology Department, University of Massachusetts, Amherst, Massachusetts, USA Rita M. P. Avancini Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Lexington, Massachusetts, USA) L. Aravind National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA William B.N. Berry Department of Geology and Geophysics, University of California, Berkeley, California, USA Meghan E. Bowser Genotypes Inc., San Francisco, California, USA James R. Brown Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA A. Burmester Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany R. N. Burns Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, UK Richard Calendar Department of Molecular and Cell Biology, University of California, Berkeley, California, USA George Chisholm Genotypes Inc., San Francisco, California, USA Jonathan B. Clark Department of Zoology, Weber State University, Ogden, Utah, USA Patrice Courvalin Unité des Agents Antibactériens, Institut Pasteur, Paris, France Pierre Darlu Inserm U 155, Kremlin-Bicêtre, France 13
Martin Day Cardiff School of Biosciences, Cardiff University, Cardiff, UK Erick Denamur Inserm U 458, Hôpital Robert Debré, Paris, France Russell F. Doolittle Center for Molecular Genetics, University of California, San Diego, La Jolla, California, USA Christophe Douady Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA Norman C. Ellstrand Department of Botany and Plant Sciences and Center for Conservation Biology, University of California, Riverside, California, USA Gayle C. Ferguson Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand Bryant E. Fong Genotypes Inc., San Francisco, California, USA M. E. Ford Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA (present address: Division of Gastroenterology and Hepatology, University of Pittsburgh School of Medicine, Pennsylvania, USA) Lynne M. Giere Genotypes Inc., San Francisco, California, USA J. Peter Gogarten Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA Sylvie Goussard Unité des Agents Antibactériens, Institut Pasteur, Paris, France Catherine Grillot-Courvalin Unité des Agents Antibactériens, Institut Pasteur, Paris, France Ruth M. Hall CSIRO Molecular Science, North Ryde, New South Wales, Australia James F. Hancock Department of Horticulture, Michigan State University, East Lansing, Michigan, USA Hyman Hartman IASB, Cambridge, Massachusetts, USA G. F. Hatfull Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Jack A. Heinemann Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand R. W. Hendrix Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Katrin Henze Institut für Botanik III, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany xiv CONTRIBUTORS 14
Nathan C. Hitzeman Genotypes Inc., San Francisco, California, USA Ronald A. Hitzeman Genotypes Inc., San Francisco, California, USA Susan Hollingshead Department of Microbiology, University of Alabama, South Birmingham, Alabama, USA Michael J. Italia Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA Clarence I. Kado Davis Crown Gall Group, University of California, Davis, California, USA Margaret G. Kidwell Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson, Arizona, USA Eugene V. Koonin National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA Valentin A. Krassilov Paleontological Institute, Moscow, Russia David J. Lampe Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania, USA) Jeffrey G. Lawrence Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Guillaume Lecointre Service de Systématique moléculaire (GDR CNRS 1005), Muséum National d’Histoire Naturelle, Paris, France Wen-Hsiung Li Ecology and Evolutionary Biology, University of Chicago, Chicago, Illinois, USA Chin Y. Loh Genotypes Inc., San Francisco, California, USA Eugene L. Madsen Department of Microbiology, Cornell University, Ithaca, New York, USA Kira S. Makarova National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA and Department of Pathology, F.E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA William Martin Institut für Botanik III, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany Ivan Matic Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France CONTRIBUTORS xv 15
Robert V. Miller Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahama, USA Gisela Mosig Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee, USA Lorraine Olendzenski Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA Honor C. Prentice Department of Systematic Botany, Lund University, Lund, Sweden Michael D. Purugganan Department of Genetics, North Carolina State University, Raleigh, North Carolina, USA Alfred Pühler Department of Genetics, University of Bielefeld, Bielefeld, Germany Miroslav Radman Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France Loren H. Rieseberg Department of Biology, Indiana University, Bloomington, Indiana, USA Steven A. Ripp Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA Hugh M. Robertson Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA Claus Schnarrenberger Institut für Pflanzenphysiologie und Mikrobiologie der FU Berlin, Berlin, Germany K. Schultze Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Joana C. Silva National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland, USA M. C. M. Smith Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, UK Jay V. Solnick Departments of Internal Medicine and Medical Microbiology and Immunology, University of California, Davis, California, USA Felipe N. Soto-Adames Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Department of Biology, University of Vermont, Burlington, Vermont, USA) Michael J. Stanhope Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA xvi CONTRIBUTORS 16
Michael Syvanen Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California, USA François Taddei Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France Andreas Tauch Department of Genetics, University of Bielefeld, Bielefeld, Germany Olivier Tenaillon Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France K. Voigt Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Kimberly K. O. Walden Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA Carole I. Weaver Genotypes Inc., San Francisco, California, USA Mark E. Welch Department of Biology, Indiana University, Bloomington, Indiana, USA Richard J. Weld Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand Donald I. Williamson Port Erin Marine Laboratory (University of Liverpool), Isle of Man, UK Yuri I. Wolf National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA A. Wöstemeyer Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany J. Wöstemeyer Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Glenn M. Young Department of Food Science and Technology, University of California, Davis, California, USA Olga Zhaxybayeva Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA CONTRIBUTORS xvii 17
S E C T I O N I Plasmids and Transfer Mechanisms in Bacteria Section 1 of this book deals with plasmids and mechanisms of gene transfer in bacteria. We will not be attempting a comprehensive review of plasmid biology, which has been the subject of many excellent reviews and is even well covered in many textbooks. Rather, a potpourri of topics will be sampled that illustrate recent developments and unexpected findings related to plasmid-mediated gene transfers. Plasmids figure prominently in the discussion of horizontal gene transfer because a large number of plasmids will stimulate conjugal transfer of bacterial DNA to cells from an extremely broad range of organisms. These include transfer to unrelated bacteria, yeasts and other fungi and plants. Chapter 1 by Heineman and Chapter 5 by Kado deal with the evolution of conjugal plasmids themselves. These vectors of horizontal transfer experience dual evolutionary pressures of survival in hosts via vertical transmission and the ability to adapt in new environments after horizontal flow. The remaining chapters in this section de- scribe some of the more recent interesting plasmid-related developments. In Chapter 2, Hall describes integrons, a site-specific recombi- nation system that serves to assemble new antibiotic resistance genes into pre-existing transposable elements. In Chapter 3, Tausch and Pühler describe an unusual antibiotic resis- tance plasmid that is a mosaic of elements found previously from throughout the bacterial kingdom. This is noteworthy because the ge- netic rearrangements and gene transfers that gave rise to this plasmid have likely occurred in the past 50 years, while the genes come from a group of distantly related organisms that last shared a common ancestor approximately 1.5–2 billion years ago. In Chapter 6, Weld and Heinemann review protein transfers, a topic that has captured attention in recent years be- cause of its importance in pathogenic mecha- nisms. Protein transfer is probably also important in ensuring survival of transferred DNA in foreign cells. As is clear, bacteria have numerous and highly adapted mechanisms in place to facilitate the transfer of DNA from donor to recipient cells. These mechanisms do not respect species boundaries. The question as to whether or not these mech- anisms operate in natural populations is the subject of the remaining three chapters in this section. It has been known for many years that conjugal plasmid transfer occurs among bac- teria in hospitals, farms and natural environ- ments. Along these lines, Madsen in Chapter 4 has an interesting story that documents the emergence of a plasmid that makes enzymes which degrade coal tars and has spread among different bacterial species in a toxic waste dump. Chapters 7 (by Day) and 8 (by Miller and Ripp) show evidence that the DNA transfer mecha- nisms of transformation and bacterial virus transduction operate efficiently in natural environments. 1 19
C H A P T E R 1 Recent History of Trans-kingdom Conjugation Gayle C. Ferguson and Jack A. Heinemann Conjugation is a mechanism of horizontal gene transfer (HGT) first observed between bacteria. The conjugative mechanism appears to be ana- logous, and sometimes homologous, to other means of transferring genes from bacteria to possibly members of every biological kingdom. As such, conjugative mechanisms of DNA transfer are necessary for a host of spectacular phenotypes such as symbiosis, virulence and antibiotic resistance. The conjugative mecha- nism is also related to the means of translocating and transferring proteins from bacteria to other species. Thus, this nearly generic form of macromolecular transport may move genes and other molecules across species boundaries. Some of these molecules may have immediate effects (e.g. through pathogenesis) and some lasting effects (e.g. through inheritance). There is even evidence that inheritable effects can be caused by transferred proteins. Interest in HGT, previously considered on the fringe, has in- creased dramatically due to the realization that HGT is not an anomaly but a biological fundamental. INTRODUCTION The idea that genes are transferred at any appre- ciable frequency between species has evolved from one scorned by molecular phylogenists to a mainstream concept. Previously, only frustrated phylogenists would dwell on the odd DNA sequence that could unlace the bootstrap analysis (Gogarten et al., 1999). Whole chromosome se- quencing of organisms, however, is beginning to validate the concept that genomes are littered with “carcasses” of DNA from other spe- cies – some genes remaining functional and neu- tral, beneficial, or deleterious to the host, and some slowly fading away into the background average G + C content of the new host. The extent of horizontal gene transfer (HGT) between organisms is difficult to determine for two main reasons. DNA sequence information is, first, limited by the simplicity of the four letter code and secondly, by the constraints on the se- quence when it must reproduce in synchrony with the host (Heinemann, 2000b; Heinemann and Roughan, 2000). Thus, the mechanisms of HGT as well as bioinformatic tools are required to quantify the extent of HGT. The renaissance in HGT thinking brought about by bioinformatics has a history and origin different from the mechanism studies. These studies identify the means by which genes move between two neighbors that may or may not share a vertical lineage. Studies describing the gene transfer mediated by viruses, plasmids, transposons and transformation are much older than bioinformatics. Mechanism studies did not make HGT a mainstream concept, though, be- cause they were considered “laboratory phe- nomenon” or “interesting exception to the rule for most genes or most organisms” by many. The mechanism studies did, however, open Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 3 All rights of reproduction in any form reserved. 21
imaginations to the potential for HGT and legiti- mized those who subjected it to serious study. This review will focus on gene transfer be- tween prokaryotes and eukaryotes by mecha- nisms that are identical, or similar, to bacterial conjugation. The review will not be a systematic account of all the literature relevant to HGT and conjugation. Instead, it will focus on publications that represent unambiguous conflations of ideas that led to HGT becoming an independent phe- nomenon for study and established bacterial conjugation as a central, general, mechanism for interkingdom gene transfer (Amábile-Cuevas and Chicurel, 1992; Heinemann, 1992). We begin with an abbreviated history of the merger be- tween HGT and crown gall disease in plants that has developed an inseparable link with bacterial conjugation. Finally, we will discuss bacterial conjugation as a paradigm of interkingdom macromolecular exchange mechanistically con- nected to pathogenesis. By the mid-twentieth century, interspecies gene transfer was recognized as an important means by which bacteria acquired antibiotic re- sistance. Those findings, as indeed most early studies in gene transfer, remained focused on the particular genes or organisms of interest. Our review of the literature suggests to us that a change in thinking about HGT was gaining momentum in the late 1960s. Subsequently, a number of studies examined HGT as a possible phenomenon in its own right, without need of allusion to important organismal adaptations, the success of pathogens (e.g. viruses and Agrobacterium tumefaciens), or the exception to the rule that all prokaryotic biology can appear to be to botanists and zoologists! THE CONVERGENCE OF INTERKINGDOM DNA TRANSFER AND CROWN GALL A. tumefaciens was clearly linked to crown gall tumors in some plants long before the 1960s (ref- erences in Stroun et al., 1970; Nester and Kosuge, 1981; Zhu et al., 2000). However, the seminal clues that the nature of the disease was inseparable from DNA transfer to the host emerged in that decade. Work by Kerr demonstrated that A. tumefaciens virulence characters were transmitted between bacteria, by an unknown mechanism (Kerr, 1969). In the late 1970s, the DNA that caused gall formation, T-DNA, would be identi- fied as a component of a conjugative plasmid, called Ti, in A. tumefaciens (Nester and Kosuge, 1981). The T-DNA was subsequently found inte- grated into plant chromosomes (Thomashow et al., 1980; Yadav et al., 1980; Zambryski et al., 1980). The search for T-DNA illustrates two different approaches to the study of interkingdom gene transfer operating simultaneously. One group of researchers, which we arbitrarily call the gen- eralists, was dominated by the sense that HGT was a phenomenon independent of the partic- ular biology of the donor and recipient organ- isms, such as the biology of the phytopathogen A. tumefaciens and its potential plant hosts. The other, which we refer to as the specialists, used the power of the causal relationship between A. tumefaciens and the gall tissue to discover HGT. The two approaches had complementary strengths and both endured the inevitable false positive and negative results that accumulate whenever techniques are pushed to their ex- treme limits of sensitivity. The path to the discovery of the discrete DNA sequences transferred from A. tumefaciens to the host, and even to other soil bacteria, was itself a study in the limits of the contemporary molecular techniques. The pioneers at the roots of the crown gall mystery during the 1960s and 1970s were also at the leading edge of molecular bi- ology and biochemistry. From such an edge, there is the risk of accumulating negative results, that is, for example, of not seeing DNA transfer (see below). New techniques also require refine- ment to distinguish between the noise at their limits of detection and true signals. The results of these early studies were consistently “equivocal, but collectively they suggested that bacterial nuc- leic acids might play a role in tumorigenesis” (Drlica and Kado, 1975). Generalists and specialists Both generalists and specialists were reporting the transfer of bacterial nucleic acids and pos- sibly proteins to eukaryotes by the late 1960s. 4 G.C. FERGUSON AND J.A. HEINEMANN 22
The nucleic acids were invariably pursued in bacteria-free tissues by hybridization (refer- ences in Drlica and Kado, 1975) or hybridization and density centrifugation (Stroun et al., 1970; Stroun and Anker, 1971, 1973). The conclusiveness of the hybridization method itself, however, was systematically chal- lenged (Drlica and Kado, 1975). Hybridization methods used to demonstrate the presence of bacterial DNA in eukaryotes were often flawed because a control measurement of hybrid thermal stabilities or dissociation profiles was omitted (Chilton et al., 1974; Drlica and Kado, 1974; Kado and Lurquin, 1976). With improved techniques applied later in the 1970s, A. tumefaciens nucleic acids were not detected in tumors (Chilton et al., 1974; Drlica and Kado, 1974). The data of some groups were unable to be reproduced at this experimental standard (for an excellent discussion on the technology of the period, see Drlica and Kado, 1974). Why did some detect nucleic acids while others did not? One possible explanation is that the sporadic claims of nucleic acid detection were artefacts generated by techniques pushed to their limits. A second possibility is that the practitio- ners of state-of-the-art techniques are important contributors to detection limits. A third possibility is experimental design. Of course, these three possibilities are not mutually exclusive and cannot be distinguished retrospectively. With the increase in rigor applied to hybrid- ization experiments came an increase in the pre- cision for calculating the detection limits of the techniques (Drlica and Kado, 1974, 1975; Kado and Lurquin, 1976). Chilton et al.’s DNA–DNA hybridization technique, for example, limited detection to one bacterial genome per three dip- loid plant genomes and “would not detect single or even multiple copies of a small specific fraction (<5%) of the bacterial … genome in tumor DNA” (Chilton et al., 1974). Such famous negative results cannot, unfortunately, be di- rectly compared with all reported positive de- tection of nucleic acids because of differences in determining the sensitivities of the techniques. Thus history cannot distinguish between spo- radic artefacts and individual experimenters as explanations for different results from all con- temporary experiments. Some groups monitored the production of bacteria-specific nucleic acids in eukaryotic tis- sues (Stroun et al., 1970). Although these studies were also not above the criticisms leveled against other hybridization studies and were not consistently reproduced (discussed in Drlica and Kado, 1975), ongoing RNA synthesis poten- tially provided access to larger quantities of nu- cleic acids complementary to the probe. In contrast, those groups searching only for trans- ferred bacterial DNA were limited by the small number of copies of those sequences in prepara- tions of eukaryotic genomes. History cannot distinguish between possible sporadic artefacts and differences in experimental design as the explanation for different data from all the dif- ferent experimenters. Some generalists introduced further confu- sion when they reported that DNA transfer oc- curred from not just A. tumefaciens, but also Escherichia coli, Bacillus subtilis and Pseudomonas fluorescens to both plants and animals. Hence, “The relationship of (these observations) to the crown gall disease (was) ambiguous” (Drlica and Kado, 1975). Since only A. tumefaciens in- duced tumors, the mechanism of putative nu- cleic acid transfers from these other bacteria may have been irrelevant to that conducted by A. tumefaciens when it induced tumors. The generalist view was to be eclipsed by the finding of particular T-DNA sequences in plants and the characterization of a mechanism that could account for its transfer. T-DNA transfer would, for a time, serve as the paradigm of interkingdom gene transfer systems. The gener- ality of HGT would be revived in the 1980s by the finding that bacterial conjugative plasmids and T-DNA were different DNA transferred by the same mechanism (Heinemann, 1991; Sprague, 1991), providing retrospective cre- dence to generalists’ claims if not vindication of early experiments. Critical experimental limits to HGT detection Until recently, interkingdom DNA transfer has been mostly observed through the isolation of phenotypically recombinant organisms (i.e. gene RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 5 23
transmission). DNA transfer can be inferred from any instance in which donor genes are recovered from recipient organisms. This is usually accom- plished by selecting recombinant phenotypes. Such phenotypes are the complex product of gene transfer and subsequent stabilization in the germ line of the recipient. Gene transfer is likely not the limiting event in most instances of gene transmission (Heinemann, 1991; Matic et al., 1996). Since inheritable phenotypes or stably maintained DNA sequences remain the easiest way to detect transferred genes, the importance of gene transmission in biasing inferences of the rate and extent of HGT cannot be ignored. In fact, the general reliance on observing recombinant phenotypes or isolating transferred DNA from offspring underestimates HGT (Chilton et al., 1974; Drlica and Kado, 1974; Heinemann and Roughan, 2000; Heinemann, 2000b). Several authors over the years have empha- sized the importance of distinguishing between gene transfer and transmission to avoid instilling a bias in experimental design and interpretation (reviewed in Heinemann, 1991, 1992). Clark and Warren (1979) made the most systematic justifica- tion for the terminology. The first authors to demonstrate the generality of interkingdom con- jugation openly acknowledged the influence of that review on their experimental design (Figure 1.1). Confusion between transfer and transmis- sion may have similarly delayed discovery of transfer of DNA from A. tumefaciens to plants out- side the bacterium’s infectious host range (Grimsley et al., 1987). 6 G.C. FERGUSON AND J.A. HEINEMANN Mix on plate Mix on plate LEU2 r e p Minimal medium LEU2 FIGURE 1.1 Illustration of the original experiment demonstrating DNA transfer from bacteria to yeast by conjugation. The rationale for the experiment was that DNA transfer was more generic than could be detected by DNA amplification or the formation of recombinant organisms, which requires DNA transmission (Heinemann and Sprague, 1989). As a test, specially constructed donor bacteria (rectangles) were mixed with genotypically marked recipient yeast (circles with “buds”) and plated on medium (large open circles) permissive to the growth of only recombinant yeast. The conjugative plasmids (open circles inside bacteria) were modified to carry either the selectable yeast LEU2 gene or both LEU2 and a DNA sequence that permits replication of extrachromosomal DNA in yeast (rep). Colonies of yeast recombinants (solid black circles) were recovered at a frequency of up to 10% (per donor bacterium) when the plasmid carried yeast-specific replication sequences. Since the DNA introduced into the conjugative plasmids was not responsible for DNA transfer (Bates et al., 1998; Heinemann and Sprague, 1989; Heinemann, 1991), these experiments unequivocally demonstrated that transmission (necessary for detecting recombinants because the DNA is subsequently inherited vertically) was a poor indicator of transfer and the absence of experimentally demonstrated transmission did not imply the absence of DNA transfer. 24
To illustrate further the importance of distin- guishing transfer from transmission, consider the recent report of a DNA virus, that infects an- imals, evolving via recombination between a DNA virus, that infects plants, and an RNA virus, that infects animals (Gibbs and Weiller, 1999). (Another remarkable intermediate in this chain of events was the likely contribution of a retroviral reverse transcriptase acting on the animal RNA virus to convert an RNA gene into DNA.) The plant virus must have been able to transfer to animals (but caused no obvious phe- notype). The many transfer events preceding the evolution of the new variant virus were not detected by selecting or observing a recombi- nant animal, and likely would not have been detected even with current DNA amplification technologies. The transmission event could be detected, but provides no quantitative informa- tion about the frequency of transfers of the orig- inal virus to animals. Furthermore, transferred nucleic acids can be retained by recombination even if whole genes are not inherited (reviewed in Heinemann, 1991; Matic et al., 1996). The extent of this recombina- tion can be masked by the selectivity of homolo- gous recombination enzymes that eliminate long tracts of dissimilar nucleotide sequences better than short tracts (Rayssiguier et al., 1989; Heinemann and Roughan, 2000). Certain envi- ronments and mutations that reduce the activity of mismatch repair systems in particular have the effect of reducing selectivity (Matic et al., 1995; Heinemann, 1999b; Vuli’c et al., 1999). Recombi- nation events resulting in the incorporation of short tracts of DNA, even over sequences of ex- treme genetic divergence, can be difficult or im- possible to identify by analysis of DNA sequences (Heinemann and Roughan, 2000). CONJUGATION AS A PARADIGM SYSTEM OF INTERKINGDOM DNA TRANSFER The first indication that bacterial conjugation de- scribed a general mechanism of interkingdom gene transfer came from the suggestion that certain DNA intermediates observed in A. tumefaciens resembled hypothetical DNA inter- mediates in bacterial conjugation (Stachel et al., 1986). In hindsight, that connection was probably better informed by inspiration than actual data, but nevertheless has withstood significant test. Conjugation Bacterial conjugation in its broadest sense has been extensively reviewed, so only a brief de- scription will be provided here (Heinemann, 1992, 1998; Frost, 2000). The focus in this review is on the paradigm conjugative systems defined by the IncP and IncF plasmid groups. Conjugation mediated by these plasmids requires, at a minimum, a cis-acting DNA se- quence called the origin of transfer (oriT). All other functions (called tra) act in trans thus al- lowing plasmids with all trans-acting functions also to transfer plasmids with no or a few trans- acting functions (Heinemann, 1992). The trans- acting gene products are divided further into those involved in DNA metabolism (and are usually specific to a particular oriT) and those in- volved in DNA transport and cell–cell interac- tions (and thus will interact with a greater range of other plasmids). The conjugative genes spe- cific to DNA metabolism introduce a nick at oriT and initiate the unwinding and concomitant transfer of DNA to a recipient cell. Both strands are used as templates for the synthesis of a com- plementary strand, one in the donor cell and one in the recipient. Single-stranded plasmid DNA (ssDNA) has been captured in recipient cells, confirming the mechanism of plasmid mobilization. The DNA is recircularized in the recipient. The transport apparatus has not been described biochemically (Heinemann, 2000a), but the genes necessary for forming the apparatus are all plasmid- encoded (Heinemann and Ankenbauer, 1993; Heinemann et al., 1996). T-DNA is interkingdom conjugation This uncontroversial model of the conjugative process grounded a model of T-DNA mobiliza- tion and transfer proposed by Stachel et al. (1986). Their experiment involved isolating RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 7 25
DNA of the T-DNA region from A. tumefaciens (not the plant) after it was induced to prepare the T-DNA for transfer. They provided con- vincing evidence that linear ssDNA strands de- fined by the left and right borders of the T-DNA region accumulated in induced bacteria, and that Ti plasmids from induced bacteria had nicks in the border sequences on the strand cor- responding to the liberated T-DNA. It appeared to Stachel et al. that the left and right borders of the T-DNA region, which are characterized as direct repeats, functioned like oriT sequences. Nicking and unwinding liber- ated only the DNA between the nicks, rather than a strand of DNA the length of the Ti plasmid. When the transfer process could not be completed, the T-DNA accumulated in the bacterium. However, the phenomenology differed from the molecular biology of conjugation in impor- tant ways. First, hypothetical ssDNA transfer in- termediates do not accumulate in bacteria that hold conjugative plasmids even when constitu- tively induced. Secondly, the conjugative ssDNA was isolated from bacterial recipients; the so-called T-DNA in the Stachel et al. study was never recovered from plants. Thirdly, there existed no evidence at the time that the DNA be- tween tandemly repeated oriTs would be liber- ated during mobilization. Whereas it was shown subsequently that tandem oriT repeats do result in mobilization-specific DNA insta- bility in some plasmids (Bhattacharjee et al., 1992; Furuya and Komano, 2000), the repeat of IncP oriTs, which are thought to be the closest relatives of the T-DNA borders (Waters et al., 1991; Waters and Guiney, 1993), does not result in mobilization-specific liberation of inter- vening DNA (Heinemann and Schreiber, per- sonal observation). Nevertheless, the model has been vindicated by several subsequent genetic tests (Lessl and Lanka, 1994; Christie, 2000). First, T-DNA re- combination experiments within plant cells pro- vide evidence that T-DNA is transferred, and enters the nucleus, single-stranded (Tinland et al., 1994). Secondly, the processing reaction be- tween the cis-acting border repeat sequences and its putative nick-ase (virD2) could be re- placed with the oriT and its cognate nick-ase (mobA) from the IncQ plasmid RSF1010 (Bu- chanan-Wollaston et al., 1987). Third, RSF1010 transmission between Agrobacteria was found to be dependent on the other Ti-encoded genes virA, virG, virB4, virB7 and virD4 (Beijersbergen et al., 1992). Thus, the vir genes, originally identified because they were necessary for viru- lence, can substitute for tra in mediation of conjugative plasmid transfer. The ability to mix and match genetic require- ments of bacterial conjugation and Ti-mediated virulence is consistent with the structural simi- larities of conjugative and virulence genes (Table 1.1). The oriT region of IncP plasmids is homologous to the T-DNA borders (Waters and Guiney, 1993; Frost, 2000), while the oriT of the Ti plasmid is homologous to the IncQ oriT. Many macromolecular transport systems appear to be composed of gene products ho- mologous to the tra functions of conjugative plasmids, including the vir genes and type IV protein secretion systems in Bordetella pertussiss, Helicobacter pylori and Legionella pneumophila (Christie, 2000; Frost, 2000) (Tables 1.1 and 1.2). CONJUGATION IS SUFFICIENT FOR INTERKINGDOM CONJUGATION A surprise to the crown gall groups was the finding that the transfer of DNA from A. tumefaciens to plants was related in part to bacte- rial conjugation. Meanwhile, yeast studies were soon to show that conjugation could account for interkingdom DNA transfer and that the ability to conjugate with eukaryotic cells is not an evo- lutionary quirk of A. tumefaciens. In 1989 we crossed bacteria with the yeast Saccharomyces cerevisiae using the same plasmids that mediated conjugation between bacteria (Heinemann and Sprague, 1989) (Figure 1.2). E. coli transferred a plasmid marked with the S. cerevisiae replication origin 2µ and LEU2 gene, to yeast. Recombinant (Leu+ ) yeast were only formed when the bacteria contained a con- jugative plasmid able to mobilize the marker plasmid in trans. Formation of Leu+ yeast recombinants was dependent on donor– recipient contact, donor viability, functional 8 G.C. FERGUSON AND J.A. HEINEMANN 26
Proposed functions of vir genes required for T-DNA transfer from A. tumefaciens to plantsb vir homologues on conjugative plasmids vir homologues involved in protein transfer/virulence vir homologues with as yet unknown function IncFb IncPc pTiC58 (tra)b IncWb IncNb B. pertussisb B. suisd B. abortise L. pneumo- phila (icm/dot)e H. pylori (cag)b L. pneumo- phila (lvh)f R. prowazekiif Wolbachia sp.g A. actinomy- cetemcomitansh virB1 Transglycosylase orf169 trbN traL virB1 virB2 Pilin subunit traA trbC trbC trwL traM ptlA virB2 lvhB2 virB3 traL trbD trbD trwM traA ptlB virB3 lvhB3 virB4 ATPase, transport activation traC trbE trbE trwK traB ptlC virB4 cagE lvhB4 virB4 virB5 Pilin subunit traE trbF trbF trwJ traC virB5 lvhB5 virB6 Candidate pore former trbL trwI traD ptlD virB6 lvhB6 virB7 Transporter assembly trwH traN ptlI virB7 rp288 virB8 trwG traE ptlE virB8 lvhB8 rp289 virB8 virB9 Transporter assembly trwF traO ptlF virB9 orf15 lvhB9 rpB9 virB9 virB10 Coupler of inner and outer membrane subcomplexes traB trbI trbI trwE traF ptlG virB10 dotG/icmE orf13 lvhB10 rpB10 virB10 virB11 ATPase, transport activator trbB trbB trwD traG ptlH virB11 dotB orf11 lvhB11 rpB11 virB11 tadA virD4 ATPase, coupler of DNA processing and transport systems traD traG trwB orf10 lvhD4 rpD4 virD4 virD2 Site-specific single- stranded nicking at the right and left borders traIi Right and left borders Site of VirD2 nicking oriTj Table adapted from Christie (1997a). a Christie (1997a, 2000). b Li et al. (1998). c O’Callaghan et al. (1999). d Frost (2000). e Segal et al. (1999). f Waters et al. (1991). g Masui et al. (2000). h Kachlany et al. (2000). i Functional homology (Pansegrau et al., 1993). j Sieira et al. (2000). TABLE 1.1 A. tumefaciens T-DNA transfer genes that are homologous to genes required for conjugation, protein transfer and virulence in a range of Gram-negative bacteriaa 27
oriT and mob genes, and was independent of ex- ogenous DNAse, indicating that the mechanism of gene transfer was not transformation. E. coli– yeast conjugation was subsequently found to be dependent on the same tra genes as required for conjugation between E. coli, with no additional plasmid-encoded requirements (Heinemann and Sprague, 1991; Bates et al., 1998). These experiments suggested that DNA transfer from E. coli to S. cerevisiae occurred by a mechanism analogous to conjugation. The range of yeast able to serve as E. coli conjugal re- cipients has been extended to at least six evolu- tionary divergent genera (Heinemann, 1991; Hayman and Bolen, 1993; Inomata et al., 1994). Unlike A. tumefaciens and plants, E. coli and yeast have no known ecological relationship and are not expected to have evolved such a specialized interaction. Therefore interkingdom gene transfer has few, if any, specific requirements evolved within the particular biology of the donor and recipient organism (although virulence and other phenotypes certainly do have specific requirements). Interkingdom conjugation is not a species-specific phenomenon E. coli is not unique in its ability to conjugate with yeast. The T-DNA from A. tumefaciens also transferred to S. cerevisiae, but by vir-dependent conjugation (Bundock et al., 1995). Using URA3 as a selectable marker with or without the 2µ replication sequence between the T-DNA bor- ders, the frequency of transmission of both replicative and integrative vectors was com- pared (Bundock et al., 1995). Where transferred T-DNA could replicate autonomously, most transconjugants inherited the vector in its en- tirety. This was attributed to a failure of VirD2 sometimes to nick the left border, effectively creating a situation where the right border was the only oriT. Other transconjugants carried recirularized dsT-DNA molecules. 10 G.C. FERGUSON AND J.A. HEINEMANN Fungi Eubacteria Plants Animals FIGURE 1.2 Bacteria transfer DNA and proteins to plant, animal and fungal cells by similar and related mechanisms. Bacteria transfer DNA (solid lines and large open circles) to both yeast and plant cells by conjugation. Bacterial DNA is integrated into eukaryotic chromosomes (double helices) upon entering the nucleus (white ellipses). Proteins (solid black circles) are transferred to animal cells during pathogenesis. Conjugative plasmids have genes homologous to some genes required for virulence in many bacterial pathogens. Some of those homologous genes are known to be required for DNA or protein transfer. 28
Interkingdom conjugation is not a plasmid-specific phenomenon Is the ability to conjugate with eukaryotic cells a particular feature of so-called “broad-host- range” plasmids, such as the IncP family? Bates et al. (1998) compared the ability of conjugation functions from three incompatibility groups to transmit a marked shuttle vector to yeast. IncP plasmids transmitted the shuttle plasmid under conditions where transmission by the narrow- host-range IncF and IncI1 plasmids was not detected (Bates et al., 1998). In contrast, all plasmids were equally capable of transmitting the shuttle plasmid to E. coli. Since recombinants were the only evidence of DNA transfer, it remains formally possible that some aspect of the IncP tra system enhances transmission by contributing to the ability of transferred DNA to be inherited. Consistent with this possibility, Heinemann and Sprague did observe F-mediated DNA transmission to yeast using an IncF plasmid derivative instead of mobilizing a shuttle plasmid in trans (Heinemann and Sprague, 1989). The higher copy number of their F plasmid derivative may have contributed to the frequency of detectable DNA transmission (Bates et al., 1998). CONJUGATION AS A CONVERGENCE OF MACROMOLECULAR TRANSPORT SYSTEMS A. tumefaciens provided an anecdotal link be- tween DNA transfer by conjugation and in pathogenesis. However, in that case, the disease was made possible by the genes transferred but DNA transfer was itself not causing the disease. It has become clear over the past decade that the DNA transport apparatus of conjugation is the ancestor, or at least a sibling (O’Callaghan et al., 1999), of other macromolecular transport sys- tems that are the raison d’être of the disease. As mentioned above, type IV protein secretion genes are homologous to conjugation genes and the transport mechanism for both protein and DNA may be the same (Winans et al., 1996; Christie, 1997a; Kirby and Isberg, 1998; Segal and Shuman, 1998a; Christie and Vogel, 2000). Bioinformatics Many homologues of the Ti virB genes (B4, B9–11 and sometimes also virD4) are found on conjugative plasmids and on chromosomes, as inferred from similarities in sequence and orga- nization. DNA transfer homologues include tra of IncN (Pohlman et al., 1994) and Ti (Li et al., 1998), trb of IncP and trw of IncW (Kado, 1994; Christie, 1997a) plasmids. The virB genes have homologues in the pertussis toxin secretion system, ptl of B. pertussis (Covacci and Rappuoli, 1993; Shirasu and Kado, 1993; Weiss et al., 1993; Farizo et al., 1996). The cag pathoge- nicity island of Helicobacter pylori, implicated in contact-mediated secretion of proteins into epi- thelial cells, is homologous to virB (Tummuru et al., 1995; Censini et al., 1996; Christie, 1997b; Covacci et al., 1997). virB homologues have also been found in the chromosome of the obli- gate intracellular parasite Rickettsia prowazekii (Andersson et al., 1998), the arthropod intracellular pathogen Wolbachia sp. (Masui et al., 2000), the human pathogen Actinobacillus actinomycetem-comitans (Kachlany et al., 2000) and are essential for virulence in the intracellular pathogens Brucella abortus and Brucella suis (O’Callaghan et al., 1999; Sieira et al., 2000). Relations between protein and DNA secre- tion systems is not restricted to vir. The icm/dot genes, essential for L. pneumophila survival and replication inside human alveolar macro- phages, are homologous to conjugation genes from various plasmids (Segal and Shuman, 1997, 1999; Purcell and Shuman, 1998; Segal et al., 1998; Vogel et al., 1998) (Table 1.2). Fourteen of the icm/dot genes are similar, both in sequence and in structural organization, to the tra region of IncI plasmid Col1b-P9 (Segal and Shuman, 1999), and icmE is homologous to trbI of IncP plasmid RK2. Mechanism The link between protein and DNA secretory systems is also suggested by mechanistic RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 11 29
studies. For example, a radiolabeled DNA primase (Rees and Wilkins, 1989, 1990) and E. coli’s RecA protein (Heinemann, 1999a) were transferred to recipients during bacterial conju- gation. In these cases, protein and DNA transfer were associated but the possibility remains that the protein and DNA need not be associated for transfer (Heinemann, 1999a). Likewise, the decreased stability of T-DNA transferred from virE2 mutant bacterial donors is complemented by in planta expression of VirE2 protein (Rossi et al., 1996) and extracellularly by virE2+ bacteria (Christie et al., 1988; Citovsky et al., 1992), suggesting that VirE2 is also transferred into plants independ- ently of T-DNA. In fact, VirE2, VirD2 and VirF may be transported to plants independently of both T-DNA and the virB genes, although tumorigenic virB-independent transfer has not been demonstrated (Chen et al., 2000). Intri- guingly, tumorigenicity is significantly inhibited when A. tumefaciens also carries the mobilizable RSF1010 plasmid (Binns et al., 1995; Stahl et al., 1998). Similarly, RSF1010 attenuates the viru- lence of L. pneumophila (Segal and Shuman, 1998b). In these two cases, the RSF1010:protein mobilization complex and the substrate of the virulence transport systems are thought to com- pete (Figure 1.3). That mutations in mobA suppress the effect of RSF1010 on L. pneumophila virulence is consis- tent with this hypothesis (Segal and Shuman, 1998b). The icm/dot genes substitute for tra sup- plied in trans to transmit RSF1010 to recipient L. pneumophila by conjugation, indicating that the RSF1010:MobA complex is a substrate for the se- cretory system encoded by icm/dot (Segal and Shuman, 1998b; Segal et al., 1998; Vogel et al., 1998). The effect of RSF1010 on virulence could be failure to transport efficiently, as yet uniden- tified, effector proteins that alter vesicle tar- geting within the macrophage because they are displaced by the RSF1010:MobA complex (Segal and Shuman, 1998a). The virB homologue lvh does not complement the effect of icmE/dotB mutations on virulence, but it did complement the effect of icmE/dotB mutations on conjugation (Segal et al., 1999). Thus, the physical require- ments for translocating the RSF1010:MobA com- plex and putative effector protein are not identical. The effects of RSF1010 on A. tumefaciens tumori- genicity are suppressed by over-expression of virB9, virB10 and virB11 (Ward et al., 1991), whose products are located in the cell mem- brane and form the putative conjugation pore (Christie, 1997a). Again, it has been suggested that an RSF1010:MobA complex may displace the T-DNA complex from the translocation apparatus due to the former’s higher copy number, the constitutive presence of its pro- cessed form, greater affinity for the trans- location complex or slow passage through the translocation pore (Binns et al., 1995; Stahl et al., 1998). The IncW plasmid pSa is an even stronger sup- pressor of tumorigenicity than RSF1010. Several lines of genetic evidence suggest that the osa gene product of pSa blocks protein VirE2 translocation (Chen and Kado, 1994, 1996; Lee et al., 1999). osa was first identified as the gene sufficient to cause pSa abolition of oncogencity (Chen and Kado, 1994). The specific effect on VirE2 rather than a protein–DNA complex is supported by the obser- vation that osa did not inhibit the conjugative transmission of the Ti plasmid. 12 G.C. FERGUSON AND J.A. HEINEMANN TABLE 1.2 tra genes homologous to icm/dot genesa L. pneumophila icm/dot ColIb-P9 (IncI1) RK2 (IncP) icmT traK icmS icmP trbA icmO trbC icmI traM icmK traN icmE trbI icmG traP icmC traQ icmD traR icmJ traT icmB traU dotA traY dotB traJ trbB dotC traI dotG traH a Adapted from Segal and Shuman (1999). 30
The osa product also does not inhibit T-DNA transfer. osa did not suppress oncogenicity when expressed in virE2 mutants as long as VirE2 was supplied by separate donors through extracellular complementation, or else it was produced by the recipient plant cell (Lee et al., 1999). The interesting ability for virE2 mutants to be complemented extracellularly by separate VirE2 donors was suppressed, however, when osa was expressed in the protein donor (Lee et al., 1999). Thus, the osa product specifically af- fects VirE2 translocation or function prior to T- DNA entry into the plant cell. The effects of pSa and RSF1010 on oncogenicity are similar but not identical. First, RSF1010 inhibits both VirE2 translocation and possibly T-DNA transfer, whereas pSa only prevents VirE2 translocation. Secondly, an RSF1010-protein complex is necessary for oncogenic suppression but only the osa gene product of pSa is required for suppression (Lee et al., 1999). Thirdly, over-expression of VirB9, VirB10 and VirB11 suppresses the RSF1010 effect on tumorigenicity but not the osa effect. These apparent dissimilarities may reflect only quantitative differences in the RSF1010 and pSa mechanisms, since RSF1010 partially inhibits oncogenicity and pSa completely abolishes tumor formation (Lee et al., 1999). However, the RSF1010 and pSa effects may have different mechanistic explanations. As discussed above, VirE2, VirD2 and VirF pro- teins are transported across the inner mem- brane by a virB24- and virD4-independent mechanism. The osa product, but not RSF1010, prevented VirE2, VirF and VirD2 from achieving normal periplasmic levels (Chen et al., 2000). This suggests that the osa product and MobA–RSF1010 could inhibit VirE2 translocation at different steps. While MobA– RSF1010 may inhibit the directed translocation of proteins through the putative outer mem- brane pore, the osa product may inhibit translocation across the inner membrane. Such a model is consistent with both the inner mem- brane localization of Osa (Chen and Kado, 1996) and the observation that VirB10, VirB11 and VirB12 over-expression did not restore tumor formation by A. tumefaciens carrying pSa (Lee et al., 1999). RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 13 Plasmid RSF1010 Effector protein Bacterial inner membrane Periplasm Bacterial outer membrane Macrophage or plant cell cytoplasm Cytoplasmic membrane MobA FIGURE 1.3 The mobilizable IncQ plasmid RSF1010 inhibits transmission of T-DNA from A. tumefaciens to plant cells. Furthermore, RSF1010 inhibits the ability of L. pneumophila to evade fusion of its phagosome with lysosomes inside the macrophage. The icm/dot genes that are required to prevent lysozome fusion are also necessary for conjugation of RSF1010. It has been proposed that icm/dot is a system that mediates secretion of proteins into the macrophage cytoplasm or phagosome during phagocytosis. The mobilized form of RSF1010 may inhibit virulence by competing with the natural substrate of these protein secretion systems. (Adapted from Segal and Shuman, 1998a.) 31
What came first, protein or DNA transfer? DNA and proteins are probably transferred be- tween species by similar mechanisms. The ef- fects of transferring non-nucleic acid molecules may sometimes be similar too; macromolecules, e.g. prions, other than nucleic acids possess gene-like qualities (Campbell, 1998; Heinemann and Roughan, 2000). Some proteins are not genes, but can influence epigenes that establish heritable phenotypes many generations after the protein has disappeared (Heinemann, 1999a). So conjugation may be a manifestation of protein secretion and, sometimes, protein se- cretion is another type of HGT. CONCLUSION HGT has established itself as a legitimate topic of study independently of the effects of the genes transferred on the biology of donor and recipient organisms. Nevertheless, the study of pathogens like A. tumefaciens and L. pneumophila, symbionts like Rhizobium meliloti, and phenotypes like antibi- otic resistance and crown gall, have each contrib- uted to the richness of the evidence supporting the notion that genes are less restricted by our no- tions of species sanctity than we have previously thought. In particular, the studies of bacterial con- jugation, crown gall disease and protein secretion have provided extensive mechanistic insight into how DNA is exchanged between kingdoms, spe- cies and siblings. Extensive similarities between genes identi- fied as either virulence or conjugation determi- nants provided an early hint that macro- molecular transport was a general phenom- enon. Those early hints have been vindicated by demonstrations of genetic interchangeability between some determinants (complementation studies) and genetic conflict between others. DNA is not special cargo but one of a number of molecules that might be transported by the same basic macromolecular transport systems. The ability to move molecules intercellularly has obvious implications for both single and multi- cellular organisms. Of immediate relevance are the diseases and recombinants that could arise from this nearly generic transport mechanism. But what of the molecules being transferred? Plasmids and viruses, for example, make excel- lent evolutionary livings transferring between or- ganisms, even evolving despite their effects on the host. Transfer alone might explain their exis- tence (Cooper and Heinemann, 2000). Did these genetic entities evolve a means to replicate by HGT, or was the existence of macromolecular transport enough for such semi-autonomous en- tities to evolve? Other kinds of molecules could transmit genetic information (Heinemann and Roughan, 2000). Could HGT be a mechanism for the evolution of genetic entities that are not nucleic acids? ACKNOWLEDGMENTS We thank A. Harker for critical reading of the manuscript and C.F. Delwichie for encouraging comments on our contribution to the first edi- tion. JAH acknowledges M. Stroun and P. Anker for their support, reprints and valuable insights. This work was supported in part by the Mar- sden Fund (Grant M1042 to JAH) and a Univer- sity of Canterbury Roper Scholarship (to GCF). REFERENCES Amábile-Cuevas, C.F. and Chicurel, M.E. (1992) Bacterial plasmids and gene flux. Cell 70: 189–199. Andersson, S.G.E., Zomorodipour, A., Andersson, J.O. et al. (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396: 133–140. Bates, S., Cashmore, A.M. and Wilkins, B.M. (1998) IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the Tra2 mating system. J. Bacteriol. 180: 6538–6543. Beijersbergen, A., den Dulk-Ras, A., Schilperoort, R.A. and Hooykaas, P.J.J. (1992) Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 256: 1324–1327. Bhattacharjee, M., Rao, X.-M. and Meyer, R.J. (1992) Role of the origin of transfer in termination of strand transfer during bacterial conjugation. J. Bacteriol. 174: 6659–6665. Binns, A.N., Beaupré, C.E. and Dale, E.M. (1995) Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J. Bacteriol. 177: 4890–4899. Buchanan-Wollaston, V., Passiatore, J.E. and Cannon, F. (1987) The mob and oriT mobilization functions of a 14 G.C. FERGUSON AND J.A. HEINEMANN 32
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C H A P T E R 2 Gene Cassettes and Integrons: Moving Single Genes Ruth M. Hall Gene cassettes are very simple, small mobile elements that generally include only a single complete gene (or open reading frame) or occa- sionally two genes and a recombination site called a 59-be that enables them to be mobilized. Movement of cassettes is achieved by site- specific recombination with the reaction catalyzed by members of the IntI-type DNA integrase family (tyrosine site-specific recombinases) that are encoded by integrons. Most commonly, cas- settes are incorporated into a specific site, an attI site, that is found in the integron adjacent to the intI gene. By repeated rounds of recombination between a 59-be and the attI site, the IntI integrase can incorporate more than one cas- sette into the same integron leading to the for- mation of short or long arrays of gene cassettes. However, because the integron-encoded IntI integrases can also recognize secondary recom- bination sites, cassettes can also be incorporated at many other locations, allowing them to be widely disseminated. Several classes of integron that are differentiated by differences in the se- quences of the IntI integrases have been found and many more are likely to be found in the future. However, the gene cassettes are shared. Among the integron classes, some are part of mobile elements, also known as inte-grons, while others are located on bacterial chromo- somes. The integrons that are themselves mobile are most important in spreading gene cassettes from strain to strain and species to spe- cies. The integrons that are an integral part of a bacterial chromosome may act as storehouses of genes for emergencies that are added to, as well as sampled and spread, by the mobile integrons. INTRODUCTION When the processes of horizontal gene transfer move DNA from one organism to another there is little impact unless the incoming DNA is stably maintained and expressed in the recip- ient organism. Several different processes can achieve this outcome and examples include the stable maintenance of a plasmid or incorpora- tion of the incoming DNA into the bacterial chromosome or into plasmids already resident in that cell. Incorporation of new DNA into an existing chromosome (bacterial or plasmid) can occur by homologous recombination, but only if homologous regions are present in both DNA species. However, other specific processes such as transposition or site-specific recombination can also lead to stable incorporation of parts or all of an incoming DNA molecule. The regions that can move in this way are generally discrete genetic elements and the ability of such mobile elements to shift their location enables them to move into entities such as plasmids and conjugative transposons that are able to move from cell to cell with ease. Because of this, translocatable elements are an important force in horizontal gene transfer in the bacterial world and any associated gene or group of genes is Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 19 All rights of reproduction in any form reserved. 37
ultimately able to gain access to many different organisms and species. In this chapter, the family of mobile elements known as gene cas- settes and their host elements, the integrons, are described. Integrons which are themselves mobile are also described. Further information on specific aspects can be found in recent re- views (Hall and Collis, 1995, 1998; Recchia and Hall, 1995a, 1997; Rowe-Magnus et al., 1999). FUNCTIONAL DEFINITION OF GENE CASSETTES AND INTEGRONS The definitions of gene cassettes and integrons are to a considerable extent interdependent as integrons are first and foremost defined by their ability to capture gene cassettes. Indeed, this is how they were found. Gene cassettes were ini- tially identified by virtue of the fact that many different gene cassettes, each containing a dif- ferent antibiotic resistance gene, can be found in the same sequence context (Cameron et al., 1986; Hall and Vockler, 1987; Ouellette et al., 1987; Wiedemann et al., 1987; Sundström et al., 1988; Hall et al., 1991). This situation allowed the identification of the approximate boundaries of gene cassettes and also of the conserved back- bone sequence into which the cassettes slot (Hall and Vockler, 1987; Sundström et al., 1988; Stokes and Hall, 1989). Within this conserved backbone, a gene was found whose product bore a significant resemblance to integrases (tyrosine site-specific recombinases) that are harbored by the genomes of some temperate phage (Hall and Vockler, 1987; Ouellette and Roy, 1987; Sundström et al., 1988; Stokes and Hall, 1989). Experimental evidence that this integrase (IntI1) was active was soon forth- coming (Martinez and de la Cruz, 1988, 1990). The term integron was originally coined to 20 R.M. HALL Empty integron Free gene cassette Integron with one cassette Integrated cassette FIGURE 2.1 Insertion of a circular gene cassette into an integron. (A) An empty integron, showing the three distinctive features: an intI gene that encodes the IntI integrase, an adjacent recombination site, attI (hatched box), and promoters Pc and Pint. (B) A gene cassette in its circular form consisting of a gene or open reading frame (ORF) and a 59-be recombination site (filled box). (C) An integron containing one gene cassette, with the boundaries of the integrated cassette shown below. IntI-catalyzed recombination between attI in the integron and the 59-be in the circular cassette results in insertion of the cassette into the integron. The ORF in the inserted cassette is now transcribed from Pc. gttrrry (A) and GTTRRRY (B) represent the 7 bp core sites surrounding the recombination crossover point in the attI site of the integron and in the 59-be of the circular cassette respectively. The configuration of these bases after incorporation of the cassette is shown in C. Further cassettes may be similarly inserted at attI, resulting in the accumulation of arrays of cassettes. 38
describe this group of elements (Stokes and Hall, 1989) but, as further types of integrons have since been found, they are now designated class 1 integrons. In 1991, all of the sequence information that was available was drawn together and the general and current definition of a gene cassette as a single gene or open reading frame coupled with a downstream 59-be recombination site emerged (Hall et al., 1991). This study also gave rise to a model for the integration and excision of gene cassettes that predicted the existence of a circular form of cassettes (Figure 2.1). Experimental studies of cassette movement, using the IntI1 integrase, were subsequently reported. Precise excision of gene cassettes was demonstrated, thus defining them experimentally (Collis and Hall, 1992a) and the circular form of cassettes was also isolated (Collis and Hall, 1992b). Demonstration of the incorporation of circular gene cassettes into an integron completed the picture (Collis et al., 1993). An interesting aspect of the latter study is that cassettes are preferentially incorporated at the attI site of the integron to become the first cas- sette in an array of gene cassettes. Finally, in 1995, a known gene cassette was found in a plasmid that does not contain an integron (Recchia and Hall, 1995b). This demon- strated that cassettes can also move into almost any location, where they become fixed, and fur- ther cassettes that have been incorporated at such secondary sites have since been reported. GENE CASSETTES Gene cassettes are the smallest of the known types of mobile elements. Generally, they GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 21 A. Free circular cassette B. Integrated linear cassette 1L 2R 2L 1R LH simple site RH simple site Coding region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AAC X · A * T GTT . . . . . . . . . . T ATG 59-be 1L 2R 2L 1R LH simple site RH simple site Coding region . . . . . . . . . . . . . . . . . . . . . . AAC X · A * TT ATG G FIGURE 2.2 Structure of gene cassettes. (A) A cassette in its free, circular form. (B) A cassette in integrated, linear form showing the coding region of the gene and the 59-be recombination site. The extent of the coding region (not to scale) is marked by start (ATG) and stop (*) codons. Boxes surround 7 bp core site sequences, related to the consensus GTTRRRY, which lie within putative IntI binding sites that include the core sites as well as flanking bases. Left hand (LH) and right hand (RH) simple sites consist of pairs of binding sites (1L and 2L; 2R and 1R respectively). The relative orientations of the core sites are indicated with arrows. Bases found in all 59-be are shown, while other bases that conform to the core site consensus are represented by dots, and an extra base in 2L is marked by an X. In any individual 59-be, 1L and 1R sites are closely related, as are 2L and 2R, but the bases between them are not. An inverted repeat, represented by a pair of arrows, lies between 2L and 2R and has a variable sequence and length. The recombination crossover point is indicated by a vertical arrow. When the circular cassette is linearized by insertion into the attI site of an integron (B), the last six bases of 1R in the circular cassette become the first six bases of the integrated cassette. 39
consist of a single gene or ORF together with a downstream IntI-specific recombination site known as a 59-be (59-base element). Occa- sionally, two ORFs are present in a single cas- sette. Each cassette is a discrete mobile element that can exist in either a free, closed-circular form or an integrated linear form (Figures 2.1 and 2.2). As cassettes contain no replication functions, the closed circular form cannot repli- cate and its role is limited to that of an interme- diate in cassette movement (Collis and Hall, 1992b). The boundaries of the linear gene cas- sette form have been precisely located (Stokes et al., 1997) and each cassette commences with a TT doublet and ends with a G residue. This is be- cause the strand exchange that occurs during cassette integration occurs between the G and TT of a completely conserved triplet that forms part of the 7 bp core site (GTTaggc or GTTRRRY) found at one end of 59-be (Stokes et al., 1997) and also in attI sites at the point of cas- sette integration (Hansson et al., 1997). The se- quence of any specific 59-be is found by joining the sequence at the right-hand (RH) end of the cassette to that at the left-hand (LH) end, to rec- reate the circular configuration (Figure 2.2). Be- cause each cassette contains a unique 59-be (see below) the 59-be are named after the gene in the cassette. Cassettes are compactly organized (Figure 2.2). In the integrated form, as few as 7 bp sepa- rate the beginning of the cassette from the initia- tion codon of the gene, leaving no space for a promoter and little space for a ribosome binding site. Commonly, the termination codon of the gene is very close to, or even within, the region ascribed to the 59-be recombination site. How- ever, occasionally a promoter is present within the cassette and translational attenuation regu- latory signals have also been found in the cmlA1 cassette and other related cmlA cassettes (Stokes and Hall, 1991). Although the cassettes that contain antibiotic resistance genes were the first to be identified, the diverse range of enzymatic functions encoded by the genes that confer resistance indicated that any gene could potentially become associated with a 59-be to form a functional gene cassette. There are now over 60 identified gene cassettes that confer resistance to antibiotics. These include genes for inner-membrane transporters (efflux proteins), β-lactamases belonging to classes A, B and D, acetyl-, adenylyl-, phosphoryl- and ribosyl- transferases with a variety of antibiotic substrate specificities, and dihydrofolate reductases (Recchia and Hall, 1995a; Hall and Collis, 1998; Mazel and Davies, 1999). Among the gene cas- settes found in the small chromosome of the Vibrio cholerae strain that has been completely se- quenced, there are also a few that contain antibi- otic resistance genes or potential resistance genes (Heidelberg et al., 2000). However, some of the gene cassettes found in V. cholerae or V. mimicus strains determine other functions. Gene products with known functions include a heat-stable toxin that is found in relatively few V. cholerae strains (Ogawa and Takeda, 1993) and a mannose- fucose-resistant haemagglutinin (Barker et al., 1994) and alipase (Rowe-Magnus et al., 2001). Potential functions for further genes have been proposed on the basis of relationships to known proteins but the majority of ORF remain unidentified (Rowe-Magnus et al., 1999; Clark et al., 2000). Among the gene cassettes that contain known antibiotic resistance genes, only one includes two open reading frames, the aacA1–orfG cassette (Accession. No. AF047479). However there are several cassettes that include two ORF found both in class 1 integrons (Accession No. AF047479) and in the Vibrio cholerae collection (Clark et al., 2000; Heidelberg et al., 2000). One of these determines the mannose-fucose- resistant haemagglutinin, but whether both ORF are needed for this activity is not known (Barker et al., 1994). A cassette from a Xantho- monas campestris isolate includes both the XbaI restriction and modification genes (Accession No. AF051092). There are also some cassettes where the orientation of the gene (or genes) is reversed. Although these exceptions remain to be adequately accounted for, a possible explana- tion for cassettes containing genes in the oppo- site to normal orientation is that the genes are not essential to V. cholerae and the sequence has drifted such that the original ORF is no longer detectable. A very large number of 59-be sites have been found (Stokes et al., 1997). Indeed, each of the cassettes found in class 1 integrons contains a 22 R.M. HALL 40
unique 59-be. Initially, 59-be were identified as a consensus sequence of 59 bp found in a few cas- settes (Cameron et al., 1986) and this alignment led to the subsequent identification, in other gene cassettes, of related but more diverged se- quence elements, some of which had different lengths (Hall et al., 1991). It is now known that 59-be can range in size from 57 to 141 bp (Recchia et al., 1995a). However, the term 59-be has found common currency despite the size variation and has been retained for all members of this family. The VCR found in V. cholerae cas- settes form a more homogeneous group (Barker et al., 1994) but are also members of the 59-be family (Recchia and Hall, 1997). Indeed 59-be that are closely related to the VCR sequence are found among the 59-be associated with antibi- otic resistance genes. INTEGRONS As described above, integrons include two dis- tinctive features, an intI gene and an adjacent attI site, that enable them to capture gene cas- settes. They do not necessarily include a gene cassette and class 1 integrons that contain no cassettes have been found in the wild and cre- ated experimentally (Bissonnette and Roy, 1992; Collis and Hall, 1992a; Rosser and Young, 1999). However, integrons generally do contain one cassette or an array of two or more gene cas- settes. The cassette array can be very long as is the case for the V. cholerae chromosomal integrons, where the sequenced strain has more than 170 cassettes (Heidelberg et al., 2000) and other strains are estimated to include at least 100 cassettes (Clark et al., 2000). As the vast majority of the known gene cas- settes contain a gene but not a promoter (Hall et al., 1991; Recchia and Hall, 1995a), an upstream promoter is required for the expression of the genes contained in cassettes. This promoter, Pc, is supplied by the integron and is the third dis- tinctive feature of an integron (Figure 2.1). In the case of the class 1 integrons, which are the commonest type of integrons found in antibi- otic-resistant clinical isolates, the Pc promoter is located just inside the beginning of the intI1 gene (Hall and Vockler, 1987; Stokes and Hall, 1989). All transcripts of the array of gene cas- settes start at Pc (Collis and Hall, 1995). The fact that the integron supplies the promoter imposes an orientation constraint on gene cassettes if their genes are to be expressed. In all cases where the gene function is known and expres- sion can be monitored, the orientation of the cassette-associated gene that is found is the one that permits expression. This orientation is achieved only if the 59-be is located down- stream of the gene. MANY CLASSES OF INTEGRON That there were, in addition to the class 1 integrons, other classes of integrons containing the same gene cassettes, but a different con- served backbone and thus a potentially a dis- tinct IntI gene, was known from the earliest studies (Cameron et al., 1986; Hall and Vockler, 1987; Wiedemann et al., 1987). In fact, the first complete gene cassettes to be sequenced were the dfrA1 and aadA1 cassettes that are respon- sible, respectively, for the resistance to tri- methoprim and to spectinomycin and strepto- mycin conferred by the transposon Tn7. How- ever, these two cassettes were identified as cas- settes only after they were also subsequently found in class 1 integrons (Cameron et al., 1986; Sundström and Sköld, 1990; Hall et al., 1991). Indeed, the relationship of the predicted prod- ucts of the intI1 gene and the partially se- quenced intI2 gene in Tn7 was identified before the nature of the gene products was known (Hall and Vockler, 1987). However, the Tn7 intI2 gene (intI2* ) is defective, because it includes an in-frame stop codon that precludes production of a functional protein and this may explain why so few examples of class 2 integrons with different arrays of gene cassettes have been re- ported thus far (Recchia and Hall, 1995a). As the level of identity between IntI1 and IntI2* was only 45%, it was obvious that these two classes of integrons (Class 1 and 2) were likely to be rep- resentative of a vast family of integrons, each encoding a related, but distinct, IntI, but re- taining the potential to share the same gene cas- settes. Subsequently, a third class of integron was found in clinical isolates of antibiotic GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 23 41
resistant Serratia marcescens in Japan (Arakawa et al., 1995) and again the cassettes are ones that had been found in class 1 integrons. INTEGRONS IN BACTERIAL CHROMOSOMES Although much is known about the impact of gene cassettes on the emergence of multiply antibiotic-resistant strains of Gram-negative bac- teria, their provenance is not restricted to antibi- otic resistance genes. The small chromosome of Vibrio cholerae has recently been shown to include an intI gene, intI4, adjacent to a long array of gene cassettes (Mazel et al., 1998; Clark et al., 1997, 2000; Heidelberg et al., 2000), and thus contains an integron. Different strains contain different cassette arrays (Clark et al., 2000). The gene cas- settes were found first (Ogawa and Takeda, 1993; Barker et al., 1994) but, as in the case of Tn7, they were not initially recognized as such. A repetitive sequence element called a VCR was identified but its similarity to 59-be was recognized later (Recchia and Hall, 1997). However, VCR were shown to be present in the chromosomes of sev- eral further Vibrio species such as V. mimicus, V. anguillarum, V. hollisae and V. metschnikvii but not in others (Ogawa and Takeda, 1993; Barker et al., 1994; Mazel et al., 1998; Clark et al., 2000). The result of these studies are not always consistent, however, they do indicate that an integron and gene cassettes are also likely to be present in many, but not necessarily all, Vibrio species. The partially sequenced intI5 gene from V. mimicus has diverged from intI4 to the same degree as other known chromosomal genes, providing evidence that an integron was a feature of the genome of the common ancestor (Clark et al., 2000). This conclusion has recently been con- firmed for other Vibrio species (Rowe-Magnus et al., 2001). MORE INTEGRONS, MORE CASSETTES It is likely that Vibrio species represent only the first case where an integron is found in the bacterial genome and, therefore, that further ex- amples of gene cassettes and new types of integrons will come to light as more bacterial genomes are sequenced. Further classes of integron may also be found on plasmids recov- ered from different environments. In fact, some genes whose products are clearly related to the IntI integrases can be found amongst the se- quences available in the partially sequenced genomes of Shewanella putrefaciens, Treponema denticola, Geobacter sulfurreducens (Rowe-Magnus et al., 1999, 2001; Nield et al., 2001). However, whether these genes indicate the presence of an integron must await the identification of gene cassettes to go with them. Recently, three new intI genes (intI6, 7 and 8) have been recovered from environmental soil samples and in two cases a potential adjacent gene cassette has been recovered, together with the intI gene (Nield et al., 2001). This confirms that integrons and gene cassettes are likely to be common in the bacterial world. SOME INTEGRONS CAN MOVE In the gene cassette/integron system, it is the cassettes that are the mobile elements. How- ever, in the context of their contribution to hori- zontal gene transfer, it is obvious that integrons and gene cassettes can have a substantial impact, as is the case with respect to antibiotic resistance, only if the integron can gain wide access to a variety of bacterial species. This occurs readily when it is located on a plasmid and this is best achieved if the integron can also translocate. Indeed, class 1 integrons are found in many different genetic contexts, mainly on different plasmids (Hall and Vockler, 1987; Stokes and Hall, 1989; Hall et al., 1994). Class 1 integrons are in fact either transposable ele- ments as exemplified by Tn402 (Rådström et al., 1994) or, more often, defective derivatives of them (Brown et al., 1996; Rådström et al., 1994; Liebert et al., 1999; Partridge et al., 2001a,b). The latter can obviously be moved so long as both outer ends are intact and a set of suitable trans- position genes are present in the same cell. Often they are found flanked by a 5 bp duplica- tion of the target site, indicating that they have 24 R.M. HALL 42
reached their current location by transposition (Brown et al., 1996; Partridge et al., 2001a,b). In a few cases, class 1 integrons that are unable to mobilize themselves have moved into, and are now found within, another transposon. This is the case for In2 which is found within Tn21 (Liebert et al., 1999), for In4, which is found in Tn1696 (Partridge et al., 2001a), and for In28, which is found in Tn1403 (Partridge et al., 2001b). The exemplar of a class 2 integron is the transposon Tn7 which contains three gene cas- settes dfrA1–sat2–aadA1 and three other known members of this group are transposons that differ in the identity of the first cassette in the array or have lost this cassette (Recchia and Hall, 1995a). The class 3 integron provides a contem- porary example of the rapid spread of resistance genes carried by self-transmissible plasmids. It was first isolated in Japan, in 1993, and has al- ready spread to several other bacterial species (Senda et al., 1996). Our preliminary evidence and that of others (Shibata et al., 1999) indicates that the class 3 integron is, not surprisingly, also a mobile element. In all of these cases, the integron is defined as the complete structure bounded by the terminal inverted repeats or that part of such a structure that remains. THE RECOMBINATION SYSTEM The components of the recombination system that effect cassette movement are the IntI-type integrases, the integron-associated attI sites and the cassette-associated 59-be sites. The known IntI integrases form a family of related proteins that share highly significant levels of identity (35–94%). They also share cer- tain features with other members of the integrase or tyrosine recombinase super-family, but pairwise identities between these other integrases and IntI integrases is generally less than 25%. The most obvious of the shared fea- tures are the two conserved domains or boxes that are normally used to identify members of this super-family (Ouellette and Roy, 1987; Sundström et al., 1988; Stokes and Hall, 1989). However a recent alignment of the C-terminal catalytic domain of all known members of the tyrosine recombinase superfamily has revealed further shared “patches” (Nunes-Düby et al., 1998). Other members of the IntI family are known (Nield et al., 2001; Rowe-Magnus et al., 2001), but whether all of them are associated with gene cassettes and are thus part of an integron remains to be established. Only the re- actions catalyzed by the IntI1 integrase have been studied in detail and these are described briefly below. However, IntI3 and IntlI4 have also been shown to be active (Hall et al., 1999; Rowe-Magnus et al., 2001; Collis and Hall, unpublished). In addition to recombination between a 59-be and attI1 site, which occurs when a cassette is in- corporated into an integron, IntI1-catalyzed re- combination between two 59-be or two attI1 sites can also occur (Martinez and de la Cruz, 1988, 1990; Hall et al., 1991; Recchia et al., 1994; Hansson et al., 1997; Stokes et al., 1997; Hall et al., 1999; Partridge et al., 2000; Collis et al., 2001). The reactions that have been reported are listed in Table 2.1. The efficiencies of the integration reac- tions have also been compared (Collis et al., 2001). Recombination between two 59-be sites occurs at high frequency but is less efficient than recombi- nation between a 59-be and the attI1 site. How- ever, excisive recombination involving two 59-be is important because it can lead to excision of the downstream cassettes in an array. Recombina- tion between two attI sites occurs at a much lower frequency than the other reactions (Hansson et al., 1997; Partridge et al., 2000) and is unlikely to be an important event in cassette movement. Re- combination between a 59-be and a secondary site (20 rs) also occurs at low frequency (Francia et al., 1993; Recchia et al., 1994) and is an important GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 25 TABLE 2.1 Recombination events catalyzed by IntI1 Participating sites Integration Excision attI1 × 59-be + + 59-be × 59-be + + attI1 × attI1 + NDa 59-be × 20 rs + –b attI1 × 20 rs + –b a Not determined. b Precise excision of a cassette located at a 20 rs is in most cases unlikely to be possible. 43
reaction that permits gene cassettes to be inte- grated at almost any position. The role of recom- bination between attI sites and 20 rs (Hansson et al., 1997; Collis et al., 2001) is less obvious. The attI sites are part of the integron back- bone and are distinguished by the fact that they are the sites into which cassettes are incorpo- rated. In known integrons, they are located up- stream of the intI gene. However the sequences of the regions adjacent to the first gene cassette in each of the four well-established integron classes are not highly conserved. The extent of the attI1 site has been established experimen- tally (Recchia et al., 1994; Hansson et al., 1997; Partridge et al., 2000). The complete attI1 site (Figure 2.3), which is required for recombina- tion with a 59-be partner, includes 56 bp from the left side of the crossover point and at least a further 9 bp to the right of the crossover (Hall et al., 1999; Partridge et al., 2000). Within this region, four binding sites for IntI1 have been found and delineated using foot-printing tech- niques (Collis et al., 1998; Gravel et al., 1998). A single molecule of IntI1 bound to the strongest binding domain protects a total of 14 bp which includes the 7 bp core site regions (Collis et al., 1998). Two of the IntI1 binding domains (1 and 2 in Figure 2.3A) are inversely oriented with re- spect to one another and form a simple site equivalent to those recognized by other tyrosine recombinases. The additional IntI1-binding do- mains (3 and 4 in Figure 2.3A) considerably en- hance the activity of attI1 in recombination with a 59-be (Recchia et al., 1994; Hall et al., 1999; Partridge et al., 2000) but are not required for recombination with a complete attI1 partner (Hansson et al., 1997; Partridge et al., 2000). They appear to bind IntI1 more strongly than sites 1 and 2 (Collis et al., 1998), and may play a role in retaining the newly synthesized mole- cules of IntI1 in the proximity of attI1. Simple sites can also be found in the expected positions in attI2, 3 and 4 (Collis et al., 1998), but whether these sites resemble attI1 in binding four molecules of their cognate integrase (IntI2, 3 or 4) remains to be established experimentally. Preliminary data suggest that each IntI prefer- entially recognizes its cognate attI site (Hall et al., 1999), but this also remains to be established rigorously. The 59-be have a different architecture from that of attI sites. All 59-be comprise two regions of 25 bp that are each related to a consensus se- quence (Collis and Hall, 1992a; Stokes et al., 1997). In any individual 59-be the consensus re- gions are imperfect inverted repeats of one an- other and are separated by a region of highly variable sequence and length that is, in most in- stances, also an inverted repeat. 59-be are rather unusual in that they include two simple sites, only one of which is the site of strand exchange (Stokes et al., 1997). The LH and RH consensus regions correspond to the bulk of these simple sites but, based on foot-printing data from attI1 (Collis et al., 1998), the IntI1-binding regions are likely to extend further (Figure 2.3) and a weak consensus is found for some of the bases in the extensions (Collis and Hall, 1992b; Stokes et al., 1997). A striking feature of this family of recom- bination sites is that the relationship between 26 R.M. HALL FIGURE 2.3 Recombination sites. The arrangement of 7 bp core sites (arrows) that form part of the larger IntI binding domains is shown. For simplicity the individual core sites are numbered (above the arrows). The vertical arrows show the position of the crossover. 44
the sequences of the LH and RH consensus re- gions is generally retained in preference to ad- herence to the consensus sequence (Hall et al., 1991; Stokes et al., 1997). This feature is yet to be adequately explained in terms of the activity of these sites. The inverted repeatedness is imper- fect in the simple site regions; the 1L and 1R core sites and the 2L and 2R core sites mirror one an- other but the bases that separate them do not and there is an extra base in 2L (Figure 2.2). The distance between 1L and 2L is 5 bp, but either 5 or 6 bp separate 2R from 1R. It remains to be established which of the differences between the LH and RH simple sites are important in ensuring that the recombination crossover occurs at 1R. CONCLUSIONS The role of gene cassettes and integrons in the emergence and spread of antibiotic resistance is well established. The same system has now been implicated in the evolution of bacterial genomes, and the extent of this involvement al- ready appears to be quite significant. Many more new classes of integrons and new gene cassettes will undoubtedly be found in the not too distant future. How gene cassettes are cre- ated and how the different integrases recognize the same cassettes as well as other intricacies of the site-specific recombination system are im- portant, but complex, questions that remain to be examined. REFERENCES Arakawa, Y., Murakami, M., Suzuki, K. et al. (1995) A novel integron-like element carrying the metallo β-lactamase gene blaIMP. Antimicrob. Agents Chemother. 39: 1612–1615. Barker, A., Clark, C.A. and Manning, P.A. (1994) Identification of VCR, a repeated sequence associated with a locus encoding a haemagglutinin in Vibrio cholerae O1. J. Bacteriol. 176: 5450–5458. Bissonnette, L. and Roy, P.H. 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Partridge, S.R., Recchia, G.D., Scaramuzzi, C. et al. (2000) Definition of the attI1 site of class 1 integrons. Microbiology 146: 2855–2864. Partridge, S.R., Brown, H.J., Stokes, H.W. and Hall, R.M. (2001a) Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrob. Agents Chemother. 45: 1263–1270. Partridge, S.R., Recchia, G.D., Stokes, H.W. and Hall, R.M. (2001b) A family of class 1 integrons related to In4 from Tn1696. Antimicrob. Agents Chemother. In press. Rådström, P., Sköld, O., Swedberg, G. et al. (1994) Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J. Bacteriol. 176: 3257–3268. Recchia, G.D., Stokes, H.W. and Hall, R.M. (1994) Characterisation of specific and secondary recombination sites recognised by the integron DNA integrase. Nucleic Acids Res. 22: 2071–2078. Recchia, G.D. and Hall, R.M. (1995a) Mobile gene cassettes: a new class of mobile element. Microbiol. 141: 3015–3027. Recchia, G.D. and Hall, R.M. (1995b) Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010. Mol. Microbiol. 15: 179–187. Recchia, G.D. and Hall, R. M. (1997) Origins of the mobile gene cassettes found in integrons. Trends Microbiol. 389: 389–394. Rowe-Magnus, D.A., Guérout, A.-M. and Mazel, D. (1999) Super-integrons. Res. Microbiol. 150: 641–651. Rowe-Magnus, D.A., Guérout, A.-M., Ploncard, P., Dychinoco, B., Davies, J. and Mazel, D. (2001). The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc. Natl Acad. Sci. USA 98: 652–657. Rosser, S.J. and Young, H.-K. (1999) Identification and characterization of class 1 integrons in bacteria from an aquatic environment. J. Antimicrob. Chemother. 44: 11–18 Senda, K., Arakawa, Y., Ichiyama, S. et al. (1996) PCR detection of metallo-β-lactamase (blaIMP) in gram- negative rods resistant to broad-spectrum β-lactams. J. Clinical Microbiol. 34: 2909–2913. Shibata, N., Kurokawa, H., Yagi, T. and Arakawa, Y. (1999) A class 3 integron carrying the IMP-1 metallo-β-lactamase gene found in Japan. 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco. Stokes, H.W. and Hall, R.M. (1989) A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol. Microbiol. 3: 1669–1683. Stokes, H.W. and Hall, R.M. (1991) Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation. Plasmid 26: 10–19. Stokes, H.W., O’Gorman, D.B., Recchia, G.D. et al. (1997) Structure and function of 59-base element re- combination sites associated with mobile gene cassettes. Mol. Microbiol. 26: 731–745. Sundström, L. and Sköld, O. 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C H A P T E R 3 A Corynebacterium Plasmid Composed of Elements from Throughout the Eubacteria Kingdom Andreas Tauch and Alfred Pühler Multiple antimicrobial resistance in human pathogens is a global medical problem. Espe- cially, Gram-positive microorganisms show alarming increases in antibiotic resistances. The complete DNA sequence of the 51-kb multiresistance plasmid pTP10 from the Gram- positive human pathogen Corynebacterium striatum M82B provides genetic information about acquired resistance mechanisms to antimicrobial agents in this species. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of a mosaic structure comprising eight DNA segments the bound- aries of which are represented by horizontal mobile elements. The DNA segments of pTP10 turned out to be virtually identical to a plasmid-encoded macrolide and lincosamide resistance region from the human pathogen Corynebacterium diphtheriae, a chromosomal DNA region from Mycobacterium tuberculosis, a mobile chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum, several transposable elements from Gram- negative phytopathogenic Pseudomonas, Xantho- monas and Erwinia species, and to a trans- posable aminoglycoside resistance region from the Gram-negative animal pathogen Pasteurella piscicida. This provides molecular evidence that natural routes exist by which antibiotic resis- tance genes from bacteria of different habitats and geographical origin can be assembled in a human pathogen. This shows that highly di- verged species that last shared a common ancestor about 2 billion years ago can still ex- change genes. Consequently, horizontal gene transfer of antibiotic resistance genes is an im- portant mechanism which limits the successful use of antimicrobials in the clinical treatment of human infections. INTRODUCTION Drug resistance in human pathogens is the result of overuse of antimicrobials in medicine and agriculture (Witte, 1998). This overuse of antibiotics has led to the rapid evolution of bac- teria that are resistant to multiple drugs such that even vancomycin and teicoplanin, the drugs of last resort, are no longer effective against some bacterial isolates. Generally, a micro- organism can have either intrinsic or acquired resistance to antibiotics (Tan et al., 2000). In- trinsic resistance is a stable genetic property, arising from mutation in the chromosomal DNA. As each mutation confers only a slight al- teration in susceptibility, microorganisms need to accumulate several mutations to become in- trinsically resistant to antibiotics. Alternatively, resistance is acquired by genetic exchange with another microorganism from the same or Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 29 All rights of reproduction in any form reserved. 47
another genus (Tan et al., 2000). Resistance genes can be transferred among bacteria and can be integrated into the bacterial chromosome to be stably inherited from one generation to the next. Additionally, antibiotic resistance genes can be maintained in an extra-chromosomal state on a bacterial plasmid. Plasmids that can transfer DNA to adjacent bacteria are known as conjugative plasmids. As conjugation can occur in a broad range of species it is one of the main mechanisms through which resistance genes are transferred between bacteria (Dröge et al., 1998, 1999). Therefore, such plasmids can be classed among the horizontal mobile elements that also include phages, integrons, trans- posons, and insertion sequences. Accordingly, the transfer of horizontal mobile elements occurs via conjugation, transduction, transposi- tion, and transformation. Today, integrated genome research offers the chance to analyze large resistance plasmids (R plasmids) and acquired antibiotic resistances at the nucleotide level. Highly automated se- quencing machines and processes have been developed to allow large-scale DNA sequencing and subsequent DNA sequence interpretation by bioinformatics. The information obtained within the scope of such projects not only sheds 30 A. TAUCH AND A. PÜHLER FIGURE 3.1 Genetic organization of the 51409-bp multiresistance plasmid pTP10 identified in C. striatum M82B. The organization of the open reading frames (ORFs) and the position of transposons and insertion sequences is presented. The eight DNA segments of pTP10 are specifically marked (I–VIII). Dotted lines represent segment boundaries corresponding to the insertion of mobile elements. The acquired antibiotic resistance genes are marked by filled boxed. Details on the DNA sequence of pTP10 are available from GenBank accession number AF139896. 48
new light on acquired antibiotic resistance mechanisms, but also on horizontal gene transfer and plasmid evolution (Perretten et al., 1997; Tauch et al., 2000). The genetic data described below focus on a plasmid project which dealt with the DNA sequence analysis of the large multiresistance plasmid pTP10 from the human pathogen Corynebacterium striatum M82B (Tauch et al., 2000). The complete DNA sequence revealed insights into how pTP10 is genetically orga- nized and, in particular, how a multiresistance plasmid from a human clinical source has evolved over time. Virtually identical DNA seg- ments have been identified in a soil bacterium and in plant, animal and human pathogens. This finding implies that horizontal gene transfer has played a central role in the evolu- tionary history of pTP10. ANALYSIS OF AN ANTIBIOTIC RESISTANCE PLASMID ISOLATED FROM CORYNEBACTERIUM STRIATUM BY INTEGRATED GENOME RESEARCH In recent years, the Gram-positive bacterium C. striatum has been recognized with increasing frequency as an important opportunistic human pathogen, especially in immuno- compromised patients and in patients under in- tensive care. In clinical diagnostics, numerous isolates of C. striatum were found to be highly re- sistant to the majority of clinically relevant anti- biotics which more and more resulted in the failure of antibiotic treatment of C. striatum- mediated human infections. C. striatum M82B was initially isolated from the bacterial flora of an otitis media patient in a Japanese hospital. It was shown to carry the R plasmid pTP10 encoding resistances to the antibiotics chlor- amphenicol, erythromycin, kanamycin, and tetracycline (Tauch et al., 1995a). Since the plasmid genome of pTP10 was determined to be only 51 kb in size, it represents an ideal model system for the analysis of acquired antibiotic resistance by integrated genome research. The total DNA sequence of the R plasmid pTP10 was determined and subsequently annotated by means of an automated genome interpretation system. In such a way the complete gene struc- ture of pTP10 was identified. The pTP10 se- quence contains 47 open reading frames (ORFs) and an additional five incomplete coding regions. Moreover, pTP10 harbors five trans- posons (Tauch et al., 1995b, 1998) and two addi- tional insertion sequences. Figure 3.1 presents a detailed map of the ORFs found on pTP10 as well as other relevant structural features. Based on the DNA sequence data, further ex- periments concentrated on the antibiotic resis- tance genes of pTP10, some of which are integral parts of horizontal mobile elements (Figure 3.1). Besides the known resistance determinants of pTP10 to chloramphenicol (cml(A), cmx(A)), erythromycin (ermLP, ermCX), kanamycin (aphA1-IAB), and tetracycline (tetAB), DNA se- quence analysis revealed the presence of a dupli- cated chloramphenicol resistance region (cml(B) and cmx(B)) and genes probably involved in bacitracin (bacA) and streptomycin resistance (strAB). However, antibiotic susceptibility studies clearly demonstrated that the bacitracin and streptomycin resistance determinants are inac- tive on pTP10 (Tauch et al., 2000). In addition, DNA sequence interpretation made it possible to deduce the respective resistance mechanisms en- coded on pTP10 and to identify virtually identical resistance genes in Gram-positive and Gram- negative bacteria of different habitats. Most inter- estingly, for tetracycline a new resistance mecha- nism could be proposed that is based on hetero- dimerization of two ABC transporters resulting in an oxytetracycline and oxacillin cross-resistance (Tauch et al., 1999). The deduced data concerning the resistance determinants of pTP10 are summa- rized in Table 3.1. HORIZONTAL GENE TRANSFER AND ACQUIRED ANTIBIOTIC RESISTANCE IN CORYNEBACTERIUM STRIATUM M82B Taking into account the DNA sequence annota- tion and the structural data, the pTP10 plasmid A PLASMID COMPOSED OF ELEMENTS FROM THROUGHOUT THE EUBACTERIA KINGDOM 31 49
was subdivided into eight genetically distinct DNA segments, six of which are involved in an- tibiotic resistance. One of the DNA segments present on pTP10 comprises the composite re- sistance transposon Tn5432 that consists of two IS1249 sequences flanking the erythromycin re- sistance gene region (Figure 3.1; I). The central part of Tn5432 was found to be virtually iden- tical at the nucleotide level to the antibiotic resis- tance gene region of plasmid pNG2 from the human pathogen Corynebacterium diphtheriae (Figure 3.2A) encoding an inducible resistance to macrolide and lincosamide antibiotics. In contrast to the genetic arrangement on pTP10, the sequenced resistance region of pNG2 is not part of a Tn5432-like mobile element (Figure 3.2A). The second DNA segment of pTP10 which is involved in antibiotic resistance is located downstream of Tn5432 and comprises a DNA region with a high G + C content (Figure 3.1; II). The gene products of the respective coding re- gions showed the highest similarity to proteins encoded by the Mycobacterium tuberculosis 32 A. TAUCH AND A. PÜHLER TABLE 3.1 Acquired antibiotic resistance gene regions of pTP10 from C. striatum M82B and its closest relatives pTP10 gene region Resistance genes Resistance mechanism Closest relative/ microorganism Erythromycin resistance ermLP, ermCX 23S rRNA methylation Corynebacterium diphtheriae (Gram-positive human pathogen) Tetracycline resistance tetA, tetB Efflux via heterodimerization of ABC transporters No similarity found Bacitracin resistance bacA Phosphorylation of undecaprenol (inactive in pTP10) Mycobacterium tuberculosis (Gram-positive human pathogen) Chloramphenicol resistance copy A and copy B cml(A), cmx(A) cml(B), cmx(B) Antibiotic export Corynebacterium glutamicum (Gram-positive soil bacterium) Streptomycin resistance strA, strB Phosphorylation (inactive in pTP10) Erwinia amylovora, Pseudomonas syringae, Xanthomonas campestris (Gram-negative plant pathogens) Kanamycin resistance aphA1-IAB Phosphorylation Pasteurella piscicida, Klebsiella pneumoniae (Gram-negative animal and human pathogens) FIGURE 3.2 (Opposite.) Comparison of pTP10 DNA segments carrying acquired antibiotic resistance gene regions with virtually identical DNA regions from Gram-positive and Gram-negative bacteria of different habitats. (A) Comparison of the macrolide and lincosamide resistance region of pTP10 (segment I) with a DNA fragment of plasmid pNG2 from the human pathogen C. diphtheriae. (B) Comparison of the chloramphenicol resistance transposon Tn5564 (segment III) with transposon Tn45 from the soil isolate C. glutamicum 1014. Both elements are virtually identical with the exception of IS1513 which inserted between cml(A) and the inverted repeat (IR) of Tn5564. (C) Comparison of the Tn3-type transposon Tn5716 present on pTP10 (segment V) with the streptomycin resistance transposon Tn5393 from plasmid pEa34 of the plant pathogen E. amylovora. (D) Comparison between the aphA1-IAB aminoglycoside resistance regions from C. striatum M82B (segment VI) and the Gram-negative fish pathogen P. piscicida. The aphA1-IAB gene is part of IS26-based composite transposons which differ in the orientation of the insertion sequences (arrows) and in the length of the spacer sequences. 50
A PLASMID COMPOSED OF ELEMENTS FROM THROUGHOUT THE EUBACTERIA KINGDOM 33 51
chromosome. Furthermore, it is noteworthy that the genetic organization of these genes on pTP10 is almost identical to the gene arrange- ment found in the M. tuberculosis chromosome. The high G + C region of pTP10 also comprises the tetAB genes (Figure 3.1) the deduced pro- teins of which are similar to chromosomally en- coded ATP-binding cassette transporters. These data strongly suggest that the DNA segment de- rived from the chromosome of a microorganism belonging to the high G + C branch of Gram- positive bacteria. The third DNA segment of pTP10 comprises the chloramphenicol resistance region that is part of the mobile element Tn5564 (Figure 3.1; III). The basic molecular structure of this transposon, comprising the resistance gene cmx(A) and the transposase gene, is nearly identical at the nucle- otide level to the chloramphenicol resistance region of plasmid pXZ10145 from the Chinese soil isolate Corynebacterium glutamicum 1014. In contrast to the basic structure present in C. glutamicum, Tn5564 carries the additional inser- tion sequence IS1513, located between the puta- tive leader sequence of cmx(A) and the left inverted repeat (Figure 3.2B). Interestingly, DNA segment IV of pTP10 is represented by a second identical copy of the basic structure of Tn5564 (Figure 3.1; IVa and IVb). This copy is disrupted by the Tn3-type mobile element Tn5716 (Figure 3.1; Va and Vb). Transposon Tn5716 contains the genetic infor- mation for a transposase, a resolvase and the linked StrAB streptomycin resistance proteins and is identical at the nucleotide level to the basic structure of Tn5393 from the Erwinia amylovora plasmid pEa34 (Figure 3.2C). Tn5393- like transposons are structurally very similar and can be distinguished by additional inser- tions of mobile elements. Although the strAB tandem pair of streptomycin resistance genes is widespread among Gram-negative bacteria, it has not been identified in a Gram-positive bac- terium to date. Moreover, the association of the strAB genes with a transposable element was exclusively found in the Gram-negative phytopathogenic bacteria E. amylovora, Pseudo- monas syringae, and Xanthomonas campestris iso- lated from American agricultural habitats where streptomycin was utilized as bactericide (Sundin and Bender, 1996). Therefore, it is most likely that the Tn5393-like DNA segment of pTP10 derived from a Gram-negative host or- ganism and was transferred to pTP10 by hori- zontal gene transfer. Interestingly, the Tn5393 variants occur on large conjugative plasmids in the three phytopathogenic genera (Sundin and Bender, 1996). The aminoglycoside resistance region of pTP10 (Figure 3.1; VI) is part of the composite transposon Tn5715 consisting of two IS26 inser- tion sequences and a resistance gene encoding an aminoglycoside-3¢,5¢¢-phosphotransferase. Both IS26 elements of Tn5715 were found to be identical to previously sequenced IS26 ele- ments from the Gram-negative species Salmo- nella ordonez, Klebsiella pneumoniae, and Pasteurella piscicida. The aminoglycoside resis- tance protein encoded by Tn5715 is identical to the AphA1-IAB protein identified in a clinical isolate of K. pneumoniae from Chile (Lee et al., 1991) and in the animal pathogen P. piscicida SP9351 from a Japanese marine fish farm (Kim and Aoki, 1994). The protein carries four amino acid substitutions when compared with the widely distributed Aph(3¢)-Ia protein from Tn903 and was associated with a significantly higher turnover of the aminoglycosides kana- mycin and neomycin in K. pneumoniae (Lee et al., 1991). The 5¢-spacer of Tn5715, located between the IS26L element and the trans- lational start of the aphA1-IAB resistance gene, and the 3¢-spacer of Tn5715 are characteristic for the aphA1-IAB gene region of P. piscicida (Figure 3.2D). This strongly indicates that both resistance regions derived from a common ancestor molecule. Due to the nucleotide sequence identity and the apparent low G + C content of the aphA1-IAB gene region (44.1%), it is obvious that DNA segment VI was trans- ferred from a Gram-negative bacterium to pTP10. Interestingly, the aphA1-IAB resistance genes from K. pneumoniae and P. piscicida are located on transferable R plasmids which might enable the rapid dissemination of this specific type of aminoglycoside resistance across spe- cies boundaries. Furthermore, the automated genome inter- pretation revealed that the pTP10 plasmid encodes two replication proteins with amino 34 A. TAUCH AND A. PÜHLER 52
Exploring the Variety of Random Documents with Different Content
being and he a god.” Kamalalawalu made answer: “Kauhiakama says Kohala is depopulated; the people are only at the beach.” To this remark of Kamalalawalu, Lanikaula replied: “You sent your son Kauhiakama to investigate as to how many people there were on Hawaii. He returned and made his report to you that there were not many people there, but Kauhiakama did not see the number of people in Kohala because he traveled on the seashore, reaching Kona from Kawaihae and arrived on the heights of Huehue. He could not have seen the people of that locality because there were only clinkers there, having proceeded along by way of Kona until he arrived at Kau. If he had traveled along the Kona route in the early morning he could not have met people at that time because the inhabitants of that section had gone to the uplands and some had gone fishing; those remaining home were only the feeble and sick, therefore the people of Kona could not have been seen by Kauhiakama on his tour. Had he gone during the he kanaka oe, a he akua kela.” I aku o Kamalalawalu: “Ka! Ua olelo mai o Kauhiakama, he leiwi wale no Kohala, eia i ka nuku na kanaka.” A no keia olelo ana aku o Kamalalawalu pela ia Lanikaula, olelo aku la o Lanikaula: “Hoouna aku nei oe i ko keiki (Kauhiakama) e hele e makaikai i ka nui o na kanaka o Hawaii, a hoi mai la, a hai mai la ia oe, aole he nui o na kanaka o Hawaii. Aka, ike ole aku la o Kauhiakama i ka nui o na kanaka o Kohala, no ka mea, ma kahakai ka hele ana; a hele aku la a hiki i Kona, hele aku la mai Kawaihae aku a hoea iluna o Huehue, aole no e ike i na kanaka olaila, no ka mea he a-a wale no; aka, hele aku la ma Kona loa a hiki i Kau, ina i ke kakahiaka nui ka hele ana ma Kona, aole e loaa kanaka ia wa, no ka mea, ua pau na kanaka o ia wahi iuka a o kekahi poe, ua pau i ka lawaia, a o ka poe koe iho he poe palupalu; a nolaila ka loaa ole o na kanaka o Kona ia Kauhiakama ma ia hele ana. Aka, ina ma ke ahiahi ka hele ana, ina ua ike i ka nui o na
evening he would surely have seen the large population of Kona because it is the largest district of Hawaii.” kanaka o Kona, no ka mea, o ka okana nui hookahi ia o Hawaii.” These observations of Lanikaula did not make much of an impression on Kamalalawalu. He still inclined to the idea of war. Lanikaula observed that Kamalalawalu was bent on going to war. He therefore spoke to Kamalalawalu again: “If you [340]intend to go to war with Lonoikamakahiki, then your grounds should be at Anaehoomalu; and should Lonoikamakahiki come to meet you, then let the battle be fought at Pohakuloa, it being a narrow place; then you will be victorious over Hawaii.” Kamalalawalu answered: “You do not know, because I was distinctly told by both Kauhipaewa and Kihapaewa that our battle field should be on Hokuula and Puuoaoaka, it being a place of eminence.” Lanikaula again said: “You are being deceived by the sons of Kumaikeau and others; you have Ma keia olelo a Lanikaula, aole nae he hoomaopopo nui o Kamalalawalu ia olelo, aka hoomau no o Kamalalawalu i kona manao kaua. A ike mai la o Lanikaula, ua paakiki loa ko Kamalalawalu manao no ke kaua, olelo aku la o Lanikaula ia Kamalalawalu: [341]“Ina i manao oe e kii ia Lonoikamakahiki e kaua, aia kou kahua e noho ai o Anaehoomalu, ina e hiki mai ke kaua a Lonoikamakahiki i o oukou la, alaila, hoihoi aku ke kaua i Pohakuloa e hoouka ai i kahi haiki, alaila lanakila oukou maluna o ka Hawaii.” I aku la o Kamalalawalu: “Aole oe i ike, no ka mea, ua olelo maopopo loa ia mai au e Kauhipaewa laua o Kihapaewa, aia ko makou kahua kaua iluna o Hokuula a me Puuoaoaka; he wahi kau iluna.” I hou aku o Lanikaula: “Puni aku la oe i na keiki a Kumaikeau ma, nolu ia mai la oe; nolaila, e hoolohe oe i ka’u; a ina e hoolohe ole oe i ka’u olelo, aole
been led astray, therefore listen to me, for if you heed not my admonitions I do not think that you will ever come home to Maui nei again.” wau e manao ana e hoi kino mai ana oe ia Maui nei.” Kamalalawalu became indignant at Lanikaula’s remarks and drove him away. But Lanikaula, out of sympathy for the king, did not cease to again give him warning: “Kamalalawalu! You are very persistent to have war. This is what I have to say to you: Better hold temple services these few days before you proceed. Propitiate the gods first, then go.” But Kamalalawalu would not harken to the words of Lanikaula, therefore he ended his remarks. Makakuikalani made the preparations of the war canoes in accordance with the strict orders of Kamalalawalu. When the canoes and the several generals, together with all the men, including the war canoes of Kamalalawalu, were ready floating in the harbor of Hamoa, Lanikaula came forth and in the presence of King Kamalalawalu and his war A no ka Lanikaula olelo ana ia Kamalalawalu pela, alaila wela ae la ko Kamalalawalu inaina no Lanikaula, a hookuke aku la. Aka, aole i hooki o Lanikaula, i kana olelo aku ia Kamalalawalu, no ka minamina no i ke alii; alaila olelo aku la no oia (Lanikaula): “E Kamalalawalu, ke paakiki loa nei oe i ke kaua; a eia ka’u ia oe. E pono ke kapu heiau i keia mau la, mamua o kou hele ana, e hoomalielie mua i ke akua, alaila hele.” Aka, o Kamalalawalu ma keia olelo ana a Lanikaula, aole no i maliu mai. Nolaila pau ae la ka Lanikaula olelo ana. Mahope iho o ka Lanikaula olelo ana ia Kamalalawalu, alaila, hoomakaukau ae la o Makakuikalani i na waa kaua, mamuli o ke kauoha ikaika a Kamalalawalu. A i ka makaukau ana o na waa a me na pukaua e ae, a me na kanaka a pau, a ike ae la ua o Lanikaula ua
canoes prophesied in chant his last words to Kamalalawalu: makaukau na waa kaua o Kamalalawalu, a e lana ana i ke awa o Hamoa; ia manawa, hele mai o Lanikaula, a wanana mai la imua o ke alii Kamalalawalu a me na waa kaua a pau, oiai e lana ana na waa o ke alii i ke kai. A penei kana wanana ma ke mele, a o ka Lanikaula olelo hope ia ia Kamalalawalu. A penei: The red koae! The white koae!68 The koae that flies steadily on, Mounting up like the stars. To me the moon is low.69 It is a god, Your god, Lono; A god that grows and shines. Puuiki, Puunui. At Puuloa, at Puupoko; At Puukahanahana, At the doings of the god of Lono. Lono the small container, Lono the large container. Puunahe the small, Puunahe the large. By Hana, you swim out, By Moe you swim in. My popolo70 is mine own, The popolo that grows by the wayside Is plucked by Kaiokane, Koae ula ke koae kea, Koae lele pauma ana; Kiekie iluna ka hoku, Haahaa i au ka malama. He akua ko akua o Lono, He akua e ulu e lama ana; Puuiki, Puunui, I Puuloa, i Puupoko, I Puukahanahana, I ka hana a ke akua o Lono; O Lono ka ipu iki, O Lono ka ipu nui, O Puunahe iki, O Puunahe nui, Na Hana au aku, Na Moe au mai, Na’u no ka’u popolo, He popolo ku kapa alanui; I aho’ hia e Kaiokane I hakaia e Kaiowahine; O kaua i Kahulikini-e,
Is watched over by Kaiowahine. We two to Kahulikini, Numberless, Vast, without number, countless Are we, O Kama. Let us two to Anaehoomalu, O my chief. He ki-ni, He kini, he lehu, he mano, Kaua, e Kama-e I Anaehoomalu kaua E kuu alii hoi-e. At the end of Lanikaula’s prophesy as made in the chant Kamalalawalu set sail with his large convoy of war canoes. It is mentioned in this tradition relative to the number of canoes of Kamalalawalu that the rear war canoes were at Hamoa, Hana, and the van at Puakea, Kohala; but at the time of this narrative the opinions of the ancients differed as to the accuracy of this. Some say that the number of canoes is greatly exaggerated. A pau ka Lanikaula olelo wanana ana ma ke mele e like me ka hoike ana maluna, alaila, holo aku la o Kamalalawalu me kona mau waa kaua he nui. Ua oleloia ma keia moolelo, o ka nui o na waa o Kamalalawalu aia ka maka hope o na waa kaua i Hamoa ma Hana, a o ka maka mua hoi o na waa, aia i Puakea ma Kohala. Aka hoi, ma ka manawa o keia moolelo, aole he like o ka manao o ka poe hahiko ma keia mea. Ua manao kekahi poe he wahahee ka mea i oleloia no ka nui o na waa. Kamalalawalu having arrived at Hawaii, Kauhipaewa and Kihapaewa were stationed at Puako, in accordance with the wishes of Lonoikamakahiki. At the first meeting that Kamalalawalu had with A hiki aku la o Kamalalawalu i Hawaii, ua hoonohoia o Kauhipaewa me Kihapaewa ma Puako, e like me ka makemake o Lonoikamakahiki. Ia manawa a Kamalalawalu i halawai mua ai me Kauhipaewa ma, olelo aku o
Kauhipaewa and others, Kumaikeau and others [342](who were men from the presence of Lonoikamakahiki) said to Kamalalawalu: “Carry the canoes inland; take the outriggers off so that should the Hawaii forces be defeated in battle they would not use the flotilla of Maui to escape. When they find that the outriggers have all been taken apart and the victors overtake them the slaughter will be yours.” Kamalalawalu did as he was told to do by the two old men. Kumaikeau ma, he mau [343]kanaka no ko Lonoikamakahiki alo, me ka olelo aku ia Kamalalawalu: “E Kamalalawalu, lawe ia na waa iuka lilo, wehewehe ke ama a me ka iako, i kaua ia a hee ka Hawaii ia oukou, malia o holo ke auhee pio, a manao o ka auwaa o ka Maui ka mea e holo ai, i hiki aku ia, ua pau ka iako i ka hemohemo, i loaa mai ia i ka lanakila, alaila na oukou no ka make.” A e like me ka olelo a kela mau elemakule ia Kamalalawalu, alaila, hana aku la o Kamalalawalu e like me ka kela mau kanaka. When Kamalalawalu arrived at Kohala, Lonoikamakahiki had his army in readiness. Kamalalawalu learning that Kanaloakuaana was still living at Waimea he concluded that his first battle should be fought with Kanaloakuaana and at Kaunooa. Kanaloakuaana was completely routed and pursued by the soldiers of Kamalalawalu, and Kauhiakama, and Kanaloakuaana was captured at Puako. At this battle the eyes of I ka manawa a Kamalalawalu i hiki aku ai ma Kohala, ua makaukau mua na puali kaua o Lonoikamakahiki. Aka, lohe ae la ua o Kamalalawalu, eia no o Kanaloakuaana i Waimea kahi i noho ai, hoouka mua iho la o Kamalalawalu me Kanaloakuaana i Kaunooa. A hee mai la o Kanaloakuaana; a alualu loa mai la ko Kamalalawalu poe koa a me Kauhiakama pu, a loaa pio iho la o Kanaloakuaana ma Puako; a
Kanaloakuaana were gouged out by the Maui forces, the eye sockets pierced by darts, and he was then killed, the eyes of Kanaloakuaana being tatued. ma ia hoouka kaua hou ana, poaloia ae la na maka o Kanaloakuaana e ko Maui kaua, a oo ia ae la na maka i ke kao hee, pepehiia iho la a make; ua kakauia nae na maka o Kanaloakuaana i ka uhi. Because of this action on the part of Kamalalawalu’s men the landing place for the canoes at Puako was called Kamakahiwa,71 and to this day is known by that name and may ever remain so to the end of this race. Because of the perpetration of this dastardly act on Kanaloakuaana the following was composed by a writer of chants, being the middle portion of a chant called “Koauli”: A oia hana ana a ko Kamalalawalu poe koa ia Kanaloakuaana, nolaila ua kapaia ka inoa oia awa pae waa ma Puako o Kamakahiwa, a o ka inoa ia o ia wahi a hiki mai i keia manawa, a hiki aku i ka hanauna hope loa o keia lahui. A no ia hana ia ana o Kanaloakuaana pela, ua hanaia e ka poe haku mele penei, oia hoi ma ka hapa waena o ke mele i oleloia o Koauli, penei: The drawing out of Kama, the ohia tree; The letting out of Kama at Waimea, The kin of Kanaloa.72 He was made black like the mud-hen. The face was blackened, Blackened was the face of Kanaloa with fire. Ke koana o Kama, ka ohia, Ko Kama kuu i Waimea, Ka io o Kanaloa, He ele he Alaea; O ka maka i kuia; I welo’a i ke kao o Kanaloa; Ko Kanaloa maka A lalapa no E uwalo wau i ka maka O Makakii;
The face of Kanaloa, With burning fire. Let me scratch the face Of Makakii. You poked at the eyes of Kamalea,73 Makahiwa, Makalau. The men were from Hoohila, Of Makakaile. The face of Makakaile the large one, the life. Kikenui of Ewa. At Ewa is the fish that knows man’s presence.74 The foreskin of Loe, consecrated in the presence of Mano The chief, heralded75 by the drum of Hawea,76 The declaration drum Of Laamaikahiki. E o mai oe i ko kamalea maka, O Makahiwa, Makalau; No Hoohila ka lau. O Makakaile. Ka maka o Makakaile nui a ola; Kikenui a Ewa No Ewa ka ia i ka maka o Paweo No Loe ka ili lolo i ka maka o Mano Ke alii ke Olowalu o ka pahu o Hawea Ha pahu hai kanaka O Laamaikahiki. This chant is dedicated to the eyes of Kanaloakuaana as indicated by the verses. O keia mele i hai ia maluna no ka maka o Kanaloakuaana, e like me ka hoakaka ana ma na pauku maluna ae o kela mele. CHAPTER XIII. MOKUNA XIII.
The Battle at Waimea. —Conquest by Lonoikamakahiki— Defeat and Death of Kamalalawalu. Ka Hoouka Kaua Ana ma Waimea.—Ka Lanakila Ana o Lonoikamakahiki.— Auhee o Kamalalawalu me Kona Make Ana. After the death of Kanaloakuaana by Kamalalawalu, and in obedience to the statements of the old men for the Maui war contingent to go to Waimea and locate at Puuoaoaka and Hokuula, Kamalalawalu and his men proceeded to the locality as indicated by them. The Maui forces followed and after locating at Hokuula awaited the [344]coming fray. On the day Kamalalawalu and his men went up to Waimea to occupy Hokuula the two deceitful old men at the time were with Kamalalawalu. In the early morning when Kamalalawalu awoke from sleep he beheld the men from Kona and those of Kau, Puna, Hilo, Hamakua and Kohala had also been assembled. Mahope iho o ka make ana o Kanaloakuaana ia Kamalalawalu ma, a e like hoi me ka olelo a na elemakule, e hoi iuka o Waimea, ma Puuoaoaka a me Hokuula e hoonoho ai ko Maui poe kaua, a nolaila ua hoi aku la o Kamalalawalu ma a ma kahi a ua mau elemakule nei i kuhikuhi ai. [345] Hoi aku la ko Maui poe a noho ma Hokuula e kali ana no ka hoouka kaua ana. I ka la a Kamalalawalu ma i pii ai iuka o Waimea a noho ma Hokuula, a o ua mau elemakule nolunolu la no kekahi me Kamalalawalu ma i kela manawa. A ma ia po a ao ae, ma ke kekahiakanui i ka manawa i ala ae ai ko Kamalalawalu hiamoe, aia hoi, ua kuahaua ia mai la na kanaka o Kona, ko Kau a o Puna a me
Hilo, o Hamakua hoi a me Kohala. Kamalalawalu looked and saw that the lava from Keohe to Kaniku was one red mass. Kamalalawalu was astonished, because the day before he observed that the lava was one mass of black, but this morning it was entirely red with people. Thereupon Kamalalawalu inquired of Kumaikeau and the others why the lava was a mass of red: “What does red portend? Does it mean war?” Kumaikeau and the others replied: “Do not think the red you see is some other red and not what you assume it to be. It is not war. That red yonder is the wind. The olauniu wind of Kalahuipuaa and Puako had been blowing in the early morning and when it is very light and gentle it hugs the lava close. This olauniu wind on the lava coming in contact with the wind from Wainaualii raises a cloud of dust covering and hiding the land in the manner you saw yesterday.” While cogitating to himself, Kamalalawalu concluded to drop the matter on Nana aku la o Kamalalawalu he ula wale la no na ke a, mai Keohe a Kaniku; ia manawa haohao no o Kamalalawalu i keia mea; no ka mea, i ka Kamalalawalu ike ana i ka la mua he uliuli ke a; a i keia kakahiaka hoi, he ula pu wale la no i na kanaka. Nolaila, ninau ae la o Kamalalawalu: “Ea, e Kumaikeau ma, ula pu hoi ke a, heaha keia ula, he kaua paha?” I aku o Kumaikeau ma: “Aole paha ia ula au e ike la, he ula e ae, a manao aku oe he kaua ia. Aole ia he kaua. Oia ula la ea, he makani, pa aku la ka makani Olauniu o Kalahuipuaa a me Puako i ka wanaao, a malamalama loa, pili-a aku la, komo aku la keia Olauniu a pili-a aku la, hui aku la me ko Wainanalii makani, ku ae la ke ehu o ka lepo, uhia aku la nalo wale ke a au i ike ai i ka la inehinei.” A no kela olelo nolu a kela mau elemakule, oki wale iho la no o Kamalalawalu, a
account of the deceit of the two old men and the loss of confidence in what Kumaikeau and the others had said, for the reason that the lava continued to be strewn with people even to the time of the setting sun. During that night and including the following morning the Kona men arrived and were assigned to occupy a position from Puupa to Haleapala. The Kau and Puna warriors were stationed from Holoholoku to Waikoloa. Those of Hilo and Hamakua were located from Mahiki to Puukanikanihia, while those of Kohala guarded from Momoualoa to Waihaka. waiho wale iloko ona ia manao, no ka mea, aole he hilinai nui i kela olelo a Kumaikeau ma, no ka mea, ua mau ka paa ana o ke a i na kanaka a hiki i ka napoo ana o ka la. Ma ia po iho, a ao ae, hiki mai la ko Kona poe a hoonoho mai la mai kai o Puupa a hiki i Haleapala. A o ko Kau hoi a me ko Puna, hoonoho ae la ka lakou poe mai Holoholoku a Waikoloa. A o ko Hilo a me ko Hamakua mai, hoonoho mai la ko lakou poe kaua mai Mahiki a Puukanikanihia. A o ko Kohala hoi, pania ia mai la e na kanaka mai Momoualoa a Waihaka. That morning Kamalalawalu observed that the lowlands were literally covered with almost countless men. Kamalalawalu then took a survey of his own men and realized that his forces were inferior in numbers. He then spoke to Kumaikeau and the others: “Kumaikeau and the rest of you, how is this and what is that large concourse of people below?” Ia kakahiaka, nana aku la o Kamalalawalu, ua uhi paa puia mai olalo i na kanaka, aole o kana mai. Alaila, nana ae la o Kamalalawalu ia lakou ua uuku loa; alaila, olelo aku la o Kamalalawalu ia Kumaikeau ma: “Ea! E Kumaikeau ma, pehea keia? Heaha keia lehulehu olalo?”
Kumaikeau and the others replied: “We have never seen so many people in Hawaii before. Do not think that because of their superior numbers they will escape us; they cannot, for the reason that their fighting will have to be from below. It is true they are more numerous, but being beneath we will defeat them.” I aku o Kumaikeau ma: “Akahi no au a ike i ka nui o na kanaka o Hawaii nei. Mai manao nae oe ia nui, e pakele ana ia kakou. Aole e pakele, aia ka lakou kaua malalo, he nui lakou, o ko lakou kaa malalo, make no ia kakou.” The following day, Lonoikamakahiki went over to meet Kamalalawalu to confer concerning the war.77 During their conference Kamalalawalu proposed to Lonoikamakahiki that war cease because he feared the greater forces of Lonoikamakahiki. But the proposal by Kamalalawalu for termination of the war did not meet Lonoikamakahiki’s approval. He had no intention of acquiescing, because he was greatly incensed at Kamalalawalu for the brutal manner in which he killed Kanaloakuaana by gouging out the eyes and other brutal acts carried into execution while the latter was still alive. I kekahi la ae, hele aku la o Lonoikamakahiki e halawai me Kamalalawalu, e kuka no ke kaua. A i ko laua kamailio ana, olelo aku o Kamalalawalu ia Lonoikamakahiki, e hoopau wale ke kaua, no ka mea, ua hopo mai la o Kamalalawalu no ka nui loa o ka Lonoikamakahiki kaua. Aka, ma kela olelo kaua a Kamalalawalu e hoopau wale ke kaua, aohe manao o Lonoikamakahiki e hoopau, e like me ka Kamalalawalu olelo, no ka mea, ua wela ko Lonoikamakahiki huhu no Kamalalawalu, no ka pepehi hoomainoino ana ia Kanaloakuaana; oia hoi, ua poaloia na maka, a ua hoomainoino ia i ko
Kanaloakuaana wa e ola okoa ana. Makakuikalani, however, upon hearing of Kamalalawalu’s proposal to Lonoikamakahiki to cease the war disapproved of it and said to Kamalalawalu not to have the [346]war cease. “Onward, and stand on the altar!78 Then will it be known which of us is a full grown child.” This determination on the part of Makakuikalani was manifested by his presence for three consecutive days before the forces of Hawaii. After the third day, the two combatting forces waged battle, Lonoikamakahiki gaining the victory over Kamalalawalu’s entire force on the same day the battle was fought, the Maui-ites being completely routed. Aka hoi, o Makakuikalani, i kona lohe ana ia Kamalalawalu ua olelo aku oia ia Lonoikamakahiki e hoopau i ke kaua, he mea makemake ole nae ia ia Makakuikalani. Oia hoi, ua olelo aku o ua Makakuikalani nei ia Kamalalawalu, aole e hoopau i ke kaua. “Ho aku imua a kau i ka nananuu; alaila ike ia na keiki makua o kakou.” A no ia manaopaa o Makakuikalani, hoike mau ae la oia imua o ko Hawaii kaua i kela la keia la pau na la ekolu. Mahope iho o na la ekolu, hoomaka iho la na aoao elua e [347]kaua, a iloko no o ua la hoouka kaua la, lanakila ae la o Lonoikamakahiki maluna o ko Kamalalawalu puali holookoa, a auhee aku la ko Maui a pau. This is the history of the battle as related by the ancients and as the narrative is preserved by them. Before the battle commenced it was customary for the old men to encourage Kamalalawalu to do battle. Whenever the two old men A penei hoi ka moolelo oia hoouka kaua ana i oleloia e ka poe kahiko, ma ka lakou malama moolelo ana. Mamua o ka hoouka kaua ana, he mea mau i na elemakule ka paipai ana ia Kamalalawalu e kaua. Aia lohe ua mau elemakule nei i na olelo
heard what Kamalalawalu and the others had to say as to what they intended doing to Lonoikamakahiki in order to be victorious in battle, the old men would wend their way to make it known to Lonoikamakahiki and the others and this duty was generally carried out during some convenient time of night. The two old men always pointed out to Kamalalawalu and the others where the battle should be fought, and the suggestions of the old men were always received with the utmost confidence by him. Therefore Kumaikeau and the two deceitful old men would in turn inform Lonoikamakahiki. The two old men never suggested any place for battle which would result advantageously to Kamalalawalu and his forces; on the contrary, it was invariably such a locality where inevitable defeat would result. a Kamalalawalu ma, no na mea a lakou e hana aku ai ia Lonoikamakahiki, ma na mea e pili ana i ke kaua e lanakila ai ko lakou aoao, a e pio ai hoi ko Lonoikamakahiki, alaila, e hele aku auanei ua mau elemakule nei e hai aku ia Lonoikamakahiki ma, ma kekahi manawa kaawale o ka po. No ka mea, na ua mau elemakule nei no e kuhikuhi aku ia Kamalalawalu ma i ke kahua kahi e hoouka ai ke kaua ana. A e like me ke kuhikuhi ana a kela mau elemakule, e lilo auanei ia i olelo na Kamalalawalu e hilinai nui ai. A no ia mea, hele aku no o Kumaikeau ma, ua mau elemakule nolu (apuka) nei a hai aku ia Lonoikamakahiki. Aole no e kuhikuhi ana ua mau elemakule nei i ke kahua kaua ma kahi e lanakila ai ko Kamalalawalu mau puali, aka, ma kahi e pio ai o Kamalalawalu ma, malaila no ka ua mau elemakule nei kahua kaua e hoonoho ai. In the early morning of the day of battle, Makakuikalani went to the I ka la o ka hoouka kaua, ma ke kakahiaka nui, hele aku la o
front with his warriors following him and planted themselves at Waikakanilua below Hokuula and Puuoaoaka at a prominence looking towards Waikoloa. Pupuakea, on observing that Makakuikalani was placing his men and self in position, he and his warriors immediately came forward prepared to give battle. It was a case where both sides were equally prepared for the fray. Makakuikalani mamua, a o kona poe kaua mahope ona, a ma Waikakanilua, malalo aku o Hokuula a me Puuoaoaka, ma ka hulei e nana iho ana ia Waikoloa. Aka hoi, o Pupuakea, i kona ike ana mai ia Makakuikalani, e hoonoho aku ana me kona poe koa, alaila, hele mai la o Pupuakea me kona poe kaua, me ka makaukau hoi no ke kaua. Aka, ua makaukau no na aoao a elua no ke kaua. Makakuikalani was a man of great height and large physique; a renowned and powerful general of Maui and was also Kamalalawalu’s younger brother. As for Pupuakea, Hawaii’s celebrated and powerful general and who was Lonoikamakahiki’s younger brother, he was only a man of small stature. Both men had been taught the art of fighting with the wooden club and were experts in its use, but their schooling was under different masters and at different places. He kanaka nui a loihi o Makakuikalani, ka pukaua ikaika kaulana o Maui, ko Kamalalawalu kaikaina. A o Pupuakea hoi, ko Hawaii pukaua ikaika kaulana, ko Lonoikamakahiki kaikaina, he wahi kanaka uuku no ia, a haahaa hoi. Ua aoia no laua a elua i ke kaka laau palau, a ua akamai no laua a elua, aka, he kumu okoa ka kekahi a me kekahi, a ua aoia no laua ma na wahi kaawale. Aka, i ka la o ka hoouka kaua ana, ua weliweli mai la ko Lonoikamakahiki poe kaua, no ka ike ana mai ia Makakuikalani.
On the day of battle the sight of Makakuikalani put Lonoikamakahiki’s forces in dreadful fear. When Pupuakea saw Makakuikalani he had no fear of him, did not tremble but stood firm ready to give battle. Aka, o Pupuakea, iloko o kona manawa i ike aku ai ia Makakuikalani, aole i komo mai iloko ona ka makau, aole no hoi oia i weliweli, aka, kupaa mau no oia e kaua aku ia Makakuikalani. While Makakuikalani and Pupuakea were standing on the battle field, Makakuikalani raised his war club and from on high struck at Pupuakea. Being short in stature he was only slightly struck but fell to the ground, however. At the instant Makakuikalani’s war club struck Pupuakea the end of it was buried deep into the ground. At the moment Pupuakea was struck by the war club and fell Makakuikalani thought that he was killed, but the latter’s master saw that Pupuakea was not dead, so [348]said to Makakuikalani: “Go back and slay him for your opponent is not dead. Your clubbing being from above only delivered a blow with the butt end.” Makakuikalani hearing the words of his teacher turned around and threw the butt end of his club, at the same time Ia Makakuikalani a me Pupuakea e ku ana ma ke kahua kaua, ia manawa, lawe ae la o Makakuikalani i kana laau palau a kiekie, a hahau iho la maluna iho o Pupuakea, a no ka haahaa o Pupuakea, ua pa lihi aku la o Pupuakea, aka, haule aku la o Pupuakea ilalo i ka honua. A o ka welau o ua laau palau la a Makakuikalani, iloko hoi o kona manawa i hahau aku ai ia Pupuakea, napoo pu aku la i ka lepo. I ka manawa i pa aku ai o Pupuakea i ka laau palau a Makakuikalani, a haule ilalo, manao ae la ua o Makakuikalani, ua make loa o Pupuakea. Aka, o ke kumu kaka laau a Makakuikalani, ka mea nana i ao o Makakuikalani, oia ka mea nana i ike mai o Pupuakea, aole i make; nolaila, olelo aku ua kumu kaka laau la a Makakuikalani: “E hoi houia aku
telling him to “Shut up! Instruction stops at home. He cannot escape, he must be dead because the club strikes true.” At the very instant that Makakuikalani faced around to talk with his teacher, he (the teacher) was dead. e hoomake, aole i make ka hoa kaua, no ka mea, he laau kau i luna, pa kano aku la kaua uhau ana.” A lohe o Makakuikalani i keia olelo ana aku a kana kumu, alaila, huli ae la oia (Makakuikalani) a wala hope ae la i ke kumu o ka laau [349]palau me ka olelo aku: “Kuli! I ka hale pau ke ao ana; aole e pakele, ua make aku la, no ka mea o ka Io ka laau.” A o ua kumu nei hoi a ua o Makakuikalani make loa aku la ia, i ka manawa no a ua o Makakuikalani i huli aku ai a kamailio. Pupuakea was lying on the ground, stunned, but somewhat recovered afterwards and raised himself up from the ground. When Makakuikalani saw that Pupuakea was still alive he rushed towards him bent on killing him. I ka manawa a Pupuakea e waiho ana i ka honua, ua maule aku la oia, a mahope loaa mai la ka mama iki ana ae, ia manawa, ala ae la o Pupuakea mai ka honua ae; ia manawa ike mai la o Makakuikalani ua ola hou o Pupuakea, alaila, holo hou mai la o Makakuikalani imua o Pupuakea, me ka manao e hoomake loa ia Pupuakea. Pupuakea observed Makakuikalani’s approach so prepared himself to slay him. When Makakuikalani drew near, A ike aku la o Pupuakea ia Makakuikalani e hele mai ana e kue hou iaia, alaila hoomakaukau ae la oia e pepehi
Pupuakea raised his club and twirled it from his right. At that moment Makakuikalani attempted also to lay his club on Pupuakea, and when his club was twirled it skidded along the ground towards the feet of Makakuikalani and being parried by Makaku, fell to the ground. When Makakuikalani swung his club from the left side it struck the back of his own neck and he was instantly killed. Pupuakea immediately stepped backward and met his master who said to him: “Go back again and slay him so he be dead.” The words of his master aroused Pupuakea’s pride and he said to his teacher: “He cannot live, he is dead.” Then looking at the palm of his hand he again said to his master: “He cannot be alive because the birthmark of Pupuakea has impressed itself thereon. The flying club through dust has killed him.” aku ia Makakuikalani. A i ke kokoke ana mai o ua Makakuikalani nei, lawe ae la o Pupuakea i kana laau palau a wili ma kona aoao akau, a i ka hoomaka hou ana o Makakuikalani e hoouka hou i kana laau palau maluna o Pupuakea, alaila, ia manawa, wili ae la o Pupuakea i kana laau, a hualepo aku la ma na wawae o Makakuikalani, a pa aku la ia Makaku, haule aku la i ka honua, a i ka wili ana mai i kana laau mai ka aoao hema mai, pa mai la ma ka hono, make iho la o Makakuikalani. Ia manawa, emi hope aku la o Pupuakea a halawai me kana kumu kaka laau nana i ao. I mai la ke kumu ia Pupuakea: “Hoi houia aku e hoomake i make.” A no ka olelo ana a ke kumu a ua o Pupuakea pela, alaila, olelo aku la o Pupuakea i kana olelo kaena imua o kana kumu: “Aole e ola! Ua make!!” Nana iho la oia i ka poho o kona lima, a olelo ae la i ke kumu ana: “Aole ia e ola, no ka mea, ua kukai ae nei ka ila o Pupuakea. Make aku la i ka laau a kaua i ka hualepo.”
After the great and renowned general of Maui had fallen the Hawaii forces continued to slaughter Kamalalawalu and the others. Upon the death of Kamalalawalu the slaughter of the Maui-ites continued for three days thereafter and those defeated who ran towards their canoes found no arms and outriggers because they had been broken. The repulsed warriors ran to Puako and noticing the paimalau79 floating in the sea mistook them for canoes. They began to waver and were again overtaken by the victors. The destruction of the remaining invaders was then complete. Referring to Kauhiakama the son of Kamalalawalu he escaped to safety. The story of his escape running thus: A haule aku la ka pukaua nui kaulana o Maui, alaila luku aku la ka Hawaii ia Kamalalawalu ma, a make aku la o Kamalalawalu. Ia make ana o ua o Kamalalawalu, lukuia aku la o Maui ekolu la, a hee aku la o Maui, a holo aku la, a na waa o lakou; aka, aole he iako, aole he ama, no ka mea, ua pau i ka haihai ia; nolaila holo aku la ke pio a ma Puako; a o ka ike i ke paimalau, kuhi he waa, a i ka hoolana ana iloko o ke kai, me ka manao, o ka waa ia, aia nae ua kahulihuli, a loaa hou aku la i ka lanakila, lukuia aku la na koena o ka Maui a pau loa i ka make. A o Kauhiakama hoi, ke keiki a Kamalalawalu, holo pio aku la oia, a pakele aku la. A penei ka moolelo o kona pakele ana. On the day that the Maui forces were defeated Kauhiakama clandestinely escaped to Kawaihae and from there his intentions were to hie to the caves, there to remain until his side was victorious and then make his appearance. I ka la o ka hee ana o ko Maui poe kaua, holo malu aku la oia a hiki i Kawaihae, a malaila mai e holo ana me ka manao e pee ma na ana, a hiki i ka wa e lanakila ai, alaila hoike ae.
Hinau, one of the generals of Lonoikamakahiki and a messenger also, had great affection for Kauhiakama, but it was previous to the time of Hinau’s assisting in the escape of Kauhiakama that he roasted some taro and, together with some dried mudfish, already roasted, proceeded to search for Kauhiakama. Hinau came to Kawaihae first and from there went to Kaiopae where for the first time he saw Kauhiakama, so Hinau hailed him and said: “Say, Kauhiakama, remain there until I reach you!” Kauhiakama looking round saw Hinau approaching, the thought of death at the hands of the victorious crossed his mind, so covering his face with his hands he wept, for he [350]was greatly depressed in spirits. Hinau came forward, however, and greeted him with a kiss on the nose, remarking: “I remained behind and roasted some taro and dried mudfish for the love of you and came to search for you.” These words of Hinau gave Kauhiakama great relief and hopes for life. A o Hinau, kekahi o na pukaua o Lonoikamakahiki, he elele no na Lonoikamakahiki, aka, ua nui loa ke aloha o Hinau ia Kauhiakama. Nolaila, mamua o ko Hinau manao ana e hoomahuka ia Kauhiakama, pulehu ae la oia i mau kalo, a moa, a paa pu ae la me na oopu maloo i pulehuia, a imi aku la ia Kauhiakama; ma Kawaihaeo ko Hinau hiki mua ana, a malaila aku a hiki i Kaiopae, ike mua aku la o Hinau ia Kauhiakama, alaila, kahea aku la: “E Kauhiakama e! Malaila iho oe a loaa aku ia’u.” I alawa ae ka hana o Kauhiakama, e hele aku ana o Hinau, alaila, manao ae la o Kauhiakama: “Make, eia ka lanakila.” Alaila, palulu ae la ua o Kauhiakama i na lima i ke poo me ka manao kaumaha i ka make, e uwe ana. Aka, hele aku la o Hinau a honi aku la i ka ihu o Kauhiakama, a uwe iho la, me ka i aku: “Ua noho au me ke aloha ia oe, a nolaila, pulehu mai nei i na wahi kalo, a me na wahi oopu maloo, a imi [351]mai nei ia oe.” A no keia olelo a Hinau, akahi no a oluolu iho la o
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  • 5. Horizontal Gene Transfer 2nd Edition Michael Syvanen Digital Instant Download Author(s): Michael Syvanen, Clarence I. Kado ISBN(s): 9780126801262, 0126801266 Edition: 2 File Details: PDF, 5.67 MB Year: 2002 Language: english
  • 6. Foreword The era of high-speed sequencing and computer- based annotating systems have generated enor- mous data bases from which many novel discov- eries are being made. Comparative genomic nucleotide and amino acid sequence analyses have revealed many sequence cassettes that appear highly conserved, thus raising the ques- tion whether these sequences were introduced by horizontal gene transfer mechanisms, or whether they were fortuitous occurrences. The prior recombinant DNA era provided valuable insights on the extent of conserved genetic mechanisms and brought to realization that genes can be moved across species barriers, a finding that was supported 20 years earlier than the recombinant DNA era by bacterial genetic ex- periments. In fact, a number of elegant bacterial genetic studies had implicated genetic transmis- sion of foreign DNA into bacteria and into plants. The natural transfer of plasmid DNA from Agrobacterium tumefaciens to plant cells, resulting in the integration of the foreign plasmid DNA into the chromosome of the plant followed by its expression to generate phenotypic change is the best case of horizontal gene transfer that occurs in nature. Certainly, bacteriophages were well known to mediate horizontal gene transfer long before the Agrobacterium story. Although this was a dramatic discovery, horizontal gene transfer among microbes did not have the impact of that mediated between a microbe and a eukaryote. The genomics era and its ever growing data bases provide vast opportunities to explore po- tential horizontal gene transfer systems that may exist between microbes and eukaryotes and be- tween lower eukaryotes and higher eukaryotes. The direction of horizontal gene transfer may be dominant in one direction, but may occur in the opposite direction (retrotransfer). In reviewing the chapters of this book, I have come to the realization that the discovery of hor- izontal gene transfer among distinct organisms has increased substantially in this new millen- nium and a “new” scientific vocabulary has been introduced. The terms “cross-species gene transfer”, “lateral gene transfer” and “horizontal gene transfer” have been used interchangeably, with the last two terms used most frequently. To avoid future confusion, the term “horizontal gene transfer” should represent the transfer of genes across distinct species, especially when interkingdom gene transfer takes place. The term “lateral gene transfer” could be retained to accommodate gene transfer between distinct species within a kingdom, viz., between pro- karyotes, or between eukaryotes. Clarence I. Kado 9
  • 7. Preface The seminal experiment that illustrated the ability of genetic information to flow between species slipped by largely unnoticed. In 1959, Tomoichiro Akiba and Kunitaro Ochia discov- ered antibiotic resistance plasmids. The most sur- prising attribute of this new class of plasmids was that they carried resistance genes to multiple anti- biotics and that they moved among different bac- terial species, spreading resistance genes, and thereby demonstrating that genetic information can flow from one species to another (Akiba et al., 1960; Ochia et al., 1959). The implications of this finding would have profound effects ranging from the applied field of genetic engineering to the very theory of evolution. Early papers prob- ing the deeper theoretical implications of hori- zontal gene transfer began to appear in the 1970s, though they were not widely acknowledged or accepted. Fritz Went, in 1971, wrote a review on similar traits that are shared by unrelated flower- ing plants thereby illustrating many examples of parallel evolution. In addition, he noted that the traits are shared among plants that occupied the same ecosystems. In this context he proposed that these unrelated plants were exchanging genes. He cited bacterial plasmid transfer as a precedent for such events. Krassilov in 1977 ar- rived at a similar model for flowering plant evo- lution based on his paleontological studies of the emergence of angiosperms in the fossil record. Anderson in 1970 and Reanney in 1976 suggested that horizontal gene transfer could affect evolu- tion in the animal kingdom, and Hartman, in 1976, suggested that horizontal gene transfer might effect speciation. There were a series of theoretical papers that cited horizontal gene transfer as an explanation for the widespread occurrence of parallelisms in the fossil record (Krassilov, 1977; Erwin and Valentine, 1984; Reanney, 1976; Jeppsson, 1984; Syvanen, 1985). Meanwhile, genetic engineering experiments began to produce startling results. In 1976, Struhl et al. placed DNA from yeast into a histidine defi- cient mutant of Escherichia coli that resulted in the restoration of histidine biosynthesis. This DNA contained a histidine biosynthesis gene from the yeast genome. What seems commonplace today was difficult to comprehend back in 1976 – genes from a eukaryotic organism artificially intro- duced into a bacterium could actually function. Davies and Jimenez in 1980 showed that a bacte- rial neomycin phosphotransferase gene would express aminoglycoside resistance in yeast, showing that a bacterial gene could be expressed in a eukaryote. As a bacteriologist, I had personally incorpo- rated the findings of Akiba and Ochia into my sci- entific world-view. I was intrigued by the implication of Struhl’s experiment. In the course of a discussion of a review of Crick’s book about the unity of the genetic code entitled Life Itself (Crick, 1981), it occurred to me that horizontal movement of genes could shed light on this ques- tion provided such gene transfer was a factor in major evolutionary transitions. If this conjecture was correct, it could provide an alternative expla- nation for not only the unity the genetic code, but many other biological unities as well. At this point, I was unaware of the works of Went, Reanney, Krassilov, Hartman and Anderson. I wrote up my ideas in 1982, and they were finally published in 1985. During this period, the field of 11
  • 8. genetic engineering was exploding. Palmiter et al. in 1983 produced the first transgenic mouse that expressed a foreign gene, the human growth hormone gene. Result after result confirmed that it was possible for genes to cross species bound- aries and to express their phenotype. These ex- periments all demonstrated that genes could be made to cross species boundaries in the labora- tory. The fundamental question that remained was whether these events occurred in nature, and whether they occurred at a frequency high enough to effect evolution. Hopefully, this collec- tion of articles will be but one of many which will begin to explore this question. By the mid-1980s, numerous mechanisms for horizontal gene transfer were firmly established, not only for bacteria but also for metazoans and, in addition, many heretofore difficult to explain biological phenomena were easily handled by a horizontal gene transfer theory. However, there was a paucity of observations giving direct sup- port to these speculations. With the rapid in- crease in the nucleic acid database over the past decade, the situation has changed. This book covers some of these more recent developments. Today, researchers in many unrelated areas are making observations related to horizontal gene transfer, which has resulted in the unusual breadth of topics included in this volume. This book does not attempt a comprehensive survey of horizontal gene transfer, but rather attempts to sample various areas with a primary focus on material from active research areas. The chap- ters in this book deal with three questions. First, can genes, or more specifically DNA move from one species to an unrelated one? Thus, a section of this book is devoted to the subject of transfer mechanisms, a phenomenon well documented in bacteria but also found in plants and animals. Obviously transfer mecha- nisms exist, the subsidiary questions are: how widespread are the mechanisms? And, do they operate in natural environments? Second, what is the evidence that horizontal gene transfer contributes to existing genotypes of species? The primary evidence supporting evolutionary significant horizontal transfers in- volves phylogenetic reasoning. This is an area where the evidence is accumulating in the gene and protein sequence databases. Two problems are repeatedly encountered – defining the to- pology of a gene tree and estimating divergence times following molecular clock assumptions. There are a number of contributions discussing results obtained from phylogenetic analysis and problems associated with this approach. The third question raised by the central hypothesis is that if the mechanisms exist and events can be documented, does horizontal gene transfer actually play any significant evolu- tionary role? Or, does a theory that incorporates migrant DNA have utility in explaining more general biological phenomena. To this end, more conjectural papers that directly address macro- evolutionary patterns and trends are presented. REFERENCES Akiba, T., Koyama, K., Ishiki, Y., Kimura, S. and Fukushima, T. (1960) The mechanism of the development of multiple- drug-resistant clones of Shigella. Jpn J. Microbiol. 4: 219. Anderson, N.G. (1970) Evolutionary significance of virus infection. Nature 227: 1346–1347. Crick, F. (1981) Life Itself: Its Origin and Nature, Simon and Schuster, New York. Davies, J. and Jimenez, A. (1980) A new selective agent for eukaryotic cloning vectors. Am. J. Tropical Med. Hygiene 29(5 Suppl): 1089–1092. Erwin, D.H. and Valentine, J.W. (1984) Hopeful monsters, transposons and metazoan radiation. Proc. Natl Acad. Sci. USA 81: 5482–5483. Hartman, H. (1976) Speculation on viruses, cells and evolut- ion. Evolution Theory 3: 159–163. Jeppsson, L. (1986) A possible mechanism in convergent evolution. Paleobiology 12: 37–44. Krassilov, V.A. (1977) The origin of angiosperms. Bot. Rev. 43: 143–176. Ochia, K., Yamanaka, T., Kimura, K. and Sawada, O. (1959) Inheritance of drug resistance (and its transfer) between Shigella strains and between Shigella and E. coli strains. Nihon Iji Shimpo 1861: 34 [in Japanese]. Palmiter, R.D., Norstedt, G., Gelinas, R.E. et al. (1983) Metallothionein–human GH fusion genes stimulate growth of mice. Science 222(4625): 809–814. Reanney, D. (1976) Extrachromosomal elements as possible agents of adaptation and development. Bacteriol. Rev. 40: 552–590. Struhl, K., Cameron, J.R. and Davis, R.W. (1976) Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc. Natl Acad. Sci. USA 73(5): 1471–1475. Syvanen, M. (1985) Cross-species gene transfer; impli- cations for a new theory of evolution. J. Theor. Biol. 112: 333–343. Went, F.W. (1971) Parallel evolution. Taxon 20: 197–226. Michael Syvanen xii PREFACE 12
  • 9. Contributors Ronald M. Adkins Biology Department, University of Massachusetts, Amherst, Massachusetts, USA Rita M. P. Avancini Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Lexington, Massachusetts, USA) L. Aravind National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA William B.N. Berry Department of Geology and Geophysics, University of California, Berkeley, California, USA Meghan E. Bowser Genotypes Inc., San Francisco, California, USA James R. Brown Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA A. Burmester Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany R. N. Burns Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, UK Richard Calendar Department of Molecular and Cell Biology, University of California, Berkeley, California, USA George Chisholm Genotypes Inc., San Francisco, California, USA Jonathan B. Clark Department of Zoology, Weber State University, Ogden, Utah, USA Patrice Courvalin Unité des Agents Antibactériens, Institut Pasteur, Paris, France Pierre Darlu Inserm U 155, Kremlin-Bicêtre, France 13
  • 10. Martin Day Cardiff School of Biosciences, Cardiff University, Cardiff, UK Erick Denamur Inserm U 458, Hôpital Robert Debré, Paris, France Russell F. Doolittle Center for Molecular Genetics, University of California, San Diego, La Jolla, California, USA Christophe Douady Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA Norman C. Ellstrand Department of Botany and Plant Sciences and Center for Conservation Biology, University of California, Riverside, California, USA Gayle C. Ferguson Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand Bryant E. Fong Genotypes Inc., San Francisco, California, USA M. E. Ford Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA (present address: Division of Gastroenterology and Hepatology, University of Pittsburgh School of Medicine, Pennsylvania, USA) Lynne M. Giere Genotypes Inc., San Francisco, California, USA J. Peter Gogarten Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA Sylvie Goussard Unité des Agents Antibactériens, Institut Pasteur, Paris, France Catherine Grillot-Courvalin Unité des Agents Antibactériens, Institut Pasteur, Paris, France Ruth M. Hall CSIRO Molecular Science, North Ryde, New South Wales, Australia James F. Hancock Department of Horticulture, Michigan State University, East Lansing, Michigan, USA Hyman Hartman IASB, Cambridge, Massachusetts, USA G. F. Hatfull Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Jack A. Heinemann Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand R. W. Hendrix Pittsburgh Bacteriophage Institute, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Katrin Henze Institut für Botanik III, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany xiv CONTRIBUTORS 14
  • 11. Nathan C. Hitzeman Genotypes Inc., San Francisco, California, USA Ronald A. Hitzeman Genotypes Inc., San Francisco, California, USA Susan Hollingshead Department of Microbiology, University of Alabama, South Birmingham, Alabama, USA Michael J. Italia Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA Clarence I. Kado Davis Crown Gall Group, University of California, Davis, California, USA Margaret G. Kidwell Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson, Arizona, USA Eugene V. Koonin National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA Valentin A. Krassilov Paleontological Institute, Moscow, Russia David J. Lampe Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania, USA) Jeffrey G. Lawrence Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Guillaume Lecointre Service de Systématique moléculaire (GDR CNRS 1005), Muséum National d’Histoire Naturelle, Paris, France Wen-Hsiung Li Ecology and Evolutionary Biology, University of Chicago, Chicago, Illinois, USA Chin Y. Loh Genotypes Inc., San Francisco, California, USA Eugene L. Madsen Department of Microbiology, Cornell University, Ithaca, New York, USA Kira S. Makarova National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA and Department of Pathology, F.E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA William Martin Institut für Botanik III, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany Ivan Matic Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France CONTRIBUTORS xv 15
  • 12. Robert V. Miller Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahama, USA Gisela Mosig Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee, USA Lorraine Olendzenski Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA Honor C. Prentice Department of Systematic Botany, Lund University, Lund, Sweden Michael D. Purugganan Department of Genetics, North Carolina State University, Raleigh, North Carolina, USA Alfred Pühler Department of Genetics, University of Bielefeld, Bielefeld, Germany Miroslav Radman Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France Loren H. Rieseberg Department of Biology, Indiana University, Bloomington, Indiana, USA Steven A. Ripp Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA Hugh M. Robertson Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA Claus Schnarrenberger Institut für Pflanzenphysiologie und Mikrobiologie der FU Berlin, Berlin, Germany K. Schultze Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Joana C. Silva National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland, USA M. C. M. Smith Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, UK Jay V. Solnick Departments of Internal Medicine and Medical Microbiology and Immunology, University of California, Davis, California, USA Felipe N. Soto-Adames Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (present address: Department of Biology, University of Vermont, Burlington, Vermont, USA) Michael J. Stanhope Bioinformatics Department, GlaxoSmithKline, Collegeville, Pennsylvania, USA xvi CONTRIBUTORS 16
  • 13. Michael Syvanen Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California, USA François Taddei Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France Andreas Tauch Department of Genetics, University of Bielefeld, Bielefeld, Germany Olivier Tenaillon Inserm E9916, Faculté de Médecine Necker-Enfants Malades, Université Paris V, Paris, France K. Voigt Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Kimberly K. O. Walden Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA Carole I. Weaver Genotypes Inc., San Francisco, California, USA Mark E. Welch Department of Biology, Indiana University, Bloomington, Indiana, USA Richard J. Weld Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand Donald I. Williamson Port Erin Marine Laboratory (University of Liverpool), Isle of Man, UK Yuri I. Wolf National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA A. Wöstemeyer Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany J. Wöstemeyer Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller Universität, Jena, Germany Glenn M. Young Department of Food Science and Technology, University of California, Davis, California, USA Olga Zhaxybayeva Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA CONTRIBUTORS xvii 17
  • 14. S E C T I O N I Plasmids and Transfer Mechanisms in Bacteria Section 1 of this book deals with plasmids and mechanisms of gene transfer in bacteria. We will not be attempting a comprehensive review of plasmid biology, which has been the subject of many excellent reviews and is even well covered in many textbooks. Rather, a potpourri of topics will be sampled that illustrate recent developments and unexpected findings related to plasmid-mediated gene transfers. Plasmids figure prominently in the discussion of horizontal gene transfer because a large number of plasmids will stimulate conjugal transfer of bacterial DNA to cells from an extremely broad range of organisms. These include transfer to unrelated bacteria, yeasts and other fungi and plants. Chapter 1 by Heineman and Chapter 5 by Kado deal with the evolution of conjugal plasmids themselves. These vectors of horizontal transfer experience dual evolutionary pressures of survival in hosts via vertical transmission and the ability to adapt in new environments after horizontal flow. The remaining chapters in this section de- scribe some of the more recent interesting plasmid-related developments. In Chapter 2, Hall describes integrons, a site-specific recombi- nation system that serves to assemble new antibiotic resistance genes into pre-existing transposable elements. In Chapter 3, Tausch and Pühler describe an unusual antibiotic resis- tance plasmid that is a mosaic of elements found previously from throughout the bacterial kingdom. This is noteworthy because the ge- netic rearrangements and gene transfers that gave rise to this plasmid have likely occurred in the past 50 years, while the genes come from a group of distantly related organisms that last shared a common ancestor approximately 1.5–2 billion years ago. In Chapter 6, Weld and Heinemann review protein transfers, a topic that has captured attention in recent years be- cause of its importance in pathogenic mecha- nisms. Protein transfer is probably also important in ensuring survival of transferred DNA in foreign cells. As is clear, bacteria have numerous and highly adapted mechanisms in place to facilitate the transfer of DNA from donor to recipient cells. These mechanisms do not respect species boundaries. The question as to whether or not these mech- anisms operate in natural populations is the subject of the remaining three chapters in this section. It has been known for many years that conjugal plasmid transfer occurs among bac- teria in hospitals, farms and natural environ- ments. Along these lines, Madsen in Chapter 4 has an interesting story that documents the emergence of a plasmid that makes enzymes which degrade coal tars and has spread among different bacterial species in a toxic waste dump. Chapters 7 (by Day) and 8 (by Miller and Ripp) show evidence that the DNA transfer mecha- nisms of transformation and bacterial virus transduction operate efficiently in natural environments. 1 19
  • 15. C H A P T E R 1 Recent History of Trans-kingdom Conjugation Gayle C. Ferguson and Jack A. Heinemann Conjugation is a mechanism of horizontal gene transfer (HGT) first observed between bacteria. The conjugative mechanism appears to be ana- logous, and sometimes homologous, to other means of transferring genes from bacteria to possibly members of every biological kingdom. As such, conjugative mechanisms of DNA transfer are necessary for a host of spectacular phenotypes such as symbiosis, virulence and antibiotic resistance. The conjugative mecha- nism is also related to the means of translocating and transferring proteins from bacteria to other species. Thus, this nearly generic form of macromolecular transport may move genes and other molecules across species boundaries. Some of these molecules may have immediate effects (e.g. through pathogenesis) and some lasting effects (e.g. through inheritance). There is even evidence that inheritable effects can be caused by transferred proteins. Interest in HGT, previously considered on the fringe, has in- creased dramatically due to the realization that HGT is not an anomaly but a biological fundamental. INTRODUCTION The idea that genes are transferred at any appre- ciable frequency between species has evolved from one scorned by molecular phylogenists to a mainstream concept. Previously, only frustrated phylogenists would dwell on the odd DNA sequence that could unlace the bootstrap analysis (Gogarten et al., 1999). Whole chromosome se- quencing of organisms, however, is beginning to validate the concept that genomes are littered with “carcasses” of DNA from other spe- cies – some genes remaining functional and neu- tral, beneficial, or deleterious to the host, and some slowly fading away into the background average G + C content of the new host. The extent of horizontal gene transfer (HGT) between organisms is difficult to determine for two main reasons. DNA sequence information is, first, limited by the simplicity of the four letter code and secondly, by the constraints on the se- quence when it must reproduce in synchrony with the host (Heinemann, 2000b; Heinemann and Roughan, 2000). Thus, the mechanisms of HGT as well as bioinformatic tools are required to quantify the extent of HGT. The renaissance in HGT thinking brought about by bioinformatics has a history and origin different from the mechanism studies. These studies identify the means by which genes move between two neighbors that may or may not share a vertical lineage. Studies describing the gene transfer mediated by viruses, plasmids, transposons and transformation are much older than bioinformatics. Mechanism studies did not make HGT a mainstream concept, though, be- cause they were considered “laboratory phe- nomenon” or “interesting exception to the rule for most genes or most organisms” by many. The mechanism studies did, however, open Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 3 All rights of reproduction in any form reserved. 21
  • 16. imaginations to the potential for HGT and legiti- mized those who subjected it to serious study. This review will focus on gene transfer be- tween prokaryotes and eukaryotes by mecha- nisms that are identical, or similar, to bacterial conjugation. The review will not be a systematic account of all the literature relevant to HGT and conjugation. Instead, it will focus on publications that represent unambiguous conflations of ideas that led to HGT becoming an independent phe- nomenon for study and established bacterial conjugation as a central, general, mechanism for interkingdom gene transfer (Amábile-Cuevas and Chicurel, 1992; Heinemann, 1992). We begin with an abbreviated history of the merger be- tween HGT and crown gall disease in plants that has developed an inseparable link with bacterial conjugation. Finally, we will discuss bacterial conjugation as a paradigm of interkingdom macromolecular exchange mechanistically con- nected to pathogenesis. By the mid-twentieth century, interspecies gene transfer was recognized as an important means by which bacteria acquired antibiotic re- sistance. Those findings, as indeed most early studies in gene transfer, remained focused on the particular genes or organisms of interest. Our review of the literature suggests to us that a change in thinking about HGT was gaining momentum in the late 1960s. Subsequently, a number of studies examined HGT as a possible phenomenon in its own right, without need of allusion to important organismal adaptations, the success of pathogens (e.g. viruses and Agrobacterium tumefaciens), or the exception to the rule that all prokaryotic biology can appear to be to botanists and zoologists! THE CONVERGENCE OF INTERKINGDOM DNA TRANSFER AND CROWN GALL A. tumefaciens was clearly linked to crown gall tumors in some plants long before the 1960s (ref- erences in Stroun et al., 1970; Nester and Kosuge, 1981; Zhu et al., 2000). However, the seminal clues that the nature of the disease was inseparable from DNA transfer to the host emerged in that decade. Work by Kerr demonstrated that A. tumefaciens virulence characters were transmitted between bacteria, by an unknown mechanism (Kerr, 1969). In the late 1970s, the DNA that caused gall formation, T-DNA, would be identi- fied as a component of a conjugative plasmid, called Ti, in A. tumefaciens (Nester and Kosuge, 1981). The T-DNA was subsequently found inte- grated into plant chromosomes (Thomashow et al., 1980; Yadav et al., 1980; Zambryski et al., 1980). The search for T-DNA illustrates two different approaches to the study of interkingdom gene transfer operating simultaneously. One group of researchers, which we arbitrarily call the gen- eralists, was dominated by the sense that HGT was a phenomenon independent of the partic- ular biology of the donor and recipient organ- isms, such as the biology of the phytopathogen A. tumefaciens and its potential plant hosts. The other, which we refer to as the specialists, used the power of the causal relationship between A. tumefaciens and the gall tissue to discover HGT. The two approaches had complementary strengths and both endured the inevitable false positive and negative results that accumulate whenever techniques are pushed to their ex- treme limits of sensitivity. The path to the discovery of the discrete DNA sequences transferred from A. tumefaciens to the host, and even to other soil bacteria, was itself a study in the limits of the contemporary molecular techniques. The pioneers at the roots of the crown gall mystery during the 1960s and 1970s were also at the leading edge of molecular bi- ology and biochemistry. From such an edge, there is the risk of accumulating negative results, that is, for example, of not seeing DNA transfer (see below). New techniques also require refine- ment to distinguish between the noise at their limits of detection and true signals. The results of these early studies were consistently “equivocal, but collectively they suggested that bacterial nuc- leic acids might play a role in tumorigenesis” (Drlica and Kado, 1975). Generalists and specialists Both generalists and specialists were reporting the transfer of bacterial nucleic acids and pos- sibly proteins to eukaryotes by the late 1960s. 4 G.C. FERGUSON AND J.A. HEINEMANN 22
  • 17. The nucleic acids were invariably pursued in bacteria-free tissues by hybridization (refer- ences in Drlica and Kado, 1975) or hybridization and density centrifugation (Stroun et al., 1970; Stroun and Anker, 1971, 1973). The conclusiveness of the hybridization method itself, however, was systematically chal- lenged (Drlica and Kado, 1975). Hybridization methods used to demonstrate the presence of bacterial DNA in eukaryotes were often flawed because a control measurement of hybrid thermal stabilities or dissociation profiles was omitted (Chilton et al., 1974; Drlica and Kado, 1974; Kado and Lurquin, 1976). With improved techniques applied later in the 1970s, A. tumefaciens nucleic acids were not detected in tumors (Chilton et al., 1974; Drlica and Kado, 1974). The data of some groups were unable to be reproduced at this experimental standard (for an excellent discussion on the technology of the period, see Drlica and Kado, 1974). Why did some detect nucleic acids while others did not? One possible explanation is that the sporadic claims of nucleic acid detection were artefacts generated by techniques pushed to their limits. A second possibility is that the practitio- ners of state-of-the-art techniques are important contributors to detection limits. A third possibility is experimental design. Of course, these three possibilities are not mutually exclusive and cannot be distinguished retrospectively. With the increase in rigor applied to hybrid- ization experiments came an increase in the pre- cision for calculating the detection limits of the techniques (Drlica and Kado, 1974, 1975; Kado and Lurquin, 1976). Chilton et al.’s DNA–DNA hybridization technique, for example, limited detection to one bacterial genome per three dip- loid plant genomes and “would not detect single or even multiple copies of a small specific fraction (<5%) of the bacterial … genome in tumor DNA” (Chilton et al., 1974). Such famous negative results cannot, unfortunately, be di- rectly compared with all reported positive de- tection of nucleic acids because of differences in determining the sensitivities of the techniques. Thus history cannot distinguish between spo- radic artefacts and individual experimenters as explanations for different results from all con- temporary experiments. Some groups monitored the production of bacteria-specific nucleic acids in eukaryotic tis- sues (Stroun et al., 1970). Although these studies were also not above the criticisms leveled against other hybridization studies and were not consistently reproduced (discussed in Drlica and Kado, 1975), ongoing RNA synthesis poten- tially provided access to larger quantities of nu- cleic acids complementary to the probe. In contrast, those groups searching only for trans- ferred bacterial DNA were limited by the small number of copies of those sequences in prepara- tions of eukaryotic genomes. History cannot distinguish between possible sporadic artefacts and differences in experimental design as the explanation for different data from all the dif- ferent experimenters. Some generalists introduced further confu- sion when they reported that DNA transfer oc- curred from not just A. tumefaciens, but also Escherichia coli, Bacillus subtilis and Pseudomonas fluorescens to both plants and animals. Hence, “The relationship of (these observations) to the crown gall disease (was) ambiguous” (Drlica and Kado, 1975). Since only A. tumefaciens in- duced tumors, the mechanism of putative nu- cleic acid transfers from these other bacteria may have been irrelevant to that conducted by A. tumefaciens when it induced tumors. The generalist view was to be eclipsed by the finding of particular T-DNA sequences in plants and the characterization of a mechanism that could account for its transfer. T-DNA transfer would, for a time, serve as the paradigm of interkingdom gene transfer systems. The gener- ality of HGT would be revived in the 1980s by the finding that bacterial conjugative plasmids and T-DNA were different DNA transferred by the same mechanism (Heinemann, 1991; Sprague, 1991), providing retrospective cre- dence to generalists’ claims if not vindication of early experiments. Critical experimental limits to HGT detection Until recently, interkingdom DNA transfer has been mostly observed through the isolation of phenotypically recombinant organisms (i.e. gene RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 5 23
  • 18. transmission). DNA transfer can be inferred from any instance in which donor genes are recovered from recipient organisms. This is usually accom- plished by selecting recombinant phenotypes. Such phenotypes are the complex product of gene transfer and subsequent stabilization in the germ line of the recipient. Gene transfer is likely not the limiting event in most instances of gene transmission (Heinemann, 1991; Matic et al., 1996). Since inheritable phenotypes or stably maintained DNA sequences remain the easiest way to detect transferred genes, the importance of gene transmission in biasing inferences of the rate and extent of HGT cannot be ignored. In fact, the general reliance on observing recombinant phenotypes or isolating transferred DNA from offspring underestimates HGT (Chilton et al., 1974; Drlica and Kado, 1974; Heinemann and Roughan, 2000; Heinemann, 2000b). Several authors over the years have empha- sized the importance of distinguishing between gene transfer and transmission to avoid instilling a bias in experimental design and interpretation (reviewed in Heinemann, 1991, 1992). Clark and Warren (1979) made the most systematic justifica- tion for the terminology. The first authors to demonstrate the generality of interkingdom con- jugation openly acknowledged the influence of that review on their experimental design (Figure 1.1). Confusion between transfer and transmis- sion may have similarly delayed discovery of transfer of DNA from A. tumefaciens to plants out- side the bacterium’s infectious host range (Grimsley et al., 1987). 6 G.C. FERGUSON AND J.A. HEINEMANN Mix on plate Mix on plate LEU2 r e p Minimal medium LEU2 FIGURE 1.1 Illustration of the original experiment demonstrating DNA transfer from bacteria to yeast by conjugation. The rationale for the experiment was that DNA transfer was more generic than could be detected by DNA amplification or the formation of recombinant organisms, which requires DNA transmission (Heinemann and Sprague, 1989). As a test, specially constructed donor bacteria (rectangles) were mixed with genotypically marked recipient yeast (circles with “buds”) and plated on medium (large open circles) permissive to the growth of only recombinant yeast. The conjugative plasmids (open circles inside bacteria) were modified to carry either the selectable yeast LEU2 gene or both LEU2 and a DNA sequence that permits replication of extrachromosomal DNA in yeast (rep). Colonies of yeast recombinants (solid black circles) were recovered at a frequency of up to 10% (per donor bacterium) when the plasmid carried yeast-specific replication sequences. Since the DNA introduced into the conjugative plasmids was not responsible for DNA transfer (Bates et al., 1998; Heinemann and Sprague, 1989; Heinemann, 1991), these experiments unequivocally demonstrated that transmission (necessary for detecting recombinants because the DNA is subsequently inherited vertically) was a poor indicator of transfer and the absence of experimentally demonstrated transmission did not imply the absence of DNA transfer. 24
  • 19. To illustrate further the importance of distin- guishing transfer from transmission, consider the recent report of a DNA virus, that infects an- imals, evolving via recombination between a DNA virus, that infects plants, and an RNA virus, that infects animals (Gibbs and Weiller, 1999). (Another remarkable intermediate in this chain of events was the likely contribution of a retroviral reverse transcriptase acting on the animal RNA virus to convert an RNA gene into DNA.) The plant virus must have been able to transfer to animals (but caused no obvious phe- notype). The many transfer events preceding the evolution of the new variant virus were not detected by selecting or observing a recombi- nant animal, and likely would not have been detected even with current DNA amplification technologies. The transmission event could be detected, but provides no quantitative informa- tion about the frequency of transfers of the orig- inal virus to animals. Furthermore, transferred nucleic acids can be retained by recombination even if whole genes are not inherited (reviewed in Heinemann, 1991; Matic et al., 1996). The extent of this recombina- tion can be masked by the selectivity of homolo- gous recombination enzymes that eliminate long tracts of dissimilar nucleotide sequences better than short tracts (Rayssiguier et al., 1989; Heinemann and Roughan, 2000). Certain envi- ronments and mutations that reduce the activity of mismatch repair systems in particular have the effect of reducing selectivity (Matic et al., 1995; Heinemann, 1999b; Vuli’c et al., 1999). Recombi- nation events resulting in the incorporation of short tracts of DNA, even over sequences of ex- treme genetic divergence, can be difficult or im- possible to identify by analysis of DNA sequences (Heinemann and Roughan, 2000). CONJUGATION AS A PARADIGM SYSTEM OF INTERKINGDOM DNA TRANSFER The first indication that bacterial conjugation de- scribed a general mechanism of interkingdom gene transfer came from the suggestion that certain DNA intermediates observed in A. tumefaciens resembled hypothetical DNA inter- mediates in bacterial conjugation (Stachel et al., 1986). In hindsight, that connection was probably better informed by inspiration than actual data, but nevertheless has withstood significant test. Conjugation Bacterial conjugation in its broadest sense has been extensively reviewed, so only a brief de- scription will be provided here (Heinemann, 1992, 1998; Frost, 2000). The focus in this review is on the paradigm conjugative systems defined by the IncP and IncF plasmid groups. Conjugation mediated by these plasmids requires, at a minimum, a cis-acting DNA se- quence called the origin of transfer (oriT). All other functions (called tra) act in trans thus al- lowing plasmids with all trans-acting functions also to transfer plasmids with no or a few trans- acting functions (Heinemann, 1992). The trans- acting gene products are divided further into those involved in DNA metabolism (and are usually specific to a particular oriT) and those in- volved in DNA transport and cell–cell interac- tions (and thus will interact with a greater range of other plasmids). The conjugative genes spe- cific to DNA metabolism introduce a nick at oriT and initiate the unwinding and concomitant transfer of DNA to a recipient cell. Both strands are used as templates for the synthesis of a com- plementary strand, one in the donor cell and one in the recipient. Single-stranded plasmid DNA (ssDNA) has been captured in recipient cells, confirming the mechanism of plasmid mobilization. The DNA is recircularized in the recipient. The transport apparatus has not been described biochemically (Heinemann, 2000a), but the genes necessary for forming the apparatus are all plasmid- encoded (Heinemann and Ankenbauer, 1993; Heinemann et al., 1996). T-DNA is interkingdom conjugation This uncontroversial model of the conjugative process grounded a model of T-DNA mobiliza- tion and transfer proposed by Stachel et al. (1986). Their experiment involved isolating RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 7 25
  • 20. DNA of the T-DNA region from A. tumefaciens (not the plant) after it was induced to prepare the T-DNA for transfer. They provided con- vincing evidence that linear ssDNA strands de- fined by the left and right borders of the T-DNA region accumulated in induced bacteria, and that Ti plasmids from induced bacteria had nicks in the border sequences on the strand cor- responding to the liberated T-DNA. It appeared to Stachel et al. that the left and right borders of the T-DNA region, which are characterized as direct repeats, functioned like oriT sequences. Nicking and unwinding liber- ated only the DNA between the nicks, rather than a strand of DNA the length of the Ti plasmid. When the transfer process could not be completed, the T-DNA accumulated in the bacterium. However, the phenomenology differed from the molecular biology of conjugation in impor- tant ways. First, hypothetical ssDNA transfer in- termediates do not accumulate in bacteria that hold conjugative plasmids even when constitu- tively induced. Secondly, the conjugative ssDNA was isolated from bacterial recipients; the so-called T-DNA in the Stachel et al. study was never recovered from plants. Thirdly, there existed no evidence at the time that the DNA be- tween tandemly repeated oriTs would be liber- ated during mobilization. Whereas it was shown subsequently that tandem oriT repeats do result in mobilization-specific DNA insta- bility in some plasmids (Bhattacharjee et al., 1992; Furuya and Komano, 2000), the repeat of IncP oriTs, which are thought to be the closest relatives of the T-DNA borders (Waters et al., 1991; Waters and Guiney, 1993), does not result in mobilization-specific liberation of inter- vening DNA (Heinemann and Schreiber, per- sonal observation). Nevertheless, the model has been vindicated by several subsequent genetic tests (Lessl and Lanka, 1994; Christie, 2000). First, T-DNA re- combination experiments within plant cells pro- vide evidence that T-DNA is transferred, and enters the nucleus, single-stranded (Tinland et al., 1994). Secondly, the processing reaction be- tween the cis-acting border repeat sequences and its putative nick-ase (virD2) could be re- placed with the oriT and its cognate nick-ase (mobA) from the IncQ plasmid RSF1010 (Bu- chanan-Wollaston et al., 1987). Third, RSF1010 transmission between Agrobacteria was found to be dependent on the other Ti-encoded genes virA, virG, virB4, virB7 and virD4 (Beijersbergen et al., 1992). Thus, the vir genes, originally identified because they were necessary for viru- lence, can substitute for tra in mediation of conjugative plasmid transfer. The ability to mix and match genetic require- ments of bacterial conjugation and Ti-mediated virulence is consistent with the structural simi- larities of conjugative and virulence genes (Table 1.1). The oriT region of IncP plasmids is homologous to the T-DNA borders (Waters and Guiney, 1993; Frost, 2000), while the oriT of the Ti plasmid is homologous to the IncQ oriT. Many macromolecular transport systems appear to be composed of gene products ho- mologous to the tra functions of conjugative plasmids, including the vir genes and type IV protein secretion systems in Bordetella pertussiss, Helicobacter pylori and Legionella pneumophila (Christie, 2000; Frost, 2000) (Tables 1.1 and 1.2). CONJUGATION IS SUFFICIENT FOR INTERKINGDOM CONJUGATION A surprise to the crown gall groups was the finding that the transfer of DNA from A. tumefaciens to plants was related in part to bacte- rial conjugation. Meanwhile, yeast studies were soon to show that conjugation could account for interkingdom DNA transfer and that the ability to conjugate with eukaryotic cells is not an evo- lutionary quirk of A. tumefaciens. In 1989 we crossed bacteria with the yeast Saccharomyces cerevisiae using the same plasmids that mediated conjugation between bacteria (Heinemann and Sprague, 1989) (Figure 1.2). E. coli transferred a plasmid marked with the S. cerevisiae replication origin 2µ and LEU2 gene, to yeast. Recombinant (Leu+ ) yeast were only formed when the bacteria contained a con- jugative plasmid able to mobilize the marker plasmid in trans. Formation of Leu+ yeast recombinants was dependent on donor– recipient contact, donor viability, functional 8 G.C. FERGUSON AND J.A. HEINEMANN 26
  • 21. Proposed functions of vir genes required for T-DNA transfer from A. tumefaciens to plantsb vir homologues on conjugative plasmids vir homologues involved in protein transfer/virulence vir homologues with as yet unknown function IncFb IncPc pTiC58 (tra)b IncWb IncNb B. pertussisb B. suisd B. abortise L. pneumo- phila (icm/dot)e H. pylori (cag)b L. pneumo- phila (lvh)f R. prowazekiif Wolbachia sp.g A. actinomy- cetemcomitansh virB1 Transglycosylase orf169 trbN traL virB1 virB2 Pilin subunit traA trbC trbC trwL traM ptlA virB2 lvhB2 virB3 traL trbD trbD trwM traA ptlB virB3 lvhB3 virB4 ATPase, transport activation traC trbE trbE trwK traB ptlC virB4 cagE lvhB4 virB4 virB5 Pilin subunit traE trbF trbF trwJ traC virB5 lvhB5 virB6 Candidate pore former trbL trwI traD ptlD virB6 lvhB6 virB7 Transporter assembly trwH traN ptlI virB7 rp288 virB8 trwG traE ptlE virB8 lvhB8 rp289 virB8 virB9 Transporter assembly trwF traO ptlF virB9 orf15 lvhB9 rpB9 virB9 virB10 Coupler of inner and outer membrane subcomplexes traB trbI trbI trwE traF ptlG virB10 dotG/icmE orf13 lvhB10 rpB10 virB10 virB11 ATPase, transport activator trbB trbB trwD traG ptlH virB11 dotB orf11 lvhB11 rpB11 virB11 tadA virD4 ATPase, coupler of DNA processing and transport systems traD traG trwB orf10 lvhD4 rpD4 virD4 virD2 Site-specific single- stranded nicking at the right and left borders traIi Right and left borders Site of VirD2 nicking oriTj Table adapted from Christie (1997a). a Christie (1997a, 2000). b Li et al. (1998). c O’Callaghan et al. (1999). d Frost (2000). e Segal et al. (1999). f Waters et al. (1991). g Masui et al. (2000). h Kachlany et al. (2000). i Functional homology (Pansegrau et al., 1993). j Sieira et al. (2000). TABLE 1.1 A. tumefaciens T-DNA transfer genes that are homologous to genes required for conjugation, protein transfer and virulence in a range of Gram-negative bacteriaa 27
  • 22. oriT and mob genes, and was independent of ex- ogenous DNAse, indicating that the mechanism of gene transfer was not transformation. E. coli– yeast conjugation was subsequently found to be dependent on the same tra genes as required for conjugation between E. coli, with no additional plasmid-encoded requirements (Heinemann and Sprague, 1991; Bates et al., 1998). These experiments suggested that DNA transfer from E. coli to S. cerevisiae occurred by a mechanism analogous to conjugation. The range of yeast able to serve as E. coli conjugal re- cipients has been extended to at least six evolu- tionary divergent genera (Heinemann, 1991; Hayman and Bolen, 1993; Inomata et al., 1994). Unlike A. tumefaciens and plants, E. coli and yeast have no known ecological relationship and are not expected to have evolved such a specialized interaction. Therefore interkingdom gene transfer has few, if any, specific requirements evolved within the particular biology of the donor and recipient organism (although virulence and other phenotypes certainly do have specific requirements). Interkingdom conjugation is not a species-specific phenomenon E. coli is not unique in its ability to conjugate with yeast. The T-DNA from A. tumefaciens also transferred to S. cerevisiae, but by vir-dependent conjugation (Bundock et al., 1995). Using URA3 as a selectable marker with or without the 2µ replication sequence between the T-DNA bor- ders, the frequency of transmission of both replicative and integrative vectors was com- pared (Bundock et al., 1995). Where transferred T-DNA could replicate autonomously, most transconjugants inherited the vector in its en- tirety. This was attributed to a failure of VirD2 sometimes to nick the left border, effectively creating a situation where the right border was the only oriT. Other transconjugants carried recirularized dsT-DNA molecules. 10 G.C. FERGUSON AND J.A. HEINEMANN Fungi Eubacteria Plants Animals FIGURE 1.2 Bacteria transfer DNA and proteins to plant, animal and fungal cells by similar and related mechanisms. Bacteria transfer DNA (solid lines and large open circles) to both yeast and plant cells by conjugation. Bacterial DNA is integrated into eukaryotic chromosomes (double helices) upon entering the nucleus (white ellipses). Proteins (solid black circles) are transferred to animal cells during pathogenesis. Conjugative plasmids have genes homologous to some genes required for virulence in many bacterial pathogens. Some of those homologous genes are known to be required for DNA or protein transfer. 28
  • 23. Interkingdom conjugation is not a plasmid-specific phenomenon Is the ability to conjugate with eukaryotic cells a particular feature of so-called “broad-host- range” plasmids, such as the IncP family? Bates et al. (1998) compared the ability of conjugation functions from three incompatibility groups to transmit a marked shuttle vector to yeast. IncP plasmids transmitted the shuttle plasmid under conditions where transmission by the narrow- host-range IncF and IncI1 plasmids was not detected (Bates et al., 1998). In contrast, all plasmids were equally capable of transmitting the shuttle plasmid to E. coli. Since recombinants were the only evidence of DNA transfer, it remains formally possible that some aspect of the IncP tra system enhances transmission by contributing to the ability of transferred DNA to be inherited. Consistent with this possibility, Heinemann and Sprague did observe F-mediated DNA transmission to yeast using an IncF plasmid derivative instead of mobilizing a shuttle plasmid in trans (Heinemann and Sprague, 1989). The higher copy number of their F plasmid derivative may have contributed to the frequency of detectable DNA transmission (Bates et al., 1998). CONJUGATION AS A CONVERGENCE OF MACROMOLECULAR TRANSPORT SYSTEMS A. tumefaciens provided an anecdotal link be- tween DNA transfer by conjugation and in pathogenesis. However, in that case, the disease was made possible by the genes transferred but DNA transfer was itself not causing the disease. It has become clear over the past decade that the DNA transport apparatus of conjugation is the ancestor, or at least a sibling (O’Callaghan et al., 1999), of other macromolecular transport sys- tems that are the raison d’être of the disease. As mentioned above, type IV protein secretion genes are homologous to conjugation genes and the transport mechanism for both protein and DNA may be the same (Winans et al., 1996; Christie, 1997a; Kirby and Isberg, 1998; Segal and Shuman, 1998a; Christie and Vogel, 2000). Bioinformatics Many homologues of the Ti virB genes (B4, B9–11 and sometimes also virD4) are found on conjugative plasmids and on chromosomes, as inferred from similarities in sequence and orga- nization. DNA transfer homologues include tra of IncN (Pohlman et al., 1994) and Ti (Li et al., 1998), trb of IncP and trw of IncW (Kado, 1994; Christie, 1997a) plasmids. The virB genes have homologues in the pertussis toxin secretion system, ptl of B. pertussis (Covacci and Rappuoli, 1993; Shirasu and Kado, 1993; Weiss et al., 1993; Farizo et al., 1996). The cag pathoge- nicity island of Helicobacter pylori, implicated in contact-mediated secretion of proteins into epi- thelial cells, is homologous to virB (Tummuru et al., 1995; Censini et al., 1996; Christie, 1997b; Covacci et al., 1997). virB homologues have also been found in the chromosome of the obli- gate intracellular parasite Rickettsia prowazekii (Andersson et al., 1998), the arthropod intracellular pathogen Wolbachia sp. (Masui et al., 2000), the human pathogen Actinobacillus actinomycetem-comitans (Kachlany et al., 2000) and are essential for virulence in the intracellular pathogens Brucella abortus and Brucella suis (O’Callaghan et al., 1999; Sieira et al., 2000). Relations between protein and DNA secre- tion systems is not restricted to vir. The icm/dot genes, essential for L. pneumophila survival and replication inside human alveolar macro- phages, are homologous to conjugation genes from various plasmids (Segal and Shuman, 1997, 1999; Purcell and Shuman, 1998; Segal et al., 1998; Vogel et al., 1998) (Table 1.2). Fourteen of the icm/dot genes are similar, both in sequence and in structural organization, to the tra region of IncI plasmid Col1b-P9 (Segal and Shuman, 1999), and icmE is homologous to trbI of IncP plasmid RK2. Mechanism The link between protein and DNA secretory systems is also suggested by mechanistic RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 11 29
  • 24. studies. For example, a radiolabeled DNA primase (Rees and Wilkins, 1989, 1990) and E. coli’s RecA protein (Heinemann, 1999a) were transferred to recipients during bacterial conju- gation. In these cases, protein and DNA transfer were associated but the possibility remains that the protein and DNA need not be associated for transfer (Heinemann, 1999a). Likewise, the decreased stability of T-DNA transferred from virE2 mutant bacterial donors is complemented by in planta expression of VirE2 protein (Rossi et al., 1996) and extracellularly by virE2+ bacteria (Christie et al., 1988; Citovsky et al., 1992), suggesting that VirE2 is also transferred into plants independ- ently of T-DNA. In fact, VirE2, VirD2 and VirF may be transported to plants independently of both T-DNA and the virB genes, although tumorigenic virB-independent transfer has not been demonstrated (Chen et al., 2000). Intri- guingly, tumorigenicity is significantly inhibited when A. tumefaciens also carries the mobilizable RSF1010 plasmid (Binns et al., 1995; Stahl et al., 1998). Similarly, RSF1010 attenuates the viru- lence of L. pneumophila (Segal and Shuman, 1998b). In these two cases, the RSF1010:protein mobilization complex and the substrate of the virulence transport systems are thought to com- pete (Figure 1.3). That mutations in mobA suppress the effect of RSF1010 on L. pneumophila virulence is consis- tent with this hypothesis (Segal and Shuman, 1998b). The icm/dot genes substitute for tra sup- plied in trans to transmit RSF1010 to recipient L. pneumophila by conjugation, indicating that the RSF1010:MobA complex is a substrate for the se- cretory system encoded by icm/dot (Segal and Shuman, 1998b; Segal et al., 1998; Vogel et al., 1998). The effect of RSF1010 on virulence could be failure to transport efficiently, as yet uniden- tified, effector proteins that alter vesicle tar- geting within the macrophage because they are displaced by the RSF1010:MobA complex (Segal and Shuman, 1998a). The virB homologue lvh does not complement the effect of icmE/dotB mutations on virulence, but it did complement the effect of icmE/dotB mutations on conjugation (Segal et al., 1999). Thus, the physical require- ments for translocating the RSF1010:MobA com- plex and putative effector protein are not identical. The effects of RSF1010 on A. tumefaciens tumori- genicity are suppressed by over-expression of virB9, virB10 and virB11 (Ward et al., 1991), whose products are located in the cell mem- brane and form the putative conjugation pore (Christie, 1997a). Again, it has been suggested that an RSF1010:MobA complex may displace the T-DNA complex from the translocation apparatus due to the former’s higher copy number, the constitutive presence of its pro- cessed form, greater affinity for the trans- location complex or slow passage through the translocation pore (Binns et al., 1995; Stahl et al., 1998). The IncW plasmid pSa is an even stronger sup- pressor of tumorigenicity than RSF1010. Several lines of genetic evidence suggest that the osa gene product of pSa blocks protein VirE2 translocation (Chen and Kado, 1994, 1996; Lee et al., 1999). osa was first identified as the gene sufficient to cause pSa abolition of oncogencity (Chen and Kado, 1994). The specific effect on VirE2 rather than a protein–DNA complex is supported by the obser- vation that osa did not inhibit the conjugative transmission of the Ti plasmid. 12 G.C. FERGUSON AND J.A. HEINEMANN TABLE 1.2 tra genes homologous to icm/dot genesa L. pneumophila icm/dot ColIb-P9 (IncI1) RK2 (IncP) icmT traK icmS icmP trbA icmO trbC icmI traM icmK traN icmE trbI icmG traP icmC traQ icmD traR icmJ traT icmB traU dotA traY dotB traJ trbB dotC traI dotG traH a Adapted from Segal and Shuman (1999). 30
  • 25. The osa product also does not inhibit T-DNA transfer. osa did not suppress oncogenicity when expressed in virE2 mutants as long as VirE2 was supplied by separate donors through extracellular complementation, or else it was produced by the recipient plant cell (Lee et al., 1999). The interesting ability for virE2 mutants to be complemented extracellularly by separate VirE2 donors was suppressed, however, when osa was expressed in the protein donor (Lee et al., 1999). Thus, the osa product specifically af- fects VirE2 translocation or function prior to T- DNA entry into the plant cell. The effects of pSa and RSF1010 on oncogenicity are similar but not identical. First, RSF1010 inhibits both VirE2 translocation and possibly T-DNA transfer, whereas pSa only prevents VirE2 translocation. Secondly, an RSF1010-protein complex is necessary for oncogenic suppression but only the osa gene product of pSa is required for suppression (Lee et al., 1999). Thirdly, over-expression of VirB9, VirB10 and VirB11 suppresses the RSF1010 effect on tumorigenicity but not the osa effect. These apparent dissimilarities may reflect only quantitative differences in the RSF1010 and pSa mechanisms, since RSF1010 partially inhibits oncogenicity and pSa completely abolishes tumor formation (Lee et al., 1999). However, the RSF1010 and pSa effects may have different mechanistic explanations. As discussed above, VirE2, VirD2 and VirF pro- teins are transported across the inner mem- brane by a virB24- and virD4-independent mechanism. The osa product, but not RSF1010, prevented VirE2, VirF and VirD2 from achieving normal periplasmic levels (Chen et al., 2000). This suggests that the osa product and MobA–RSF1010 could inhibit VirE2 translocation at different steps. While MobA– RSF1010 may inhibit the directed translocation of proteins through the putative outer mem- brane pore, the osa product may inhibit translocation across the inner membrane. Such a model is consistent with both the inner mem- brane localization of Osa (Chen and Kado, 1996) and the observation that VirB10, VirB11 and VirB12 over-expression did not restore tumor formation by A. tumefaciens carrying pSa (Lee et al., 1999). RECENT HISTORY OF TRANS-KINGDOM CONJUGATION 13 Plasmid RSF1010 Effector protein Bacterial inner membrane Periplasm Bacterial outer membrane Macrophage or plant cell cytoplasm Cytoplasmic membrane MobA FIGURE 1.3 The mobilizable IncQ plasmid RSF1010 inhibits transmission of T-DNA from A. tumefaciens to plant cells. Furthermore, RSF1010 inhibits the ability of L. pneumophila to evade fusion of its phagosome with lysosomes inside the macrophage. The icm/dot genes that are required to prevent lysozome fusion are also necessary for conjugation of RSF1010. It has been proposed that icm/dot is a system that mediates secretion of proteins into the macrophage cytoplasm or phagosome during phagocytosis. The mobilized form of RSF1010 may inhibit virulence by competing with the natural substrate of these protein secretion systems. (Adapted from Segal and Shuman, 1998a.) 31
  • 26. What came first, protein or DNA transfer? DNA and proteins are probably transferred be- tween species by similar mechanisms. The ef- fects of transferring non-nucleic acid molecules may sometimes be similar too; macromolecules, e.g. prions, other than nucleic acids possess gene-like qualities (Campbell, 1998; Heinemann and Roughan, 2000). Some proteins are not genes, but can influence epigenes that establish heritable phenotypes many generations after the protein has disappeared (Heinemann, 1999a). So conjugation may be a manifestation of protein secretion and, sometimes, protein se- cretion is another type of HGT. CONCLUSION HGT has established itself as a legitimate topic of study independently of the effects of the genes transferred on the biology of donor and recipient organisms. Nevertheless, the study of pathogens like A. tumefaciens and L. pneumophila, symbionts like Rhizobium meliloti, and phenotypes like antibi- otic resistance and crown gall, have each contrib- uted to the richness of the evidence supporting the notion that genes are less restricted by our no- tions of species sanctity than we have previously thought. In particular, the studies of bacterial con- jugation, crown gall disease and protein secretion have provided extensive mechanistic insight into how DNA is exchanged between kingdoms, spe- cies and siblings. Extensive similarities between genes identi- fied as either virulence or conjugation determi- nants provided an early hint that macro- molecular transport was a general phenom- enon. Those early hints have been vindicated by demonstrations of genetic interchangeability between some determinants (complementation studies) and genetic conflict between others. DNA is not special cargo but one of a number of molecules that might be transported by the same basic macromolecular transport systems. The ability to move molecules intercellularly has obvious implications for both single and multi- cellular organisms. Of immediate relevance are the diseases and recombinants that could arise from this nearly generic transport mechanism. But what of the molecules being transferred? Plasmids and viruses, for example, make excel- lent evolutionary livings transferring between or- ganisms, even evolving despite their effects on the host. Transfer alone might explain their exis- tence (Cooper and Heinemann, 2000). Did these genetic entities evolve a means to replicate by HGT, or was the existence of macromolecular transport enough for such semi-autonomous en- tities to evolve? Other kinds of molecules could transmit genetic information (Heinemann and Roughan, 2000). Could HGT be a mechanism for the evolution of genetic entities that are not nucleic acids? ACKNOWLEDGMENTS We thank A. Harker for critical reading of the manuscript and C.F. Delwichie for encouraging comments on our contribution to the first edi- tion. JAH acknowledges M. Stroun and P. Anker for their support, reprints and valuable insights. This work was supported in part by the Mar- sden Fund (Grant M1042 to JAH) and a Univer- sity of Canterbury Roper Scholarship (to GCF). REFERENCES Amábile-Cuevas, C.F. and Chicurel, M.E. (1992) Bacterial plasmids and gene flux. Cell 70: 189–199. Andersson, S.G.E., Zomorodipour, A., Andersson, J.O. et al. (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396: 133–140. Bates, S., Cashmore, A.M. and Wilkins, B.M. (1998) IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the Tra2 mating system. J. Bacteriol. 180: 6538–6543. Beijersbergen, A., den Dulk-Ras, A., Schilperoort, R.A. and Hooykaas, P.J.J. (1992) Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 256: 1324–1327. Bhattacharjee, M., Rao, X.-M. and Meyer, R.J. (1992) Role of the origin of transfer in termination of strand transfer during bacterial conjugation. J. Bacteriol. 174: 6659–6665. Binns, A.N., Beaupré, C.E. and Dale, E.M. (1995) Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J. Bacteriol. 177: 4890–4899. Buchanan-Wollaston, V., Passiatore, J.E. and Cannon, F. (1987) The mob and oriT mobilization functions of a 14 G.C. FERGUSON AND J.A. HEINEMANN 32
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  • 30. C H A P T E R 2 Gene Cassettes and Integrons: Moving Single Genes Ruth M. Hall Gene cassettes are very simple, small mobile elements that generally include only a single complete gene (or open reading frame) or occa- sionally two genes and a recombination site called a 59-be that enables them to be mobilized. Movement of cassettes is achieved by site- specific recombination with the reaction catalyzed by members of the IntI-type DNA integrase family (tyrosine site-specific recombinases) that are encoded by integrons. Most commonly, cas- settes are incorporated into a specific site, an attI site, that is found in the integron adjacent to the intI gene. By repeated rounds of recombination between a 59-be and the attI site, the IntI integrase can incorporate more than one cas- sette into the same integron leading to the for- mation of short or long arrays of gene cassettes. However, because the integron-encoded IntI integrases can also recognize secondary recom- bination sites, cassettes can also be incorporated at many other locations, allowing them to be widely disseminated. Several classes of integron that are differentiated by differences in the se- quences of the IntI integrases have been found and many more are likely to be found in the future. However, the gene cassettes are shared. Among the integron classes, some are part of mobile elements, also known as inte-grons, while others are located on bacterial chromo- somes. The integrons that are themselves mobile are most important in spreading gene cassettes from strain to strain and species to spe- cies. The integrons that are an integral part of a bacterial chromosome may act as storehouses of genes for emergencies that are added to, as well as sampled and spread, by the mobile integrons. INTRODUCTION When the processes of horizontal gene transfer move DNA from one organism to another there is little impact unless the incoming DNA is stably maintained and expressed in the recip- ient organism. Several different processes can achieve this outcome and examples include the stable maintenance of a plasmid or incorpora- tion of the incoming DNA into the bacterial chromosome or into plasmids already resident in that cell. Incorporation of new DNA into an existing chromosome (bacterial or plasmid) can occur by homologous recombination, but only if homologous regions are present in both DNA species. However, other specific processes such as transposition or site-specific recombination can also lead to stable incorporation of parts or all of an incoming DNA molecule. The regions that can move in this way are generally discrete genetic elements and the ability of such mobile elements to shift their location enables them to move into entities such as plasmids and conjugative transposons that are able to move from cell to cell with ease. Because of this, translocatable elements are an important force in horizontal gene transfer in the bacterial world and any associated gene or group of genes is Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 19 All rights of reproduction in any form reserved. 37
  • 31. ultimately able to gain access to many different organisms and species. In this chapter, the family of mobile elements known as gene cas- settes and their host elements, the integrons, are described. Integrons which are themselves mobile are also described. Further information on specific aspects can be found in recent re- views (Hall and Collis, 1995, 1998; Recchia and Hall, 1995a, 1997; Rowe-Magnus et al., 1999). FUNCTIONAL DEFINITION OF GENE CASSETTES AND INTEGRONS The definitions of gene cassettes and integrons are to a considerable extent interdependent as integrons are first and foremost defined by their ability to capture gene cassettes. Indeed, this is how they were found. Gene cassettes were ini- tially identified by virtue of the fact that many different gene cassettes, each containing a dif- ferent antibiotic resistance gene, can be found in the same sequence context (Cameron et al., 1986; Hall and Vockler, 1987; Ouellette et al., 1987; Wiedemann et al., 1987; Sundström et al., 1988; Hall et al., 1991). This situation allowed the identification of the approximate boundaries of gene cassettes and also of the conserved back- bone sequence into which the cassettes slot (Hall and Vockler, 1987; Sundström et al., 1988; Stokes and Hall, 1989). Within this conserved backbone, a gene was found whose product bore a significant resemblance to integrases (tyrosine site-specific recombinases) that are harbored by the genomes of some temperate phage (Hall and Vockler, 1987; Ouellette and Roy, 1987; Sundström et al., 1988; Stokes and Hall, 1989). Experimental evidence that this integrase (IntI1) was active was soon forth- coming (Martinez and de la Cruz, 1988, 1990). The term integron was originally coined to 20 R.M. HALL Empty integron Free gene cassette Integron with one cassette Integrated cassette FIGURE 2.1 Insertion of a circular gene cassette into an integron. (A) An empty integron, showing the three distinctive features: an intI gene that encodes the IntI integrase, an adjacent recombination site, attI (hatched box), and promoters Pc and Pint. (B) A gene cassette in its circular form consisting of a gene or open reading frame (ORF) and a 59-be recombination site (filled box). (C) An integron containing one gene cassette, with the boundaries of the integrated cassette shown below. IntI-catalyzed recombination between attI in the integron and the 59-be in the circular cassette results in insertion of the cassette into the integron. The ORF in the inserted cassette is now transcribed from Pc. gttrrry (A) and GTTRRRY (B) represent the 7 bp core sites surrounding the recombination crossover point in the attI site of the integron and in the 59-be of the circular cassette respectively. The configuration of these bases after incorporation of the cassette is shown in C. Further cassettes may be similarly inserted at attI, resulting in the accumulation of arrays of cassettes. 38
  • 32. describe this group of elements (Stokes and Hall, 1989) but, as further types of integrons have since been found, they are now designated class 1 integrons. In 1991, all of the sequence information that was available was drawn together and the general and current definition of a gene cassette as a single gene or open reading frame coupled with a downstream 59-be recombination site emerged (Hall et al., 1991). This study also gave rise to a model for the integration and excision of gene cassettes that predicted the existence of a circular form of cassettes (Figure 2.1). Experimental studies of cassette movement, using the IntI1 integrase, were subsequently reported. Precise excision of gene cassettes was demonstrated, thus defining them experimentally (Collis and Hall, 1992a) and the circular form of cassettes was also isolated (Collis and Hall, 1992b). Demonstration of the incorporation of circular gene cassettes into an integron completed the picture (Collis et al., 1993). An interesting aspect of the latter study is that cassettes are preferentially incorporated at the attI site of the integron to become the first cas- sette in an array of gene cassettes. Finally, in 1995, a known gene cassette was found in a plasmid that does not contain an integron (Recchia and Hall, 1995b). This demon- strated that cassettes can also move into almost any location, where they become fixed, and fur- ther cassettes that have been incorporated at such secondary sites have since been reported. GENE CASSETTES Gene cassettes are the smallest of the known types of mobile elements. Generally, they GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 21 A. Free circular cassette B. Integrated linear cassette 1L 2R 2L 1R LH simple site RH simple site Coding region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AAC X · A * T GTT . . . . . . . . . . T ATG 59-be 1L 2R 2L 1R LH simple site RH simple site Coding region . . . . . . . . . . . . . . . . . . . . . . AAC X · A * TT ATG G FIGURE 2.2 Structure of gene cassettes. (A) A cassette in its free, circular form. (B) A cassette in integrated, linear form showing the coding region of the gene and the 59-be recombination site. The extent of the coding region (not to scale) is marked by start (ATG) and stop (*) codons. Boxes surround 7 bp core site sequences, related to the consensus GTTRRRY, which lie within putative IntI binding sites that include the core sites as well as flanking bases. Left hand (LH) and right hand (RH) simple sites consist of pairs of binding sites (1L and 2L; 2R and 1R respectively). The relative orientations of the core sites are indicated with arrows. Bases found in all 59-be are shown, while other bases that conform to the core site consensus are represented by dots, and an extra base in 2L is marked by an X. In any individual 59-be, 1L and 1R sites are closely related, as are 2L and 2R, but the bases between them are not. An inverted repeat, represented by a pair of arrows, lies between 2L and 2R and has a variable sequence and length. The recombination crossover point is indicated by a vertical arrow. When the circular cassette is linearized by insertion into the attI site of an integron (B), the last six bases of 1R in the circular cassette become the first six bases of the integrated cassette. 39
  • 33. consist of a single gene or ORF together with a downstream IntI-specific recombination site known as a 59-be (59-base element). Occa- sionally, two ORFs are present in a single cas- sette. Each cassette is a discrete mobile element that can exist in either a free, closed-circular form or an integrated linear form (Figures 2.1 and 2.2). As cassettes contain no replication functions, the closed circular form cannot repli- cate and its role is limited to that of an interme- diate in cassette movement (Collis and Hall, 1992b). The boundaries of the linear gene cas- sette form have been precisely located (Stokes et al., 1997) and each cassette commences with a TT doublet and ends with a G residue. This is be- cause the strand exchange that occurs during cassette integration occurs between the G and TT of a completely conserved triplet that forms part of the 7 bp core site (GTTaggc or GTTRRRY) found at one end of 59-be (Stokes et al., 1997) and also in attI sites at the point of cas- sette integration (Hansson et al., 1997). The se- quence of any specific 59-be is found by joining the sequence at the right-hand (RH) end of the cassette to that at the left-hand (LH) end, to rec- reate the circular configuration (Figure 2.2). Be- cause each cassette contains a unique 59-be (see below) the 59-be are named after the gene in the cassette. Cassettes are compactly organized (Figure 2.2). In the integrated form, as few as 7 bp sepa- rate the beginning of the cassette from the initia- tion codon of the gene, leaving no space for a promoter and little space for a ribosome binding site. Commonly, the termination codon of the gene is very close to, or even within, the region ascribed to the 59-be recombination site. How- ever, occasionally a promoter is present within the cassette and translational attenuation regu- latory signals have also been found in the cmlA1 cassette and other related cmlA cassettes (Stokes and Hall, 1991). Although the cassettes that contain antibiotic resistance genes were the first to be identified, the diverse range of enzymatic functions encoded by the genes that confer resistance indicated that any gene could potentially become associated with a 59-be to form a functional gene cassette. There are now over 60 identified gene cassettes that confer resistance to antibiotics. These include genes for inner-membrane transporters (efflux proteins), β-lactamases belonging to classes A, B and D, acetyl-, adenylyl-, phosphoryl- and ribosyl- transferases with a variety of antibiotic substrate specificities, and dihydrofolate reductases (Recchia and Hall, 1995a; Hall and Collis, 1998; Mazel and Davies, 1999). Among the gene cas- settes found in the small chromosome of the Vibrio cholerae strain that has been completely se- quenced, there are also a few that contain antibi- otic resistance genes or potential resistance genes (Heidelberg et al., 2000). However, some of the gene cassettes found in V. cholerae or V. mimicus strains determine other functions. Gene products with known functions include a heat-stable toxin that is found in relatively few V. cholerae strains (Ogawa and Takeda, 1993) and a mannose- fucose-resistant haemagglutinin (Barker et al., 1994) and alipase (Rowe-Magnus et al., 2001). Potential functions for further genes have been proposed on the basis of relationships to known proteins but the majority of ORF remain unidentified (Rowe-Magnus et al., 1999; Clark et al., 2000). Among the gene cassettes that contain known antibiotic resistance genes, only one includes two open reading frames, the aacA1–orfG cassette (Accession. No. AF047479). However there are several cassettes that include two ORF found both in class 1 integrons (Accession No. AF047479) and in the Vibrio cholerae collection (Clark et al., 2000; Heidelberg et al., 2000). One of these determines the mannose-fucose- resistant haemagglutinin, but whether both ORF are needed for this activity is not known (Barker et al., 1994). A cassette from a Xantho- monas campestris isolate includes both the XbaI restriction and modification genes (Accession No. AF051092). There are also some cassettes where the orientation of the gene (or genes) is reversed. Although these exceptions remain to be adequately accounted for, a possible explana- tion for cassettes containing genes in the oppo- site to normal orientation is that the genes are not essential to V. cholerae and the sequence has drifted such that the original ORF is no longer detectable. A very large number of 59-be sites have been found (Stokes et al., 1997). Indeed, each of the cassettes found in class 1 integrons contains a 22 R.M. HALL 40
  • 34. unique 59-be. Initially, 59-be were identified as a consensus sequence of 59 bp found in a few cas- settes (Cameron et al., 1986) and this alignment led to the subsequent identification, in other gene cassettes, of related but more diverged se- quence elements, some of which had different lengths (Hall et al., 1991). It is now known that 59-be can range in size from 57 to 141 bp (Recchia et al., 1995a). However, the term 59-be has found common currency despite the size variation and has been retained for all members of this family. The VCR found in V. cholerae cas- settes form a more homogeneous group (Barker et al., 1994) but are also members of the 59-be family (Recchia and Hall, 1997). Indeed 59-be that are closely related to the VCR sequence are found among the 59-be associated with antibi- otic resistance genes. INTEGRONS As described above, integrons include two dis- tinctive features, an intI gene and an adjacent attI site, that enable them to capture gene cas- settes. They do not necessarily include a gene cassette and class 1 integrons that contain no cassettes have been found in the wild and cre- ated experimentally (Bissonnette and Roy, 1992; Collis and Hall, 1992a; Rosser and Young, 1999). However, integrons generally do contain one cassette or an array of two or more gene cas- settes. The cassette array can be very long as is the case for the V. cholerae chromosomal integrons, where the sequenced strain has more than 170 cassettes (Heidelberg et al., 2000) and other strains are estimated to include at least 100 cassettes (Clark et al., 2000). As the vast majority of the known gene cas- settes contain a gene but not a promoter (Hall et al., 1991; Recchia and Hall, 1995a), an upstream promoter is required for the expression of the genes contained in cassettes. This promoter, Pc, is supplied by the integron and is the third dis- tinctive feature of an integron (Figure 2.1). In the case of the class 1 integrons, which are the commonest type of integrons found in antibi- otic-resistant clinical isolates, the Pc promoter is located just inside the beginning of the intI1 gene (Hall and Vockler, 1987; Stokes and Hall, 1989). All transcripts of the array of gene cas- settes start at Pc (Collis and Hall, 1995). The fact that the integron supplies the promoter imposes an orientation constraint on gene cassettes if their genes are to be expressed. In all cases where the gene function is known and expres- sion can be monitored, the orientation of the cassette-associated gene that is found is the one that permits expression. This orientation is achieved only if the 59-be is located down- stream of the gene. MANY CLASSES OF INTEGRON That there were, in addition to the class 1 integrons, other classes of integrons containing the same gene cassettes, but a different con- served backbone and thus a potentially a dis- tinct IntI gene, was known from the earliest studies (Cameron et al., 1986; Hall and Vockler, 1987; Wiedemann et al., 1987). In fact, the first complete gene cassettes to be sequenced were the dfrA1 and aadA1 cassettes that are respon- sible, respectively, for the resistance to tri- methoprim and to spectinomycin and strepto- mycin conferred by the transposon Tn7. How- ever, these two cassettes were identified as cas- settes only after they were also subsequently found in class 1 integrons (Cameron et al., 1986; Sundström and Sköld, 1990; Hall et al., 1991). Indeed, the relationship of the predicted prod- ucts of the intI1 gene and the partially se- quenced intI2 gene in Tn7 was identified before the nature of the gene products was known (Hall and Vockler, 1987). However, the Tn7 intI2 gene (intI2* ) is defective, because it includes an in-frame stop codon that precludes production of a functional protein and this may explain why so few examples of class 2 integrons with different arrays of gene cassettes have been re- ported thus far (Recchia and Hall, 1995a). As the level of identity between IntI1 and IntI2* was only 45%, it was obvious that these two classes of integrons (Class 1 and 2) were likely to be rep- resentative of a vast family of integrons, each encoding a related, but distinct, IntI, but re- taining the potential to share the same gene cas- settes. Subsequently, a third class of integron was found in clinical isolates of antibiotic GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 23 41
  • 35. resistant Serratia marcescens in Japan (Arakawa et al., 1995) and again the cassettes are ones that had been found in class 1 integrons. INTEGRONS IN BACTERIAL CHROMOSOMES Although much is known about the impact of gene cassettes on the emergence of multiply antibiotic-resistant strains of Gram-negative bac- teria, their provenance is not restricted to antibi- otic resistance genes. The small chromosome of Vibrio cholerae has recently been shown to include an intI gene, intI4, adjacent to a long array of gene cassettes (Mazel et al., 1998; Clark et al., 1997, 2000; Heidelberg et al., 2000), and thus contains an integron. Different strains contain different cassette arrays (Clark et al., 2000). The gene cas- settes were found first (Ogawa and Takeda, 1993; Barker et al., 1994) but, as in the case of Tn7, they were not initially recognized as such. A repetitive sequence element called a VCR was identified but its similarity to 59-be was recognized later (Recchia and Hall, 1997). However, VCR were shown to be present in the chromosomes of sev- eral further Vibrio species such as V. mimicus, V. anguillarum, V. hollisae and V. metschnikvii but not in others (Ogawa and Takeda, 1993; Barker et al., 1994; Mazel et al., 1998; Clark et al., 2000). The result of these studies are not always consistent, however, they do indicate that an integron and gene cassettes are also likely to be present in many, but not necessarily all, Vibrio species. The partially sequenced intI5 gene from V. mimicus has diverged from intI4 to the same degree as other known chromosomal genes, providing evidence that an integron was a feature of the genome of the common ancestor (Clark et al., 2000). This conclusion has recently been con- firmed for other Vibrio species (Rowe-Magnus et al., 2001). MORE INTEGRONS, MORE CASSETTES It is likely that Vibrio species represent only the first case where an integron is found in the bacterial genome and, therefore, that further ex- amples of gene cassettes and new types of integrons will come to light as more bacterial genomes are sequenced. Further classes of integron may also be found on plasmids recov- ered from different environments. In fact, some genes whose products are clearly related to the IntI integrases can be found amongst the se- quences available in the partially sequenced genomes of Shewanella putrefaciens, Treponema denticola, Geobacter sulfurreducens (Rowe-Magnus et al., 1999, 2001; Nield et al., 2001). However, whether these genes indicate the presence of an integron must await the identification of gene cassettes to go with them. Recently, three new intI genes (intI6, 7 and 8) have been recovered from environmental soil samples and in two cases a potential adjacent gene cassette has been recovered, together with the intI gene (Nield et al., 2001). This confirms that integrons and gene cassettes are likely to be common in the bacterial world. SOME INTEGRONS CAN MOVE In the gene cassette/integron system, it is the cassettes that are the mobile elements. How- ever, in the context of their contribution to hori- zontal gene transfer, it is obvious that integrons and gene cassettes can have a substantial impact, as is the case with respect to antibiotic resistance, only if the integron can gain wide access to a variety of bacterial species. This occurs readily when it is located on a plasmid and this is best achieved if the integron can also translocate. Indeed, class 1 integrons are found in many different genetic contexts, mainly on different plasmids (Hall and Vockler, 1987; Stokes and Hall, 1989; Hall et al., 1994). Class 1 integrons are in fact either transposable ele- ments as exemplified by Tn402 (Rådström et al., 1994) or, more often, defective derivatives of them (Brown et al., 1996; Rådström et al., 1994; Liebert et al., 1999; Partridge et al., 2001a,b). The latter can obviously be moved so long as both outer ends are intact and a set of suitable trans- position genes are present in the same cell. Often they are found flanked by a 5 bp duplica- tion of the target site, indicating that they have 24 R.M. HALL 42
  • 36. reached their current location by transposition (Brown et al., 1996; Partridge et al., 2001a,b). In a few cases, class 1 integrons that are unable to mobilize themselves have moved into, and are now found within, another transposon. This is the case for In2 which is found within Tn21 (Liebert et al., 1999), for In4, which is found in Tn1696 (Partridge et al., 2001a), and for In28, which is found in Tn1403 (Partridge et al., 2001b). The exemplar of a class 2 integron is the transposon Tn7 which contains three gene cas- settes dfrA1–sat2–aadA1 and three other known members of this group are transposons that differ in the identity of the first cassette in the array or have lost this cassette (Recchia and Hall, 1995a). The class 3 integron provides a contem- porary example of the rapid spread of resistance genes carried by self-transmissible plasmids. It was first isolated in Japan, in 1993, and has al- ready spread to several other bacterial species (Senda et al., 1996). Our preliminary evidence and that of others (Shibata et al., 1999) indicates that the class 3 integron is, not surprisingly, also a mobile element. In all of these cases, the integron is defined as the complete structure bounded by the terminal inverted repeats or that part of such a structure that remains. THE RECOMBINATION SYSTEM The components of the recombination system that effect cassette movement are the IntI-type integrases, the integron-associated attI sites and the cassette-associated 59-be sites. The known IntI integrases form a family of related proteins that share highly significant levels of identity (35–94%). They also share cer- tain features with other members of the integrase or tyrosine recombinase super-family, but pairwise identities between these other integrases and IntI integrases is generally less than 25%. The most obvious of the shared fea- tures are the two conserved domains or boxes that are normally used to identify members of this super-family (Ouellette and Roy, 1987; Sundström et al., 1988; Stokes and Hall, 1989). However a recent alignment of the C-terminal catalytic domain of all known members of the tyrosine recombinase superfamily has revealed further shared “patches” (Nunes-Düby et al., 1998). Other members of the IntI family are known (Nield et al., 2001; Rowe-Magnus et al., 2001), but whether all of them are associated with gene cassettes and are thus part of an integron remains to be established. Only the re- actions catalyzed by the IntI1 integrase have been studied in detail and these are described briefly below. However, IntI3 and IntlI4 have also been shown to be active (Hall et al., 1999; Rowe-Magnus et al., 2001; Collis and Hall, unpublished). In addition to recombination between a 59-be and attI1 site, which occurs when a cassette is in- corporated into an integron, IntI1-catalyzed re- combination between two 59-be or two attI1 sites can also occur (Martinez and de la Cruz, 1988, 1990; Hall et al., 1991; Recchia et al., 1994; Hansson et al., 1997; Stokes et al., 1997; Hall et al., 1999; Partridge et al., 2000; Collis et al., 2001). The reactions that have been reported are listed in Table 2.1. The efficiencies of the integration reac- tions have also been compared (Collis et al., 2001). Recombination between two 59-be sites occurs at high frequency but is less efficient than recombi- nation between a 59-be and the attI1 site. How- ever, excisive recombination involving two 59-be is important because it can lead to excision of the downstream cassettes in an array. Recombina- tion between two attI sites occurs at a much lower frequency than the other reactions (Hansson et al., 1997; Partridge et al., 2000) and is unlikely to be an important event in cassette movement. Re- combination between a 59-be and a secondary site (20 rs) also occurs at low frequency (Francia et al., 1993; Recchia et al., 1994) and is an important GENE CASSETTES AND INTEGRONS: MOVING SINGLE GENES 25 TABLE 2.1 Recombination events catalyzed by IntI1 Participating sites Integration Excision attI1 × 59-be + + 59-be × 59-be + + attI1 × attI1 + NDa 59-be × 20 rs + –b attI1 × 20 rs + –b a Not determined. b Precise excision of a cassette located at a 20 rs is in most cases unlikely to be possible. 43
  • 37. reaction that permits gene cassettes to be inte- grated at almost any position. The role of recom- bination between attI sites and 20 rs (Hansson et al., 1997; Collis et al., 2001) is less obvious. The attI sites are part of the integron back- bone and are distinguished by the fact that they are the sites into which cassettes are incorpo- rated. In known integrons, they are located up- stream of the intI gene. However the sequences of the regions adjacent to the first gene cassette in each of the four well-established integron classes are not highly conserved. The extent of the attI1 site has been established experimen- tally (Recchia et al., 1994; Hansson et al., 1997; Partridge et al., 2000). The complete attI1 site (Figure 2.3), which is required for recombina- tion with a 59-be partner, includes 56 bp from the left side of the crossover point and at least a further 9 bp to the right of the crossover (Hall et al., 1999; Partridge et al., 2000). Within this region, four binding sites for IntI1 have been found and delineated using foot-printing tech- niques (Collis et al., 1998; Gravel et al., 1998). A single molecule of IntI1 bound to the strongest binding domain protects a total of 14 bp which includes the 7 bp core site regions (Collis et al., 1998). Two of the IntI1 binding domains (1 and 2 in Figure 2.3A) are inversely oriented with re- spect to one another and form a simple site equivalent to those recognized by other tyrosine recombinases. The additional IntI1-binding do- mains (3 and 4 in Figure 2.3A) considerably en- hance the activity of attI1 in recombination with a 59-be (Recchia et al., 1994; Hall et al., 1999; Partridge et al., 2000) but are not required for recombination with a complete attI1 partner (Hansson et al., 1997; Partridge et al., 2000). They appear to bind IntI1 more strongly than sites 1 and 2 (Collis et al., 1998), and may play a role in retaining the newly synthesized mole- cules of IntI1 in the proximity of attI1. Simple sites can also be found in the expected positions in attI2, 3 and 4 (Collis et al., 1998), but whether these sites resemble attI1 in binding four molecules of their cognate integrase (IntI2, 3 or 4) remains to be established experimentally. Preliminary data suggest that each IntI prefer- entially recognizes its cognate attI site (Hall et al., 1999), but this also remains to be established rigorously. The 59-be have a different architecture from that of attI sites. All 59-be comprise two regions of 25 bp that are each related to a consensus se- quence (Collis and Hall, 1992a; Stokes et al., 1997). In any individual 59-be the consensus re- gions are imperfect inverted repeats of one an- other and are separated by a region of highly variable sequence and length that is, in most in- stances, also an inverted repeat. 59-be are rather unusual in that they include two simple sites, only one of which is the site of strand exchange (Stokes et al., 1997). The LH and RH consensus regions correspond to the bulk of these simple sites but, based on foot-printing data from attI1 (Collis et al., 1998), the IntI1-binding regions are likely to extend further (Figure 2.3) and a weak consensus is found for some of the bases in the extensions (Collis and Hall, 1992b; Stokes et al., 1997). A striking feature of this family of recom- bination sites is that the relationship between 26 R.M. HALL FIGURE 2.3 Recombination sites. The arrangement of 7 bp core sites (arrows) that form part of the larger IntI binding domains is shown. For simplicity the individual core sites are numbered (above the arrows). The vertical arrows show the position of the crossover. 44
  • 38. the sequences of the LH and RH consensus re- gions is generally retained in preference to ad- herence to the consensus sequence (Hall et al., 1991; Stokes et al., 1997). This feature is yet to be adequately explained in terms of the activity of these sites. The inverted repeatedness is imper- fect in the simple site regions; the 1L and 1R core sites and the 2L and 2R core sites mirror one an- other but the bases that separate them do not and there is an extra base in 2L (Figure 2.2). The distance between 1L and 2L is 5 bp, but either 5 or 6 bp separate 2R from 1R. It remains to be established which of the differences between the LH and RH simple sites are important in ensuring that the recombination crossover occurs at 1R. CONCLUSIONS The role of gene cassettes and integrons in the emergence and spread of antibiotic resistance is well established. The same system has now been implicated in the evolution of bacterial genomes, and the extent of this involvement al- ready appears to be quite significant. Many more new classes of integrons and new gene cassettes will undoubtedly be found in the not too distant future. How gene cassettes are cre- ated and how the different integrases recognize the same cassettes as well as other intricacies of the site-specific recombination system are im- portant, but complex, questions that remain to be examined. REFERENCES Arakawa, Y., Murakami, M., Suzuki, K. et al. (1995) A novel integron-like element carrying the metallo β-lactamase gene blaIMP. Antimicrob. Agents Chemother. 39: 1612–1615. Barker, A., Clark, C.A. and Manning, P.A. (1994) Identification of VCR, a repeated sequence associated with a locus encoding a haemagglutinin in Vibrio cholerae O1. J. Bacteriol. 176: 5450–5458. Bissonnette, L. and Roy, P.H. (1992) Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. J. Bacteriol. 174: 1248–1257. Brown, H.J., Stokes, H.W. and Hall, R.M. (1996) The integrons In0, In2 and In5 are defective transposon derivatives. J. Bacteriol. 178: 4429–4437. Cameron, F.H., Groot Obbink, D.J., Ackerman, V.P. et al. (1986) Nucleotide sequence of the AAD(2¢¢) amino- glycoside adenylyltransferase determinant aadB. Evo- lutionary relationship of this region with those surrounding aadA in R538–1 and dhfrII in R388. Nucleic Acids Res. 14: 8625–8635. Clark, C.A., Purins, L., Kaewrakon, P. and Manning, P. (1997) VCR repetitive sequence elements in the Vibrio cholerae chromosome constitute a mega-integron. Mol. Microbiol. 26: 1137–1138. Clark, C.A., Purins, L., Kaewrakon, P. et al. (2000) The Vibrio cholerae O1 chromosomal integron. Microbiology 146: 2605–2612. Collis, C.M. and Hall, R.M. (1992a) Site-specific deletion and rearrangement of integron insert genes catalysed by the integron DNA integrase. J. Bacteriol. 174, 1574–1585. Collis, C.M. and Hall, R.M. (1992b) Gene cassettes from the insert region of integrons are excised as covalently closed circles. Mol. Microbiol. 6: 2875–2885. Collis, C.M. and Hall, R.M. (1995) Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob. Agents Chemother. 39: 155–162. Collis, C.M., Grammaticopoulos, G., Briton, J., et al. (1993) Site-specific insertion of gene cassettes into integrons. Mol. Microbiol. 9: 41–52. Collis, C.M., Kim, M.-J., Stokes, H.W. et al. (1998) Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites. Mol. Microbiol. 29: 477–490. Collis, C.M., Recchia, G.D., Kim, M.-J. et al. (2001) Efficiency of recombination reactions catalysed by the class 1 integron integrase IntI1. J. Bacteriol. 183: 2535–2542. Francia, M.V., de la Cruz, F. and García Lobo, M. (1993) Secondary sites for integration mediated by the Tn21 integrase. Mol. Microbiol. 10: 823–828. Gravel, A., Fournier, B. and Roy, P.H. (1998) DNA complexes obtained with the integron integrase IntI1 at the attI1 site. Nucleic Acids Res. 26: 4347–4355. Hall, R.M. and Collis, C.M. (1995) Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol. Microbiol. 15: 593–600. Hall, R.M. and Collis, C.M. (1998) Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist. Updates 1:109–119. Hall, R.M. and Vockler, C. (1987) The region of the IncN plasmid R46 coding for resistance to β-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. Nucleic Acids Res 15: 7491–7501. Hall, R.M., Brookes, D.E. and Stokes, H.W. 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  • 39. Hansson, K., Sköld, O. and Sundström, L. (1997) Non- palindromic attI sites of integrons are capable of site- specific recombination with one another and with secondary targets. Mol. Microbiol. 26: 441–453. Heidelberg, J.F., Elsen, J.A., Nelson, W.C. et al. (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406: 477–483. Liebert, C.A., Hall, R.M and Summers, A.O. (1999) Transposon Tn21, flagship of the floating genome. Microb. Mol. Biol. Rev. 63: 507–522. Martinez, E., and de la Cruz, F. (1988) Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol. Gen. Genet. 211: 320–325. Martinez, E. and de la Cruz, F. (1990) Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J. 9: 1275–1281. Mazel, D. and Davies, J. (1999) Antibiotic resistance in microbes. Cell. Mol. Life Sci. 56: 742–754. Mazel, D., Dychinco, B., Webb, V.A. and Davies, J. (1998) A distinctive class of integron in the Vibrio cholerae genome. Science 280: 605–608. Nield, S.B., Holmes, A.J., Gillings, M.R. et al. (2001) Recovery of new integron classes from environmental DNA. FEMS Microbiol. Lett. Submitted. Nunes-Düby, S.E., Kwon, H.J., Tirumalai, R. et al. (1998) Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res. 26: 391–406. Ogawa, A. and Takeda, T. (1993) The gene encoding the heat-stable enterotoxin of Vibrio cholerae is flanked by 123 base pair direct repeats. Microbiol. Immunol. 37: 607–616. Ouellette, M. and Roy, P.H. (1987) Homology of ORFs from Tn2603 and from R46 to site-specific recombinases. Nucleic Acids Res. 15: 10055. Ouellette, M., Bissonnette, L. and Roy, P.H. (1987) Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 β-lactamase gene. Proc. Natl Acad. Sci. USA 84: 7378–7382. Partridge, S.R., Recchia, G.D., Scaramuzzi, C. et al. (2000) Definition of the attI1 site of class 1 integrons. Microbiology 146: 2855–2864. Partridge, S.R., Brown, H.J., Stokes, H.W. and Hall, R.M. (2001a) Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrob. Agents Chemother. 45: 1263–1270. Partridge, S.R., Recchia, G.D., Stokes, H.W. and Hall, R.M. (2001b) A family of class 1 integrons related to In4 from Tn1696. Antimicrob. Agents Chemother. In press. Rådström, P., Sköld, O., Swedberg, G. et al. (1994) Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J. Bacteriol. 176: 3257–3268. Recchia, G.D., Stokes, H.W. and Hall, R.M. (1994) Characterisation of specific and secondary recombination sites recognised by the integron DNA integrase. Nucleic Acids Res. 22: 2071–2078. Recchia, G.D. and Hall, R.M. (1995a) Mobile gene cassettes: a new class of mobile element. Microbiol. 141: 3015–3027. Recchia, G.D. and Hall, R.M. (1995b) Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010. Mol. Microbiol. 15: 179–187. Recchia, G.D. and Hall, R. M. (1997) Origins of the mobile gene cassettes found in integrons. Trends Microbiol. 389: 389–394. Rowe-Magnus, D.A., Guérout, A.-M. and Mazel, D. (1999) Super-integrons. Res. Microbiol. 150: 641–651. Rowe-Magnus, D.A., Guérout, A.-M., Ploncard, P., Dychinoco, B., Davies, J. and Mazel, D. (2001). The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc. Natl Acad. Sci. USA 98: 652–657. Rosser, S.J. and Young, H.-K. (1999) Identification and characterization of class 1 integrons in bacteria from an aquatic environment. J. Antimicrob. Chemother. 44: 11–18 Senda, K., Arakawa, Y., Ichiyama, S. et al. (1996) PCR detection of metallo-β-lactamase (blaIMP) in gram- negative rods resistant to broad-spectrum β-lactams. J. Clinical Microbiol. 34: 2909–2913. Shibata, N., Kurokawa, H., Yagi, T. and Arakawa, Y. (1999) A class 3 integron carrying the IMP-1 metallo-β-lactamase gene found in Japan. 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco. Stokes, H.W. and Hall, R.M. (1989) A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol. Microbiol. 3: 1669–1683. Stokes, H.W. and Hall, R.M. (1991) Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation. Plasmid 26: 10–19. Stokes, H.W., O’Gorman, D.B., Recchia, G.D. et al. (1997) Structure and function of 59-base element re- combination sites associated with mobile gene cassettes. Mol. Microbiol. 26: 731–745. Sundström, L. and Sköld, O. (1990) The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings. Antimicrob. Agents Chemother. 34: 642–650. Sundström, L., Rådström, P., Swedberg, G. and Sköld, O. (1988) Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol. Gen. Genet. 213: 191–201 Wiedemann, B., Meyer, J.F. and Zühlsdorf, M.T. (1987) Insertions of resistance genes into Tn21-like transposons. J. Antimicrob. Chemother. 18: 85–92. 28 R.M. HALL 46
  • 40. C H A P T E R 3 A Corynebacterium Plasmid Composed of Elements from Throughout the Eubacteria Kingdom Andreas Tauch and Alfred Pühler Multiple antimicrobial resistance in human pathogens is a global medical problem. Espe- cially, Gram-positive microorganisms show alarming increases in antibiotic resistances. The complete DNA sequence of the 51-kb multiresistance plasmid pTP10 from the Gram- positive human pathogen Corynebacterium striatum M82B provides genetic information about acquired resistance mechanisms to antimicrobial agents in this species. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of a mosaic structure comprising eight DNA segments the bound- aries of which are represented by horizontal mobile elements. The DNA segments of pTP10 turned out to be virtually identical to a plasmid-encoded macrolide and lincosamide resistance region from the human pathogen Corynebacterium diphtheriae, a chromosomal DNA region from Mycobacterium tuberculosis, a mobile chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum, several transposable elements from Gram- negative phytopathogenic Pseudomonas, Xantho- monas and Erwinia species, and to a trans- posable aminoglycoside resistance region from the Gram-negative animal pathogen Pasteurella piscicida. This provides molecular evidence that natural routes exist by which antibiotic resis- tance genes from bacteria of different habitats and geographical origin can be assembled in a human pathogen. This shows that highly di- verged species that last shared a common ancestor about 2 billion years ago can still ex- change genes. Consequently, horizontal gene transfer of antibiotic resistance genes is an im- portant mechanism which limits the successful use of antimicrobials in the clinical treatment of human infections. INTRODUCTION Drug resistance in human pathogens is the result of overuse of antimicrobials in medicine and agriculture (Witte, 1998). This overuse of antibiotics has led to the rapid evolution of bac- teria that are resistant to multiple drugs such that even vancomycin and teicoplanin, the drugs of last resort, are no longer effective against some bacterial isolates. Generally, a micro- organism can have either intrinsic or acquired resistance to antibiotics (Tan et al., 2000). In- trinsic resistance is a stable genetic property, arising from mutation in the chromosomal DNA. As each mutation confers only a slight al- teration in susceptibility, microorganisms need to accumulate several mutations to become in- trinsically resistant to antibiotics. Alternatively, resistance is acquired by genetic exchange with another microorganism from the same or Horizontal Gene Transfer Copyright © 2002 by Academic Press. ISBN: 0-12-680126-6 29 All rights of reproduction in any form reserved. 47
  • 41. another genus (Tan et al., 2000). Resistance genes can be transferred among bacteria and can be integrated into the bacterial chromosome to be stably inherited from one generation to the next. Additionally, antibiotic resistance genes can be maintained in an extra-chromosomal state on a bacterial plasmid. Plasmids that can transfer DNA to adjacent bacteria are known as conjugative plasmids. As conjugation can occur in a broad range of species it is one of the main mechanisms through which resistance genes are transferred between bacteria (Dröge et al., 1998, 1999). Therefore, such plasmids can be classed among the horizontal mobile elements that also include phages, integrons, trans- posons, and insertion sequences. Accordingly, the transfer of horizontal mobile elements occurs via conjugation, transduction, transposi- tion, and transformation. Today, integrated genome research offers the chance to analyze large resistance plasmids (R plasmids) and acquired antibiotic resistances at the nucleotide level. Highly automated se- quencing machines and processes have been developed to allow large-scale DNA sequencing and subsequent DNA sequence interpretation by bioinformatics. The information obtained within the scope of such projects not only sheds 30 A. TAUCH AND A. PÜHLER FIGURE 3.1 Genetic organization of the 51409-bp multiresistance plasmid pTP10 identified in C. striatum M82B. The organization of the open reading frames (ORFs) and the position of transposons and insertion sequences is presented. The eight DNA segments of pTP10 are specifically marked (I–VIII). Dotted lines represent segment boundaries corresponding to the insertion of mobile elements. The acquired antibiotic resistance genes are marked by filled boxed. Details on the DNA sequence of pTP10 are available from GenBank accession number AF139896. 48
  • 42. new light on acquired antibiotic resistance mechanisms, but also on horizontal gene transfer and plasmid evolution (Perretten et al., 1997; Tauch et al., 2000). The genetic data described below focus on a plasmid project which dealt with the DNA sequence analysis of the large multiresistance plasmid pTP10 from the human pathogen Corynebacterium striatum M82B (Tauch et al., 2000). The complete DNA sequence revealed insights into how pTP10 is genetically orga- nized and, in particular, how a multiresistance plasmid from a human clinical source has evolved over time. Virtually identical DNA seg- ments have been identified in a soil bacterium and in plant, animal and human pathogens. This finding implies that horizontal gene transfer has played a central role in the evolu- tionary history of pTP10. ANALYSIS OF AN ANTIBIOTIC RESISTANCE PLASMID ISOLATED FROM CORYNEBACTERIUM STRIATUM BY INTEGRATED GENOME RESEARCH In recent years, the Gram-positive bacterium C. striatum has been recognized with increasing frequency as an important opportunistic human pathogen, especially in immuno- compromised patients and in patients under in- tensive care. In clinical diagnostics, numerous isolates of C. striatum were found to be highly re- sistant to the majority of clinically relevant anti- biotics which more and more resulted in the failure of antibiotic treatment of C. striatum- mediated human infections. C. striatum M82B was initially isolated from the bacterial flora of an otitis media patient in a Japanese hospital. It was shown to carry the R plasmid pTP10 encoding resistances to the antibiotics chlor- amphenicol, erythromycin, kanamycin, and tetracycline (Tauch et al., 1995a). Since the plasmid genome of pTP10 was determined to be only 51 kb in size, it represents an ideal model system for the analysis of acquired antibiotic resistance by integrated genome research. The total DNA sequence of the R plasmid pTP10 was determined and subsequently annotated by means of an automated genome interpretation system. In such a way the complete gene struc- ture of pTP10 was identified. The pTP10 se- quence contains 47 open reading frames (ORFs) and an additional five incomplete coding regions. Moreover, pTP10 harbors five trans- posons (Tauch et al., 1995b, 1998) and two addi- tional insertion sequences. Figure 3.1 presents a detailed map of the ORFs found on pTP10 as well as other relevant structural features. Based on the DNA sequence data, further ex- periments concentrated on the antibiotic resis- tance genes of pTP10, some of which are integral parts of horizontal mobile elements (Figure 3.1). Besides the known resistance determinants of pTP10 to chloramphenicol (cml(A), cmx(A)), erythromycin (ermLP, ermCX), kanamycin (aphA1-IAB), and tetracycline (tetAB), DNA se- quence analysis revealed the presence of a dupli- cated chloramphenicol resistance region (cml(B) and cmx(B)) and genes probably involved in bacitracin (bacA) and streptomycin resistance (strAB). However, antibiotic susceptibility studies clearly demonstrated that the bacitracin and streptomycin resistance determinants are inac- tive on pTP10 (Tauch et al., 2000). In addition, DNA sequence interpretation made it possible to deduce the respective resistance mechanisms en- coded on pTP10 and to identify virtually identical resistance genes in Gram-positive and Gram- negative bacteria of different habitats. Most inter- estingly, for tetracycline a new resistance mecha- nism could be proposed that is based on hetero- dimerization of two ABC transporters resulting in an oxytetracycline and oxacillin cross-resistance (Tauch et al., 1999). The deduced data concerning the resistance determinants of pTP10 are summa- rized in Table 3.1. HORIZONTAL GENE TRANSFER AND ACQUIRED ANTIBIOTIC RESISTANCE IN CORYNEBACTERIUM STRIATUM M82B Taking into account the DNA sequence annota- tion and the structural data, the pTP10 plasmid A PLASMID COMPOSED OF ELEMENTS FROM THROUGHOUT THE EUBACTERIA KINGDOM 31 49
  • 43. was subdivided into eight genetically distinct DNA segments, six of which are involved in an- tibiotic resistance. One of the DNA segments present on pTP10 comprises the composite re- sistance transposon Tn5432 that consists of two IS1249 sequences flanking the erythromycin re- sistance gene region (Figure 3.1; I). The central part of Tn5432 was found to be virtually iden- tical at the nucleotide level to the antibiotic resis- tance gene region of plasmid pNG2 from the human pathogen Corynebacterium diphtheriae (Figure 3.2A) encoding an inducible resistance to macrolide and lincosamide antibiotics. In contrast to the genetic arrangement on pTP10, the sequenced resistance region of pNG2 is not part of a Tn5432-like mobile element (Figure 3.2A). The second DNA segment of pTP10 which is involved in antibiotic resistance is located downstream of Tn5432 and comprises a DNA region with a high G + C content (Figure 3.1; II). The gene products of the respective coding re- gions showed the highest similarity to proteins encoded by the Mycobacterium tuberculosis 32 A. TAUCH AND A. PÜHLER TABLE 3.1 Acquired antibiotic resistance gene regions of pTP10 from C. striatum M82B and its closest relatives pTP10 gene region Resistance genes Resistance mechanism Closest relative/ microorganism Erythromycin resistance ermLP, ermCX 23S rRNA methylation Corynebacterium diphtheriae (Gram-positive human pathogen) Tetracycline resistance tetA, tetB Efflux via heterodimerization of ABC transporters No similarity found Bacitracin resistance bacA Phosphorylation of undecaprenol (inactive in pTP10) Mycobacterium tuberculosis (Gram-positive human pathogen) Chloramphenicol resistance copy A and copy B cml(A), cmx(A) cml(B), cmx(B) Antibiotic export Corynebacterium glutamicum (Gram-positive soil bacterium) Streptomycin resistance strA, strB Phosphorylation (inactive in pTP10) Erwinia amylovora, Pseudomonas syringae, Xanthomonas campestris (Gram-negative plant pathogens) Kanamycin resistance aphA1-IAB Phosphorylation Pasteurella piscicida, Klebsiella pneumoniae (Gram-negative animal and human pathogens) FIGURE 3.2 (Opposite.) Comparison of pTP10 DNA segments carrying acquired antibiotic resistance gene regions with virtually identical DNA regions from Gram-positive and Gram-negative bacteria of different habitats. (A) Comparison of the macrolide and lincosamide resistance region of pTP10 (segment I) with a DNA fragment of plasmid pNG2 from the human pathogen C. diphtheriae. (B) Comparison of the chloramphenicol resistance transposon Tn5564 (segment III) with transposon Tn45 from the soil isolate C. glutamicum 1014. Both elements are virtually identical with the exception of IS1513 which inserted between cml(A) and the inverted repeat (IR) of Tn5564. (C) Comparison of the Tn3-type transposon Tn5716 present on pTP10 (segment V) with the streptomycin resistance transposon Tn5393 from plasmid pEa34 of the plant pathogen E. amylovora. (D) Comparison between the aphA1-IAB aminoglycoside resistance regions from C. striatum M82B (segment VI) and the Gram-negative fish pathogen P. piscicida. The aphA1-IAB gene is part of IS26-based composite transposons which differ in the orientation of the insertion sequences (arrows) and in the length of the spacer sequences. 50
  • 44. A PLASMID COMPOSED OF ELEMENTS FROM THROUGHOUT THE EUBACTERIA KINGDOM 33 51
  • 45. chromosome. Furthermore, it is noteworthy that the genetic organization of these genes on pTP10 is almost identical to the gene arrange- ment found in the M. tuberculosis chromosome. The high G + C region of pTP10 also comprises the tetAB genes (Figure 3.1) the deduced pro- teins of which are similar to chromosomally en- coded ATP-binding cassette transporters. These data strongly suggest that the DNA segment de- rived from the chromosome of a microorganism belonging to the high G + C branch of Gram- positive bacteria. The third DNA segment of pTP10 comprises the chloramphenicol resistance region that is part of the mobile element Tn5564 (Figure 3.1; III). The basic molecular structure of this transposon, comprising the resistance gene cmx(A) and the transposase gene, is nearly identical at the nucle- otide level to the chloramphenicol resistance region of plasmid pXZ10145 from the Chinese soil isolate Corynebacterium glutamicum 1014. In contrast to the basic structure present in C. glutamicum, Tn5564 carries the additional inser- tion sequence IS1513, located between the puta- tive leader sequence of cmx(A) and the left inverted repeat (Figure 3.2B). Interestingly, DNA segment IV of pTP10 is represented by a second identical copy of the basic structure of Tn5564 (Figure 3.1; IVa and IVb). This copy is disrupted by the Tn3-type mobile element Tn5716 (Figure 3.1; Va and Vb). Transposon Tn5716 contains the genetic infor- mation for a transposase, a resolvase and the linked StrAB streptomycin resistance proteins and is identical at the nucleotide level to the basic structure of Tn5393 from the Erwinia amylovora plasmid pEa34 (Figure 3.2C). Tn5393- like transposons are structurally very similar and can be distinguished by additional inser- tions of mobile elements. Although the strAB tandem pair of streptomycin resistance genes is widespread among Gram-negative bacteria, it has not been identified in a Gram-positive bac- terium to date. Moreover, the association of the strAB genes with a transposable element was exclusively found in the Gram-negative phytopathogenic bacteria E. amylovora, Pseudo- monas syringae, and Xanthomonas campestris iso- lated from American agricultural habitats where streptomycin was utilized as bactericide (Sundin and Bender, 1996). Therefore, it is most likely that the Tn5393-like DNA segment of pTP10 derived from a Gram-negative host or- ganism and was transferred to pTP10 by hori- zontal gene transfer. Interestingly, the Tn5393 variants occur on large conjugative plasmids in the three phytopathogenic genera (Sundin and Bender, 1996). The aminoglycoside resistance region of pTP10 (Figure 3.1; VI) is part of the composite transposon Tn5715 consisting of two IS26 inser- tion sequences and a resistance gene encoding an aminoglycoside-3¢,5¢¢-phosphotransferase. Both IS26 elements of Tn5715 were found to be identical to previously sequenced IS26 ele- ments from the Gram-negative species Salmo- nella ordonez, Klebsiella pneumoniae, and Pasteurella piscicida. The aminoglycoside resis- tance protein encoded by Tn5715 is identical to the AphA1-IAB protein identified in a clinical isolate of K. pneumoniae from Chile (Lee et al., 1991) and in the animal pathogen P. piscicida SP9351 from a Japanese marine fish farm (Kim and Aoki, 1994). The protein carries four amino acid substitutions when compared with the widely distributed Aph(3¢)-Ia protein from Tn903 and was associated with a significantly higher turnover of the aminoglycosides kana- mycin and neomycin in K. pneumoniae (Lee et al., 1991). The 5¢-spacer of Tn5715, located between the IS26L element and the trans- lational start of the aphA1-IAB resistance gene, and the 3¢-spacer of Tn5715 are characteristic for the aphA1-IAB gene region of P. piscicida (Figure 3.2D). This strongly indicates that both resistance regions derived from a common ancestor molecule. Due to the nucleotide sequence identity and the apparent low G + C content of the aphA1-IAB gene region (44.1%), it is obvious that DNA segment VI was trans- ferred from a Gram-negative bacterium to pTP10. Interestingly, the aphA1-IAB resistance genes from K. pneumoniae and P. piscicida are located on transferable R plasmids which might enable the rapid dissemination of this specific type of aminoglycoside resistance across spe- cies boundaries. Furthermore, the automated genome inter- pretation revealed that the pTP10 plasmid encodes two replication proteins with amino 34 A. TAUCH AND A. PÜHLER 52
  • 46. Exploring the Variety of Random Documents with Different Content
  • 47. being and he a god.” Kamalalawalu made answer: “Kauhiakama says Kohala is depopulated; the people are only at the beach.” To this remark of Kamalalawalu, Lanikaula replied: “You sent your son Kauhiakama to investigate as to how many people there were on Hawaii. He returned and made his report to you that there were not many people there, but Kauhiakama did not see the number of people in Kohala because he traveled on the seashore, reaching Kona from Kawaihae and arrived on the heights of Huehue. He could not have seen the people of that locality because there were only clinkers there, having proceeded along by way of Kona until he arrived at Kau. If he had traveled along the Kona route in the early morning he could not have met people at that time because the inhabitants of that section had gone to the uplands and some had gone fishing; those remaining home were only the feeble and sick, therefore the people of Kona could not have been seen by Kauhiakama on his tour. Had he gone during the he kanaka oe, a he akua kela.” I aku o Kamalalawalu: “Ka! Ua olelo mai o Kauhiakama, he leiwi wale no Kohala, eia i ka nuku na kanaka.” A no keia olelo ana aku o Kamalalawalu pela ia Lanikaula, olelo aku la o Lanikaula: “Hoouna aku nei oe i ko keiki (Kauhiakama) e hele e makaikai i ka nui o na kanaka o Hawaii, a hoi mai la, a hai mai la ia oe, aole he nui o na kanaka o Hawaii. Aka, ike ole aku la o Kauhiakama i ka nui o na kanaka o Kohala, no ka mea, ma kahakai ka hele ana; a hele aku la a hiki i Kona, hele aku la mai Kawaihae aku a hoea iluna o Huehue, aole no e ike i na kanaka olaila, no ka mea he a-a wale no; aka, hele aku la ma Kona loa a hiki i Kau, ina i ke kakahiaka nui ka hele ana ma Kona, aole e loaa kanaka ia wa, no ka mea, ua pau na kanaka o ia wahi iuka a o kekahi poe, ua pau i ka lawaia, a o ka poe koe iho he poe palupalu; a nolaila ka loaa ole o na kanaka o Kona ia Kauhiakama ma ia hele ana. Aka, ina ma ke ahiahi ka hele ana, ina ua ike i ka nui o na
  • 48. evening he would surely have seen the large population of Kona because it is the largest district of Hawaii.” kanaka o Kona, no ka mea, o ka okana nui hookahi ia o Hawaii.” These observations of Lanikaula did not make much of an impression on Kamalalawalu. He still inclined to the idea of war. Lanikaula observed that Kamalalawalu was bent on going to war. He therefore spoke to Kamalalawalu again: “If you [340]intend to go to war with Lonoikamakahiki, then your grounds should be at Anaehoomalu; and should Lonoikamakahiki come to meet you, then let the battle be fought at Pohakuloa, it being a narrow place; then you will be victorious over Hawaii.” Kamalalawalu answered: “You do not know, because I was distinctly told by both Kauhipaewa and Kihapaewa that our battle field should be on Hokuula and Puuoaoaka, it being a place of eminence.” Lanikaula again said: “You are being deceived by the sons of Kumaikeau and others; you have Ma keia olelo a Lanikaula, aole nae he hoomaopopo nui o Kamalalawalu ia olelo, aka hoomau no o Kamalalawalu i kona manao kaua. A ike mai la o Lanikaula, ua paakiki loa ko Kamalalawalu manao no ke kaua, olelo aku la o Lanikaula ia Kamalalawalu: [341]“Ina i manao oe e kii ia Lonoikamakahiki e kaua, aia kou kahua e noho ai o Anaehoomalu, ina e hiki mai ke kaua a Lonoikamakahiki i o oukou la, alaila, hoihoi aku ke kaua i Pohakuloa e hoouka ai i kahi haiki, alaila lanakila oukou maluna o ka Hawaii.” I aku la o Kamalalawalu: “Aole oe i ike, no ka mea, ua olelo maopopo loa ia mai au e Kauhipaewa laua o Kihapaewa, aia ko makou kahua kaua iluna o Hokuula a me Puuoaoaka; he wahi kau iluna.” I hou aku o Lanikaula: “Puni aku la oe i na keiki a Kumaikeau ma, nolu ia mai la oe; nolaila, e hoolohe oe i ka’u; a ina e hoolohe ole oe i ka’u olelo, aole
  • 49. been led astray, therefore listen to me, for if you heed not my admonitions I do not think that you will ever come home to Maui nei again.” wau e manao ana e hoi kino mai ana oe ia Maui nei.” Kamalalawalu became indignant at Lanikaula’s remarks and drove him away. But Lanikaula, out of sympathy for the king, did not cease to again give him warning: “Kamalalawalu! You are very persistent to have war. This is what I have to say to you: Better hold temple services these few days before you proceed. Propitiate the gods first, then go.” But Kamalalawalu would not harken to the words of Lanikaula, therefore he ended his remarks. Makakuikalani made the preparations of the war canoes in accordance with the strict orders of Kamalalawalu. When the canoes and the several generals, together with all the men, including the war canoes of Kamalalawalu, were ready floating in the harbor of Hamoa, Lanikaula came forth and in the presence of King Kamalalawalu and his war A no ka Lanikaula olelo ana ia Kamalalawalu pela, alaila wela ae la ko Kamalalawalu inaina no Lanikaula, a hookuke aku la. Aka, aole i hooki o Lanikaula, i kana olelo aku ia Kamalalawalu, no ka minamina no i ke alii; alaila olelo aku la no oia (Lanikaula): “E Kamalalawalu, ke paakiki loa nei oe i ke kaua; a eia ka’u ia oe. E pono ke kapu heiau i keia mau la, mamua o kou hele ana, e hoomalielie mua i ke akua, alaila hele.” Aka, o Kamalalawalu ma keia olelo ana a Lanikaula, aole no i maliu mai. Nolaila pau ae la ka Lanikaula olelo ana. Mahope iho o ka Lanikaula olelo ana ia Kamalalawalu, alaila, hoomakaukau ae la o Makakuikalani i na waa kaua, mamuli o ke kauoha ikaika a Kamalalawalu. A i ka makaukau ana o na waa a me na pukaua e ae, a me na kanaka a pau, a ike ae la ua o Lanikaula ua
  • 50. canoes prophesied in chant his last words to Kamalalawalu: makaukau na waa kaua o Kamalalawalu, a e lana ana i ke awa o Hamoa; ia manawa, hele mai o Lanikaula, a wanana mai la imua o ke alii Kamalalawalu a me na waa kaua a pau, oiai e lana ana na waa o ke alii i ke kai. A penei kana wanana ma ke mele, a o ka Lanikaula olelo hope ia ia Kamalalawalu. A penei: The red koae! The white koae!68 The koae that flies steadily on, Mounting up like the stars. To me the moon is low.69 It is a god, Your god, Lono; A god that grows and shines. Puuiki, Puunui. At Puuloa, at Puupoko; At Puukahanahana, At the doings of the god of Lono. Lono the small container, Lono the large container. Puunahe the small, Puunahe the large. By Hana, you swim out, By Moe you swim in. My popolo70 is mine own, The popolo that grows by the wayside Is plucked by Kaiokane, Koae ula ke koae kea, Koae lele pauma ana; Kiekie iluna ka hoku, Haahaa i au ka malama. He akua ko akua o Lono, He akua e ulu e lama ana; Puuiki, Puunui, I Puuloa, i Puupoko, I Puukahanahana, I ka hana a ke akua o Lono; O Lono ka ipu iki, O Lono ka ipu nui, O Puunahe iki, O Puunahe nui, Na Hana au aku, Na Moe au mai, Na’u no ka’u popolo, He popolo ku kapa alanui; I aho’ hia e Kaiokane I hakaia e Kaiowahine; O kaua i Kahulikini-e,
  • 51. Is watched over by Kaiowahine. We two to Kahulikini, Numberless, Vast, without number, countless Are we, O Kama. Let us two to Anaehoomalu, O my chief. He ki-ni, He kini, he lehu, he mano, Kaua, e Kama-e I Anaehoomalu kaua E kuu alii hoi-e. At the end of Lanikaula’s prophesy as made in the chant Kamalalawalu set sail with his large convoy of war canoes. It is mentioned in this tradition relative to the number of canoes of Kamalalawalu that the rear war canoes were at Hamoa, Hana, and the van at Puakea, Kohala; but at the time of this narrative the opinions of the ancients differed as to the accuracy of this. Some say that the number of canoes is greatly exaggerated. A pau ka Lanikaula olelo wanana ana ma ke mele e like me ka hoike ana maluna, alaila, holo aku la o Kamalalawalu me kona mau waa kaua he nui. Ua oleloia ma keia moolelo, o ka nui o na waa o Kamalalawalu aia ka maka hope o na waa kaua i Hamoa ma Hana, a o ka maka mua hoi o na waa, aia i Puakea ma Kohala. Aka hoi, ma ka manawa o keia moolelo, aole he like o ka manao o ka poe hahiko ma keia mea. Ua manao kekahi poe he wahahee ka mea i oleloia no ka nui o na waa. Kamalalawalu having arrived at Hawaii, Kauhipaewa and Kihapaewa were stationed at Puako, in accordance with the wishes of Lonoikamakahiki. At the first meeting that Kamalalawalu had with A hiki aku la o Kamalalawalu i Hawaii, ua hoonohoia o Kauhipaewa me Kihapaewa ma Puako, e like me ka makemake o Lonoikamakahiki. Ia manawa a Kamalalawalu i halawai mua ai me Kauhipaewa ma, olelo aku o
  • 52. Kauhipaewa and others, Kumaikeau and others [342](who were men from the presence of Lonoikamakahiki) said to Kamalalawalu: “Carry the canoes inland; take the outriggers off so that should the Hawaii forces be defeated in battle they would not use the flotilla of Maui to escape. When they find that the outriggers have all been taken apart and the victors overtake them the slaughter will be yours.” Kamalalawalu did as he was told to do by the two old men. Kumaikeau ma, he mau [343]kanaka no ko Lonoikamakahiki alo, me ka olelo aku ia Kamalalawalu: “E Kamalalawalu, lawe ia na waa iuka lilo, wehewehe ke ama a me ka iako, i kaua ia a hee ka Hawaii ia oukou, malia o holo ke auhee pio, a manao o ka auwaa o ka Maui ka mea e holo ai, i hiki aku ia, ua pau ka iako i ka hemohemo, i loaa mai ia i ka lanakila, alaila na oukou no ka make.” A e like me ka olelo a kela mau elemakule ia Kamalalawalu, alaila, hana aku la o Kamalalawalu e like me ka kela mau kanaka. When Kamalalawalu arrived at Kohala, Lonoikamakahiki had his army in readiness. Kamalalawalu learning that Kanaloakuaana was still living at Waimea he concluded that his first battle should be fought with Kanaloakuaana and at Kaunooa. Kanaloakuaana was completely routed and pursued by the soldiers of Kamalalawalu, and Kauhiakama, and Kanaloakuaana was captured at Puako. At this battle the eyes of I ka manawa a Kamalalawalu i hiki aku ai ma Kohala, ua makaukau mua na puali kaua o Lonoikamakahiki. Aka, lohe ae la ua o Kamalalawalu, eia no o Kanaloakuaana i Waimea kahi i noho ai, hoouka mua iho la o Kamalalawalu me Kanaloakuaana i Kaunooa. A hee mai la o Kanaloakuaana; a alualu loa mai la ko Kamalalawalu poe koa a me Kauhiakama pu, a loaa pio iho la o Kanaloakuaana ma Puako; a
  • 53. Kanaloakuaana were gouged out by the Maui forces, the eye sockets pierced by darts, and he was then killed, the eyes of Kanaloakuaana being tatued. ma ia hoouka kaua hou ana, poaloia ae la na maka o Kanaloakuaana e ko Maui kaua, a oo ia ae la na maka i ke kao hee, pepehiia iho la a make; ua kakauia nae na maka o Kanaloakuaana i ka uhi. Because of this action on the part of Kamalalawalu’s men the landing place for the canoes at Puako was called Kamakahiwa,71 and to this day is known by that name and may ever remain so to the end of this race. Because of the perpetration of this dastardly act on Kanaloakuaana the following was composed by a writer of chants, being the middle portion of a chant called “Koauli”: A oia hana ana a ko Kamalalawalu poe koa ia Kanaloakuaana, nolaila ua kapaia ka inoa oia awa pae waa ma Puako o Kamakahiwa, a o ka inoa ia o ia wahi a hiki mai i keia manawa, a hiki aku i ka hanauna hope loa o keia lahui. A no ia hana ia ana o Kanaloakuaana pela, ua hanaia e ka poe haku mele penei, oia hoi ma ka hapa waena o ke mele i oleloia o Koauli, penei: The drawing out of Kama, the ohia tree; The letting out of Kama at Waimea, The kin of Kanaloa.72 He was made black like the mud-hen. The face was blackened, Blackened was the face of Kanaloa with fire. Ke koana o Kama, ka ohia, Ko Kama kuu i Waimea, Ka io o Kanaloa, He ele he Alaea; O ka maka i kuia; I welo’a i ke kao o Kanaloa; Ko Kanaloa maka A lalapa no E uwalo wau i ka maka O Makakii;
  • 54. The face of Kanaloa, With burning fire. Let me scratch the face Of Makakii. You poked at the eyes of Kamalea,73 Makahiwa, Makalau. The men were from Hoohila, Of Makakaile. The face of Makakaile the large one, the life. Kikenui of Ewa. At Ewa is the fish that knows man’s presence.74 The foreskin of Loe, consecrated in the presence of Mano The chief, heralded75 by the drum of Hawea,76 The declaration drum Of Laamaikahiki. E o mai oe i ko kamalea maka, O Makahiwa, Makalau; No Hoohila ka lau. O Makakaile. Ka maka o Makakaile nui a ola; Kikenui a Ewa No Ewa ka ia i ka maka o Paweo No Loe ka ili lolo i ka maka o Mano Ke alii ke Olowalu o ka pahu o Hawea Ha pahu hai kanaka O Laamaikahiki. This chant is dedicated to the eyes of Kanaloakuaana as indicated by the verses. O keia mele i hai ia maluna no ka maka o Kanaloakuaana, e like me ka hoakaka ana ma na pauku maluna ae o kela mele. CHAPTER XIII. MOKUNA XIII.
  • 55. The Battle at Waimea. —Conquest by Lonoikamakahiki— Defeat and Death of Kamalalawalu. Ka Hoouka Kaua Ana ma Waimea.—Ka Lanakila Ana o Lonoikamakahiki.— Auhee o Kamalalawalu me Kona Make Ana. After the death of Kanaloakuaana by Kamalalawalu, and in obedience to the statements of the old men for the Maui war contingent to go to Waimea and locate at Puuoaoaka and Hokuula, Kamalalawalu and his men proceeded to the locality as indicated by them. The Maui forces followed and after locating at Hokuula awaited the [344]coming fray. On the day Kamalalawalu and his men went up to Waimea to occupy Hokuula the two deceitful old men at the time were with Kamalalawalu. In the early morning when Kamalalawalu awoke from sleep he beheld the men from Kona and those of Kau, Puna, Hilo, Hamakua and Kohala had also been assembled. Mahope iho o ka make ana o Kanaloakuaana ia Kamalalawalu ma, a e like hoi me ka olelo a na elemakule, e hoi iuka o Waimea, ma Puuoaoaka a me Hokuula e hoonoho ai ko Maui poe kaua, a nolaila ua hoi aku la o Kamalalawalu ma a ma kahi a ua mau elemakule nei i kuhikuhi ai. [345] Hoi aku la ko Maui poe a noho ma Hokuula e kali ana no ka hoouka kaua ana. I ka la a Kamalalawalu ma i pii ai iuka o Waimea a noho ma Hokuula, a o ua mau elemakule nolunolu la no kekahi me Kamalalawalu ma i kela manawa. A ma ia po a ao ae, ma ke kekahiakanui i ka manawa i ala ae ai ko Kamalalawalu hiamoe, aia hoi, ua kuahaua ia mai la na kanaka o Kona, ko Kau a o Puna a me
  • 56. Hilo, o Hamakua hoi a me Kohala. Kamalalawalu looked and saw that the lava from Keohe to Kaniku was one red mass. Kamalalawalu was astonished, because the day before he observed that the lava was one mass of black, but this morning it was entirely red with people. Thereupon Kamalalawalu inquired of Kumaikeau and the others why the lava was a mass of red: “What does red portend? Does it mean war?” Kumaikeau and the others replied: “Do not think the red you see is some other red and not what you assume it to be. It is not war. That red yonder is the wind. The olauniu wind of Kalahuipuaa and Puako had been blowing in the early morning and when it is very light and gentle it hugs the lava close. This olauniu wind on the lava coming in contact with the wind from Wainaualii raises a cloud of dust covering and hiding the land in the manner you saw yesterday.” While cogitating to himself, Kamalalawalu concluded to drop the matter on Nana aku la o Kamalalawalu he ula wale la no na ke a, mai Keohe a Kaniku; ia manawa haohao no o Kamalalawalu i keia mea; no ka mea, i ka Kamalalawalu ike ana i ka la mua he uliuli ke a; a i keia kakahiaka hoi, he ula pu wale la no i na kanaka. Nolaila, ninau ae la o Kamalalawalu: “Ea, e Kumaikeau ma, ula pu hoi ke a, heaha keia ula, he kaua paha?” I aku o Kumaikeau ma: “Aole paha ia ula au e ike la, he ula e ae, a manao aku oe he kaua ia. Aole ia he kaua. Oia ula la ea, he makani, pa aku la ka makani Olauniu o Kalahuipuaa a me Puako i ka wanaao, a malamalama loa, pili-a aku la, komo aku la keia Olauniu a pili-a aku la, hui aku la me ko Wainanalii makani, ku ae la ke ehu o ka lepo, uhia aku la nalo wale ke a au i ike ai i ka la inehinei.” A no kela olelo nolu a kela mau elemakule, oki wale iho la no o Kamalalawalu, a
  • 57. account of the deceit of the two old men and the loss of confidence in what Kumaikeau and the others had said, for the reason that the lava continued to be strewn with people even to the time of the setting sun. During that night and including the following morning the Kona men arrived and were assigned to occupy a position from Puupa to Haleapala. The Kau and Puna warriors were stationed from Holoholoku to Waikoloa. Those of Hilo and Hamakua were located from Mahiki to Puukanikanihia, while those of Kohala guarded from Momoualoa to Waihaka. waiho wale iloko ona ia manao, no ka mea, aole he hilinai nui i kela olelo a Kumaikeau ma, no ka mea, ua mau ka paa ana o ke a i na kanaka a hiki i ka napoo ana o ka la. Ma ia po iho, a ao ae, hiki mai la ko Kona poe a hoonoho mai la mai kai o Puupa a hiki i Haleapala. A o ko Kau hoi a me ko Puna, hoonoho ae la ka lakou poe mai Holoholoku a Waikoloa. A o ko Hilo a me ko Hamakua mai, hoonoho mai la ko lakou poe kaua mai Mahiki a Puukanikanihia. A o ko Kohala hoi, pania ia mai la e na kanaka mai Momoualoa a Waihaka. That morning Kamalalawalu observed that the lowlands were literally covered with almost countless men. Kamalalawalu then took a survey of his own men and realized that his forces were inferior in numbers. He then spoke to Kumaikeau and the others: “Kumaikeau and the rest of you, how is this and what is that large concourse of people below?” Ia kakahiaka, nana aku la o Kamalalawalu, ua uhi paa puia mai olalo i na kanaka, aole o kana mai. Alaila, nana ae la o Kamalalawalu ia lakou ua uuku loa; alaila, olelo aku la o Kamalalawalu ia Kumaikeau ma: “Ea! E Kumaikeau ma, pehea keia? Heaha keia lehulehu olalo?”
  • 58. Kumaikeau and the others replied: “We have never seen so many people in Hawaii before. Do not think that because of their superior numbers they will escape us; they cannot, for the reason that their fighting will have to be from below. It is true they are more numerous, but being beneath we will defeat them.” I aku o Kumaikeau ma: “Akahi no au a ike i ka nui o na kanaka o Hawaii nei. Mai manao nae oe ia nui, e pakele ana ia kakou. Aole e pakele, aia ka lakou kaua malalo, he nui lakou, o ko lakou kaa malalo, make no ia kakou.” The following day, Lonoikamakahiki went over to meet Kamalalawalu to confer concerning the war.77 During their conference Kamalalawalu proposed to Lonoikamakahiki that war cease because he feared the greater forces of Lonoikamakahiki. But the proposal by Kamalalawalu for termination of the war did not meet Lonoikamakahiki’s approval. He had no intention of acquiescing, because he was greatly incensed at Kamalalawalu for the brutal manner in which he killed Kanaloakuaana by gouging out the eyes and other brutal acts carried into execution while the latter was still alive. I kekahi la ae, hele aku la o Lonoikamakahiki e halawai me Kamalalawalu, e kuka no ke kaua. A i ko laua kamailio ana, olelo aku o Kamalalawalu ia Lonoikamakahiki, e hoopau wale ke kaua, no ka mea, ua hopo mai la o Kamalalawalu no ka nui loa o ka Lonoikamakahiki kaua. Aka, ma kela olelo kaua a Kamalalawalu e hoopau wale ke kaua, aohe manao o Lonoikamakahiki e hoopau, e like me ka Kamalalawalu olelo, no ka mea, ua wela ko Lonoikamakahiki huhu no Kamalalawalu, no ka pepehi hoomainoino ana ia Kanaloakuaana; oia hoi, ua poaloia na maka, a ua hoomainoino ia i ko
  • 59. Kanaloakuaana wa e ola okoa ana. Makakuikalani, however, upon hearing of Kamalalawalu’s proposal to Lonoikamakahiki to cease the war disapproved of it and said to Kamalalawalu not to have the [346]war cease. “Onward, and stand on the altar!78 Then will it be known which of us is a full grown child.” This determination on the part of Makakuikalani was manifested by his presence for three consecutive days before the forces of Hawaii. After the third day, the two combatting forces waged battle, Lonoikamakahiki gaining the victory over Kamalalawalu’s entire force on the same day the battle was fought, the Maui-ites being completely routed. Aka hoi, o Makakuikalani, i kona lohe ana ia Kamalalawalu ua olelo aku oia ia Lonoikamakahiki e hoopau i ke kaua, he mea makemake ole nae ia ia Makakuikalani. Oia hoi, ua olelo aku o ua Makakuikalani nei ia Kamalalawalu, aole e hoopau i ke kaua. “Ho aku imua a kau i ka nananuu; alaila ike ia na keiki makua o kakou.” A no ia manaopaa o Makakuikalani, hoike mau ae la oia imua o ko Hawaii kaua i kela la keia la pau na la ekolu. Mahope iho o na la ekolu, hoomaka iho la na aoao elua e [347]kaua, a iloko no o ua la hoouka kaua la, lanakila ae la o Lonoikamakahiki maluna o ko Kamalalawalu puali holookoa, a auhee aku la ko Maui a pau. This is the history of the battle as related by the ancients and as the narrative is preserved by them. Before the battle commenced it was customary for the old men to encourage Kamalalawalu to do battle. Whenever the two old men A penei hoi ka moolelo oia hoouka kaua ana i oleloia e ka poe kahiko, ma ka lakou malama moolelo ana. Mamua o ka hoouka kaua ana, he mea mau i na elemakule ka paipai ana ia Kamalalawalu e kaua. Aia lohe ua mau elemakule nei i na olelo
  • 60. heard what Kamalalawalu and the others had to say as to what they intended doing to Lonoikamakahiki in order to be victorious in battle, the old men would wend their way to make it known to Lonoikamakahiki and the others and this duty was generally carried out during some convenient time of night. The two old men always pointed out to Kamalalawalu and the others where the battle should be fought, and the suggestions of the old men were always received with the utmost confidence by him. Therefore Kumaikeau and the two deceitful old men would in turn inform Lonoikamakahiki. The two old men never suggested any place for battle which would result advantageously to Kamalalawalu and his forces; on the contrary, it was invariably such a locality where inevitable defeat would result. a Kamalalawalu ma, no na mea a lakou e hana aku ai ia Lonoikamakahiki, ma na mea e pili ana i ke kaua e lanakila ai ko lakou aoao, a e pio ai hoi ko Lonoikamakahiki, alaila, e hele aku auanei ua mau elemakule nei e hai aku ia Lonoikamakahiki ma, ma kekahi manawa kaawale o ka po. No ka mea, na ua mau elemakule nei no e kuhikuhi aku ia Kamalalawalu ma i ke kahua kahi e hoouka ai ke kaua ana. A e like me ke kuhikuhi ana a kela mau elemakule, e lilo auanei ia i olelo na Kamalalawalu e hilinai nui ai. A no ia mea, hele aku no o Kumaikeau ma, ua mau elemakule nolu (apuka) nei a hai aku ia Lonoikamakahiki. Aole no e kuhikuhi ana ua mau elemakule nei i ke kahua kaua ma kahi e lanakila ai ko Kamalalawalu mau puali, aka, ma kahi e pio ai o Kamalalawalu ma, malaila no ka ua mau elemakule nei kahua kaua e hoonoho ai. In the early morning of the day of battle, Makakuikalani went to the I ka la o ka hoouka kaua, ma ke kakahiaka nui, hele aku la o
  • 61. front with his warriors following him and planted themselves at Waikakanilua below Hokuula and Puuoaoaka at a prominence looking towards Waikoloa. Pupuakea, on observing that Makakuikalani was placing his men and self in position, he and his warriors immediately came forward prepared to give battle. It was a case where both sides were equally prepared for the fray. Makakuikalani mamua, a o kona poe kaua mahope ona, a ma Waikakanilua, malalo aku o Hokuula a me Puuoaoaka, ma ka hulei e nana iho ana ia Waikoloa. Aka hoi, o Pupuakea, i kona ike ana mai ia Makakuikalani, e hoonoho aku ana me kona poe koa, alaila, hele mai la o Pupuakea me kona poe kaua, me ka makaukau hoi no ke kaua. Aka, ua makaukau no na aoao a elua no ke kaua. Makakuikalani was a man of great height and large physique; a renowned and powerful general of Maui and was also Kamalalawalu’s younger brother. As for Pupuakea, Hawaii’s celebrated and powerful general and who was Lonoikamakahiki’s younger brother, he was only a man of small stature. Both men had been taught the art of fighting with the wooden club and were experts in its use, but their schooling was under different masters and at different places. He kanaka nui a loihi o Makakuikalani, ka pukaua ikaika kaulana o Maui, ko Kamalalawalu kaikaina. A o Pupuakea hoi, ko Hawaii pukaua ikaika kaulana, ko Lonoikamakahiki kaikaina, he wahi kanaka uuku no ia, a haahaa hoi. Ua aoia no laua a elua i ke kaka laau palau, a ua akamai no laua a elua, aka, he kumu okoa ka kekahi a me kekahi, a ua aoia no laua ma na wahi kaawale. Aka, i ka la o ka hoouka kaua ana, ua weliweli mai la ko Lonoikamakahiki poe kaua, no ka ike ana mai ia Makakuikalani.
  • 62. On the day of battle the sight of Makakuikalani put Lonoikamakahiki’s forces in dreadful fear. When Pupuakea saw Makakuikalani he had no fear of him, did not tremble but stood firm ready to give battle. Aka, o Pupuakea, iloko o kona manawa i ike aku ai ia Makakuikalani, aole i komo mai iloko ona ka makau, aole no hoi oia i weliweli, aka, kupaa mau no oia e kaua aku ia Makakuikalani. While Makakuikalani and Pupuakea were standing on the battle field, Makakuikalani raised his war club and from on high struck at Pupuakea. Being short in stature he was only slightly struck but fell to the ground, however. At the instant Makakuikalani’s war club struck Pupuakea the end of it was buried deep into the ground. At the moment Pupuakea was struck by the war club and fell Makakuikalani thought that he was killed, but the latter’s master saw that Pupuakea was not dead, so [348]said to Makakuikalani: “Go back and slay him for your opponent is not dead. Your clubbing being from above only delivered a blow with the butt end.” Makakuikalani hearing the words of his teacher turned around and threw the butt end of his club, at the same time Ia Makakuikalani a me Pupuakea e ku ana ma ke kahua kaua, ia manawa, lawe ae la o Makakuikalani i kana laau palau a kiekie, a hahau iho la maluna iho o Pupuakea, a no ka haahaa o Pupuakea, ua pa lihi aku la o Pupuakea, aka, haule aku la o Pupuakea ilalo i ka honua. A o ka welau o ua laau palau la a Makakuikalani, iloko hoi o kona manawa i hahau aku ai ia Pupuakea, napoo pu aku la i ka lepo. I ka manawa i pa aku ai o Pupuakea i ka laau palau a Makakuikalani, a haule ilalo, manao ae la ua o Makakuikalani, ua make loa o Pupuakea. Aka, o ke kumu kaka laau a Makakuikalani, ka mea nana i ao o Makakuikalani, oia ka mea nana i ike mai o Pupuakea, aole i make; nolaila, olelo aku ua kumu kaka laau la a Makakuikalani: “E hoi houia aku
  • 63. telling him to “Shut up! Instruction stops at home. He cannot escape, he must be dead because the club strikes true.” At the very instant that Makakuikalani faced around to talk with his teacher, he (the teacher) was dead. e hoomake, aole i make ka hoa kaua, no ka mea, he laau kau i luna, pa kano aku la kaua uhau ana.” A lohe o Makakuikalani i keia olelo ana aku a kana kumu, alaila, huli ae la oia (Makakuikalani) a wala hope ae la i ke kumu o ka laau [349]palau me ka olelo aku: “Kuli! I ka hale pau ke ao ana; aole e pakele, ua make aku la, no ka mea o ka Io ka laau.” A o ua kumu nei hoi a ua o Makakuikalani make loa aku la ia, i ka manawa no a ua o Makakuikalani i huli aku ai a kamailio. Pupuakea was lying on the ground, stunned, but somewhat recovered afterwards and raised himself up from the ground. When Makakuikalani saw that Pupuakea was still alive he rushed towards him bent on killing him. I ka manawa a Pupuakea e waiho ana i ka honua, ua maule aku la oia, a mahope loaa mai la ka mama iki ana ae, ia manawa, ala ae la o Pupuakea mai ka honua ae; ia manawa ike mai la o Makakuikalani ua ola hou o Pupuakea, alaila, holo hou mai la o Makakuikalani imua o Pupuakea, me ka manao e hoomake loa ia Pupuakea. Pupuakea observed Makakuikalani’s approach so prepared himself to slay him. When Makakuikalani drew near, A ike aku la o Pupuakea ia Makakuikalani e hele mai ana e kue hou iaia, alaila hoomakaukau ae la oia e pepehi
  • 64. Pupuakea raised his club and twirled it from his right. At that moment Makakuikalani attempted also to lay his club on Pupuakea, and when his club was twirled it skidded along the ground towards the feet of Makakuikalani and being parried by Makaku, fell to the ground. When Makakuikalani swung his club from the left side it struck the back of his own neck and he was instantly killed. Pupuakea immediately stepped backward and met his master who said to him: “Go back again and slay him so he be dead.” The words of his master aroused Pupuakea’s pride and he said to his teacher: “He cannot live, he is dead.” Then looking at the palm of his hand he again said to his master: “He cannot be alive because the birthmark of Pupuakea has impressed itself thereon. The flying club through dust has killed him.” aku ia Makakuikalani. A i ke kokoke ana mai o ua Makakuikalani nei, lawe ae la o Pupuakea i kana laau palau a wili ma kona aoao akau, a i ka hoomaka hou ana o Makakuikalani e hoouka hou i kana laau palau maluna o Pupuakea, alaila, ia manawa, wili ae la o Pupuakea i kana laau, a hualepo aku la ma na wawae o Makakuikalani, a pa aku la ia Makaku, haule aku la i ka honua, a i ka wili ana mai i kana laau mai ka aoao hema mai, pa mai la ma ka hono, make iho la o Makakuikalani. Ia manawa, emi hope aku la o Pupuakea a halawai me kana kumu kaka laau nana i ao. I mai la ke kumu ia Pupuakea: “Hoi houia aku e hoomake i make.” A no ka olelo ana a ke kumu a ua o Pupuakea pela, alaila, olelo aku la o Pupuakea i kana olelo kaena imua o kana kumu: “Aole e ola! Ua make!!” Nana iho la oia i ka poho o kona lima, a olelo ae la i ke kumu ana: “Aole ia e ola, no ka mea, ua kukai ae nei ka ila o Pupuakea. Make aku la i ka laau a kaua i ka hualepo.”
  • 65. After the great and renowned general of Maui had fallen the Hawaii forces continued to slaughter Kamalalawalu and the others. Upon the death of Kamalalawalu the slaughter of the Maui-ites continued for three days thereafter and those defeated who ran towards their canoes found no arms and outriggers because they had been broken. The repulsed warriors ran to Puako and noticing the paimalau79 floating in the sea mistook them for canoes. They began to waver and were again overtaken by the victors. The destruction of the remaining invaders was then complete. Referring to Kauhiakama the son of Kamalalawalu he escaped to safety. The story of his escape running thus: A haule aku la ka pukaua nui kaulana o Maui, alaila luku aku la ka Hawaii ia Kamalalawalu ma, a make aku la o Kamalalawalu. Ia make ana o ua o Kamalalawalu, lukuia aku la o Maui ekolu la, a hee aku la o Maui, a holo aku la, a na waa o lakou; aka, aole he iako, aole he ama, no ka mea, ua pau i ka haihai ia; nolaila holo aku la ke pio a ma Puako; a o ka ike i ke paimalau, kuhi he waa, a i ka hoolana ana iloko o ke kai, me ka manao, o ka waa ia, aia nae ua kahulihuli, a loaa hou aku la i ka lanakila, lukuia aku la na koena o ka Maui a pau loa i ka make. A o Kauhiakama hoi, ke keiki a Kamalalawalu, holo pio aku la oia, a pakele aku la. A penei ka moolelo o kona pakele ana. On the day that the Maui forces were defeated Kauhiakama clandestinely escaped to Kawaihae and from there his intentions were to hie to the caves, there to remain until his side was victorious and then make his appearance. I ka la o ka hee ana o ko Maui poe kaua, holo malu aku la oia a hiki i Kawaihae, a malaila mai e holo ana me ka manao e pee ma na ana, a hiki i ka wa e lanakila ai, alaila hoike ae.
  • 66. Hinau, one of the generals of Lonoikamakahiki and a messenger also, had great affection for Kauhiakama, but it was previous to the time of Hinau’s assisting in the escape of Kauhiakama that he roasted some taro and, together with some dried mudfish, already roasted, proceeded to search for Kauhiakama. Hinau came to Kawaihae first and from there went to Kaiopae where for the first time he saw Kauhiakama, so Hinau hailed him and said: “Say, Kauhiakama, remain there until I reach you!” Kauhiakama looking round saw Hinau approaching, the thought of death at the hands of the victorious crossed his mind, so covering his face with his hands he wept, for he [350]was greatly depressed in spirits. Hinau came forward, however, and greeted him with a kiss on the nose, remarking: “I remained behind and roasted some taro and dried mudfish for the love of you and came to search for you.” These words of Hinau gave Kauhiakama great relief and hopes for life. A o Hinau, kekahi o na pukaua o Lonoikamakahiki, he elele no na Lonoikamakahiki, aka, ua nui loa ke aloha o Hinau ia Kauhiakama. Nolaila, mamua o ko Hinau manao ana e hoomahuka ia Kauhiakama, pulehu ae la oia i mau kalo, a moa, a paa pu ae la me na oopu maloo i pulehuia, a imi aku la ia Kauhiakama; ma Kawaihaeo ko Hinau hiki mua ana, a malaila aku a hiki i Kaiopae, ike mua aku la o Hinau ia Kauhiakama, alaila, kahea aku la: “E Kauhiakama e! Malaila iho oe a loaa aku ia’u.” I alawa ae ka hana o Kauhiakama, e hele aku ana o Hinau, alaila, manao ae la o Kauhiakama: “Make, eia ka lanakila.” Alaila, palulu ae la ua o Kauhiakama i na lima i ke poo me ka manao kaumaha i ka make, e uwe ana. Aka, hele aku la o Hinau a honi aku la i ka ihu o Kauhiakama, a uwe iho la, me ka i aku: “Ua noho au me ke aloha ia oe, a nolaila, pulehu mai nei i na wahi kalo, a me na wahi oopu maloo, a imi [351]mai nei ia oe.” A no keia olelo a Hinau, akahi no a oluolu iho la o
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