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Host Bacteria Interactions Methods and Protocols 1st
Edition Annette C. Vergunst Digital Instant Download
Author(s): Annette C. Vergunst, David O'Callaghan (eds.)
ISBN(s): 9781493912605, 1493912607
Edition: 1
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Year: 2014
Language: english

Host-Bacteria
Interactions
Annette C.Vergunst
David O‘Callaghan Editors
Methods and Protocols
Methods in
Molecular Biology 1197

ME T H O D S I N MO L E C U L A R BI O LO G Y
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Host-Bacteria Interactions
Methods and Protocols
Edited by
Annette C. Vergunst and David O’Callaghan
INSERM,U1047,Nîmes,FranceandUniversitéMontpellier1,UFRMédecine,Nîmes,France

ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1260-5 ISBN 978-1-4939-1261-2 (eBook)
DOI 10.1007/978-1-4939-1261-2
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Editors
Annette C. Vergunst
INSERM, U1047
Nîmes, France
and
Université Montpellier 1
UFR Médecine
Nîmes, France
David O’Callaghan
INSERM, U1047
Nîmes, France
and
Université Montpellier 1
UFR Médecine
Nîmes, France

v
Bacterial infections are a complex interplay between host and pathogen. Over the last
20 years, there have been great advances in our understanding of the pathogenesis of infec-
tious disease by integrating detailed knowledge of bacterial genetics, cell biology, immunol-
ogy, and host physiology. This has led to the development of the new field of “cellular
microbiology.” Early studies began with well-studied organisms such as Salmonella, Shigella,
and Listeria and used simple cell culture models such as HeLa cells; however, this rapidly
extended to other pathogens and to a wide array of cell types. Cellular microbiology has
been instrumental in the identification of bacterial virulence factors required for interaction
with the host, their cellular targets, and how the interactions can modulate host cell biology
in favor of the pathogen. Recent advances now make it possible to study in great detail the
infection in vivo, at the cellular, tissue, and whole animal level.
In this volume we have brought together a set of cutting edge protocols that cover
aspects of the investigation of host–bacteria interactions using mammalian and novel non-
mammalian infection models, cell biology, OMICS, and bacterial genetics. Our aim is to
provide a pathway through the techniques that can be used to investigate different aspects
of the physiopathology of bacterial infections, from the whole animal to tissue, cellular, and
molecular levels. The pathogens used in the protocols are mainly, but not exclusively facul-
tative and obligate intracellular bacteria, for which we are trying to decipher how the intra-
cellular stages of a pathogen contribute to disease. However, the protocols are generally
applicable to most other pathogens. Since the principal goal of the book is to provide
researchers with a comprehensive account of the practical steps necessary for carrying out
each protocol successfully, the Methods section contains detailed step-by-step descriptions
of every protocol. The Notes section complements the Methods with tips based on the
authors first-hand experience explaining the “tricks of the trade” and the best ways to deal
with any problem or difficulty that might arise.
From the earliest times, infection models have been instrumental in understanding
infectious disease. Over recent years, there has been a move away from classical models
using mammals and mammalian cells to nonvertebrate systems. In this volume, chapters in
Part I will describe how to use Galleria (wax moth) larvae (Chapter 1) or Drosophila as
infection models (Chapter 2), whereas the non-animal models using amoeba (Chapter 9)
or plants (Chapters 6 and 11) are included in Part II. The zebrafish has recently emerged
as a model where we can exploit both the genetic tractability and optical transparency of
developing embryos to follow the infection and assess the role of both host and pathogen
factors in real time at the cellular and whole animal level (Chapter 3). Advances in live imag-
ing techniques have also allowed the development of mammalian systems where lumines-
cent bacteria or cells can be seen in the body using highly sensitive cameras (Chapter 4).
Two photon microscopy now allows the observation of events at the cellular level in living
tissue (Chapter 5).
To fully understand bacterial virulence, it is essential to investigate the host–pathogen
interaction at the cellular and molecular level (Part II). Using plant or yeast cells as a
surrogate model, we can identify and characterize the bacterial proteins, or effectors,
Preface

vi
translocated into host cells through bacterial secretion systems to understand how the
bacterium tries to manipulate host defense mechanisms to create its own niche (Chapter 6).
We present a protocol that can be used to identify the host targets of a bacterial virulence
factor, either when exposed on the pathogen surface or injected into the cell (Chapter 7).
We also present protocols to show how pathogens modulate key host cell processes
including protein degradation through the proteasome (Chapter 8), phosphoinositide
dynamics (Chapter 9), and apoptosis (Chapter 10). Using bimolecular fluorescence com-
plementation, in vivo interactions between host and bacterial proteins can be identified
(Chapter 11). There is also a protocol to examine how bacterial pathogens can modulate
innate immune signaling through the TLR pathway (Chapter 12).
Technological advances have led to an explosion in the quantity and complexity of
OMICS data that can be generated (Part III). A Drosophila cell line can be used for siRNA
screens (Chapter 13) to identify host factors required for the infection. Two chapters
describe the purification of bacteria for proteomic or RNAseq from infected cells (Chapter 14),
and the isolation of host phagocytes for RNAseq from zebra fish embryos using FACS sort-
ing (Chapter 15). We also include a protocol for rapid sample production for high-
throughput proteomic analysis and data extraction (Chapter 16). Exploiting the masses of
data generated in these studies requires powerful bioinformatics support. Chapter 17
describes PATRIC, an NIH-funded database dedicated to OMICS data from pathogens.
Genetic manipulation of the bacterial pathogen is crucial to elucidate the molecular
basis of bacteria–host interactions (Part IV). We include three protocols (Chapters 18–20)
describing techniques to manipulate bacteria that are either highly recalcitrant or obligate
intracellular.
We would like to thank all the contributors, who are leading researchers in the field and
have either developed, or are expert users of the presented methods, for providing their
comprehensive protocols and tips for this volume. We would like to take the opportunity
to thank Dr. John Walker, the Editor-in-Chief of the Methods in Molecular Biology series, for
giving us the opportunity to edit this volume and his constant support.
We hope you enjoy this volume of Methods in Molecular Biology.
Nîmes, France Annette C. Vergunst
David O’Callaghan
Preface
Preface

vii
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I INFECTION MODELS TO STUDY BACTERIAL VIRULENCE
1 Galleria mellonella as an Infection Model for Select Agents . . . . . . . . . . . . . . . 3
Nicolas Sprynski, Eric Valade, and Fabienne Neulat-Ripoll
2 Drosophila as a Model for Intestinal Infections. . . . . . . . . . . . . . . . . . . . . . . . . 11
Matthieu Lestradet, Kwang-Zin Lee, and Dominique Ferrandon
3 Zebrafish Embryos as a Model to Study Bacterial Virulence. . . . . . . . . . . . . . . 41
Jennifer Mesureur and Annette C. Vergunst
4 Studying Host-Pathogen Interaction Events in Living Mice
Visualized in Real Time Using Biophotonic Imaging. . . . . . . . . . . . . . . . . . . . 67
Gary Splitter, Jerome Harms, Erik Petersen, Diogo Magnani,
Marina Durward, Gireesh Rajashekara, and Girish Radhakrishnan
5 Intravital Two-Photon Imaging to Understand Bacterial
Infections of the Mammalian Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Ferdinand X. Choong and Agneta Richter-Dahlfors
PART II CELLULAR ASPECTS OF HOST-BACTERIA INTERACTIONS
6 Cre Reporter Assay for Translocation (CRAfT): A Tool
for the Study of Protein Translocation into Host Cells. . . . . . . . . . . . . . . . . . . 103
Amke den Dulk-Ras, Annette C. Vergunst, and Paul J.J. Hooykaas
7 Detection of the Interaction Between Host and Bacterial Proteins:
Eukaryotic Nucleolin Interacts with Francisella Elongation Factor Tu . . . . . . . 123
Monique Barel and Alain Charbit
8 Hijacking the Host Proteasome for the Temporal Degradation
of Bacterial Effectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Tomoko Kubori, Andree M. Hubber, and Hiroki Nagai
9 Live Cell Imaging of Phosphoinositide Dynamics During
Legionella Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Stephen Weber and Hubert Hilbi
10 Investigating Interference with Apoptosis Induction by Bacterial Proteins . . . . 169
Hua Niu and Yasuko Rikihisa
11 Bimolecular Fluorescence Complementation for Imaging
Protein Interactions in Plant Hosts of Microbial Pathogens . . . . . . . . . . . . . . . 185
Lan-Ying Lee and Stanton B. Gelvin
Contents

viii
12 Investigating TLR Signaling Responses in Murine Dendritic Cells
Upon Bacterial Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Suzana Pinto Salcedo and Lena Alexopoulou
PART III OMICS AND LARGE SCALE SCREENING
13 siRNA Screens Using Drosophila Cells to Identify
Host Factors Required for Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Aseem Pandey, Sheng Li Ding, Thomas A. Ficht, and Paul de Figueiredo
14 Purification of Intracellular Bacteria: Isolation of Viable Brucella
abortus from Host Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Esteban Chaves-Olarte, Pamela Altamirano-Silva,
Caterina Guzmán-Verri, and Edgardo Moreno
15 RNA Sequencing of FACS-Sorted Immune Cell Populations
from Zebrafish Infection Models to Identify Cell Specific Responses
to Intracellular Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Julien Rougeot, Ania Zakrzewska, Zakia Kanwal, Hans J. Jansen,
Herman P. Spaink, and Annemarie H. Meijer
16 Taking the Shortcut for High-Throughput Shotgun Proteomic
Analysis of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Erica Marie Hartmann, François Allain, Jean-Charles Gaillard,
Olivier Pible, and Jean Armengaud
17 Comparative Genomic Analysis at the PATRIC, A Bioinformatic
Resource Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Alice R. Wattam, Joseph L. Gabbard, Maulik Shukla,
and Bruno W. Sobral
PART IV APPROACHES FOR DIFFICULT BACTERIA
18 A Markerless Deletion Method for Genetic Manipulation
of Burkholderia cenocepacia and Other Multidrug-Resistant
Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Daniel F. Aubert, Mohamad A. Hamad, and Miguel A. Valvano
19 Gene Inactivation in Coxiella burnetii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Paul A. Beare and Robert A. Heinzen
20 A Chemical Mutagenesis Approach to Identify Virulence Determinants
in the Obligate Intracellular Pathogen Chlamydia trachomatis. . . . . . . . . . . . . 347
Bidong Nguyen and Raphael Valdivia
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Contents
Contents

ix
LENA ALEXOPOULOU • Centre d’Immunologie de Marseille-Luminy, Aix-Marseille
Université UM 2, Marseille, France
FRANÇOIS ALLAIN • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze,
France; Bertin Technologies, Montigny-le-Bretonneu, France
PAMELA ALTAMIRANO-SILVA • Programa de Investigación en Enfermedades Tropicales,
Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica;
Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología,
Universidad de Costa Rica, San José, Costa Rica; Centro de Investigación en Estructuras
Microscópicas, Universidad de Costa Rica, San José, Costa Rica
JEAN ARMENGAUD • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze,
France
DANIEL F. AUBERT • Centre for Human Immunology, University of Western Ontario,
London, ON, Canada; Department of Microbiology and Immunology, University of
Western Ontario, London, ON, Canada
MONIQUE BAREL • INSERM U1002, Unité de Pathogénie des Infections Systémiques,
Université Paris Descartes, Paris, France
PAUL A. BEARE • Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Hamilton, MT, USA
ALAIN CHARBIT • INSERM U1002, Unité de Pathogénie des Infections Systémiques,
Université Paris Descartes, Paris, France
ESTEBAN CHAVES-OLARTE • Programa de Investigación en Enfermedades Tropicales,
Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica;
Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología,
Universidad de Costa Rica, San José, Costa Rica; Centro de Investigación en
Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica
FERDINAND X. CHOONG • Department of Neuroscience, Swedish Medical
Nanoscience Center, Karolinska Institutet, Stockholm, Sweden
SHENG LI DING • Department of Plant Pathology and Microbiology,
Texas A&M University, College Station, TX, USA; Norman Borlaug Center,
Texas A&M University, College Station, TX, USA; Department of Molecular
and Cellular Medicine, College of Medicine, Texas A&M Health Science Center,
College Station, TX, USA
AMKE DEN DULK-RAS • Sylvius Laboratory, Institute of Biology Leiden (IBL),
Leiden University, Leiden, The Netherlands
MARINA DURWARD • Department of Pathobiological Sciences, University of
Wisconsin-Madison, Madison, WI, USA
DOMINIQUE FERRANDON • UPR9022 du CNRS, Université de Strasbourg, Strasbourg,
France
THOMAS A. FICHT • Department of Veterinary Pathobiology, Texas A&M University,
Contributors

x
PAUL DE FIGUEIREDO • Department of Veterinary Pathobiology, Texas A&M University,
College Station, TX, USA; Department of Plant Pathology and Microbiology,
Texas A&M University, College Station, TX, USA; Norman Borlaug Center,
Texas A&M University, College Station, TX, USA; Department of Microbial
and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center,
JOSEPH L. GABBARD • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg,
VA, USA
JEAN-CHARLES GAILLARD • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze,
France
STANTON B. GELVIN • Department of Biological Sciences, Purdue University, West Lafayette,
IN, USA
CATERINA GUZMÁN-VERRI • Programa de Investigación en Enfermedades Tropicales,
Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica
MOHAMAD A. HAMAD • Centre for Human Immunology, University of Western Ontario,
London, ON, Canada; Department of Microbiology and Immunology,
University of Western Ontario, London, ON, Canada
JEROME HARMS • Department of Pathobiological Sciences, University of Wisconsin-Madison,
Madison, WI, USA
ERICA MARIE HARTMANN • CEA, DSV, IBEB, Lab Biochim System Perturb,
Bagnols-sur-Cèze, France
ROBERT A. HEINZEN • Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Hamilton, MT, USA
HUBERT HILBI • Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich,
Germany
PAUL J.J. HOOYKAAS • Sylvius Laboratory, Institute of Biology Leiden (IBL), Leiden
University, Leiden, The Netherlands
ANDREE M. HUBBER • Research Institute for Microbial Diseases, Osaka University, Osaka,
Japan
HANS J. JANSEN • Institute of Biology, Leiden University, Leiden, The Netherlands
ZAKIA KANWAL • Institute of Biology, Leiden University, Leiden, The Netherlands
TOMOKO KUBORI • Research Institute for Microbial Diseases, Osaka University, Osaka,
Japan
KWANG-ZIN LEE • UPR9022 du CNRS, Université de Strasbourg, Strasbourg, France
LAN-YING LEE • Department of Biological Sciences, Purdue University, West Lafayette,
IN, USA
MATTHIEU LESTRADET • UPR9022 du CNRS, Université de Strasbourg, Strasbourg, France
DIOGO MAGNANI • University of Miami, Miami, FL, USA
ANNEMARIE H. MEIJER • Institute of Biology, Leiden University, Leiden, The Netherlands
JENNIFER MESUREUR • INSERM, U1047, Nîmes, France and Université Montpellier 1,
UFR Médecine, Nîmes, France
EDGARDO MORENO • Programa de Investigación en Enfermedades Tropicales, Escuela de
Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; Instituto Clodomiro
Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
HIROKI NAGAI • Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
FABIENNE NEULAT-RIPOLL • Unité de Bactériologie, Institut de Recherche Biomédicale
des Armées, La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie,
IRBA, Aix-Marseille Université, Marseille, France
Contributors
Contributors

xi
BIDONG NGUYEN • Department of Molecular Genetics and Microbiology,
Duke University Medical Center, Durham, NC, USA
HUA NIU • Department of Veterinary Biosciences, The Ohio State University,
Columbus, OH, USA
ASEEM PANDEY • Department of Veterinary Pathobiology, Texas A&M University,
ERIK PETERSEN • Department of Medicine, University of Washington, Seattle, WA, USA
OLIVIER PIBLE • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France
GIRISH RADHAKRISHNAN • National Institute of Animal Biotechnology, University of
Hyderabad, Hyderabad, India
GIREESH RAJASHEKARA • Ohio State University, Wooster, OH, USA
AGNETA RICHTER-DAHLFORS • Department of Neuroscience, Swedish Medical
Nanoscience Center, Karolinska Institutet, Stockholm, Sweden
YASUKO RIKIHISA • Department of Veterinary Biosciences, The Ohio State University,
Columbus, OH, USA
JULIEN ROUGEOT • Institute of Biology, Leiden University, Leiden, The Netherlands
SUZANA PINTO SALCEDO • Bases Moléculaires et Structurales des Systèmes Infectieux,
CNRS UMR 5086, Institut de Biologie et Chimie des Protéines, IBCP,
Université Lyon 1, Lyon, France
MAULIK SHUKLA • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
BRUNO W. SOBRAL • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg,
VA, USA
HERMAN P. SPAINK • Institute of Biology, Leiden University, Leiden, The Netherlands
GARY SPLITTER • Department of Pathobiological Sciences, University of Wisconsin-Madison,
Madison, WI, USA
NICOLAS SPRYNSKI • Unité de Bactériologie, Institut de Recherche Biomédicale des Armées,
La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie, IRBA,
Aix-Marseille Université, Marseille, France; Antabio, Labège, France
ERIC VALADE • Unité de Bactériologie, Institut de Recherche Biomédicale des Armées,
La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie, IRBA,
Aix-Marseille Université, Marseille, France; Ecole du Val-de-Grâce, Paris, France
RAPHAEL VALDIVIA • Department of Molecular Genetics and Microbiology, Duke University
Medical Center, Durham, NC, USA
MIGUEL A. VALVANO • Centre for Human Immunology, University of Western Ontario,
London, ON, Canada; Department of Microbiology and Immunology, University of
Western Ontario, London, ON, Canada; Centre for Infection and Immunity,
Queen’s University Belfast, Belfast, UK
ANNETTE C. VERGUNST • INSERM, U1047, Nîmes, France and Université Montpellier 1,
UFR Médecine, Nîmes, France
ALICE R. WATTAM • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
STEPHEN WEBER • Max von Pettenkofer Institute, Ludwig-Maximilians University,
Munich, Germany
ANIA ZAKRZEWSKA • Institute of Biology, Leiden University, Leiden, The Netherlands
Contributors
Contributors

Part I
Infection Models to Study Bacterial Virulence

Exploring the Variety of Random
Documents with Different Content

FROM “BYRON”
In men whom men condemn as ill
I find so much of goodness still,
In men whom men pronounce divine
I find so much of sin and blot,
I do not dare to draw a line
Between the two, where God has not.

Edward Rowland Sill
Edward Rowland Sill was born at Windsor, Connecticut, in 1841. In 1861 he was
graduated from Yale and shortly thereafter his poor health compelled him West.
After various unsuccessful experiments, he drifted into teaching, first in the high
schools in Ohio, later in the English department of the University of California. His
uncertain physical condition added to his mental uncertainty. Unable to ally
himself either with the lethargic, conservative forces whom he hated or with the
radicals whom he distrusted, Sill became an uncomfortable solitary; half
rebellious, half resigned. During the last decade of his life, his brooding
seriousness was less pronounced, a lighter irony took the place of his dark
reflections.
The Hermitage, his first volume, was published in 1867, a later edition
(including later poems) appearing in 1889. His two posthumous books are Poems
(1887) and Hermione and Other Poems (1899).
Sill died, after bringing something of the Eastern culture to the West, in 1887.

SOLITUDE
All alone—alone,
Calm, as on a kingly throne,
Take thy place in the crowded land,
Self-centred in free self-command.
Let thy manhood leave behind
The narrow ways of the lesser mind:
What to thee are its little cares,
The feeble love or the spite it bears?
Let the noisy crowd go by:
In thy lonely watch on high,
Far from the chattering tongues of men,
Sitting above their call or ken,
Free from links of manner and form
Thou shalt learn of the wingéd storm—
God shall speak to thee out of the sky.

DARE YOU?
Doubting Thomas and loving John,
Behind the others walking on:—
“Tell me now, John, dare you be
One of the minority?
To be lonely in your thought,
Never visited nor sought,
Shunned with secret shrug, to go
Thro’ the world, esteemed its foe;
To be singled out and hissed,
Pointed at as one unblessed,
Warned against in whispers faint,
Lest the children catch a taint;
To bear off your titles well,—
Heretic and infidel?
If you dare, come now with me,
Fearless, confident, and free.”
“Thomas, do you dare to be
Of the great majority?
To be only, as the rest,
With Heaven’s creature comforts blessed;
To accept, in humble part,
Truth that shines on every heart;
Never to be set on high,
Where the envious curses fly;
Never name or fame to find,
Still outstripped in soul and mind;
To be hid, unless to God,
As one grass-blade in the sod,
Underfoot with millions trod?
If you dare, come with us, be
Lost in love’s great unity.”

Sidney Lanier
Sidney Lanier was born at Macon, Georgia, February 3, 1842. His was a family of
musicians (Lanier himself was a skilful performer on various instruments), and it
is not surprising that his verse emphasizes—even overstresses—the influence of
music on poetry. He attended Oglethorpe College, graduating at the age of eighteen
(1860), and, a year later, volunteered as a private in the Confederate army. After
several months’ imprisonment (he had been captured while acting as signal officer
on a blockade-runner), Lanier was released in February, 1865, returning from
Point Lookout to Georgia on foot, accompanied only by his flute, from which he
refused to be separated. His physical health, never the most robust, had been
frightfully impaired by his incarceration, and he was already suffering from
tuberculosis, the rest of his life being spent in an unequal struggle against it.
He was now only twenty-three years old and the problem of choosing a vocation
was complicated by his marriage in 1867. He spent five years in the study and
practice of law, during which time he wrote comparatively little verse. But the law
could not hold him; he felt premonitions of death and realized he must devote his
talents to art before it was too late. He was fortunate enough to obtain a position as
flautist with the Peabody Symphony Orchestra in 1873 in Baltimore, where he had
free access to the music and literature he craved. Here he wrote all of his best
poetry. In 1879, he was made lecturer on English in Johns Hopkins University, and
it was for his courses there that he wrote his chief prose work, a brilliant if not
conclusive study, The Science of English Verse. Besides his poetry, he wrote several
books for boys, the two most popular being The Boy’s Froissart (1878) and The
Boy’s King Arthur (1880).
Lanier’s poetry, charming though most of it is, suffers from his all too frequent
theorizing, his too conscious effort to bring verse over into the province of pure
music. He thought almost entirely, even in his most intellectual conceptions, in
terms of musical form. His main theory that English verse has for its essential
basis not accent but a strict musical quantity is a wholly erroneous conclusion,
possible only to one who could write “whatever turn I have for art is purely musical
—poetry being with me a mere tangent into which I shoot.” Lanier is at his best in
his ballads, although a few of his lyrics have a similar spontaneity. In spite of the
fact that he had rather novel schemes of rhythm and stanza-structure, much of his
work is marred by strained effects, elaborate conceits and a kind of verse that
approaches mere pattern-making. But such a vigorous ballad as “The Song of the

Chattahoochee,” lyrics like “Night and Day” and “The Stirrup Cup,” and parts of
the symphonic “Hymns of the Marshes” are sure of a place in American literature.
Never a great figure, he was one of the most interesting and spiritual of our minor
poets.
Lanier died, a victim of his disease, in the mountains of North Carolina,
September 7, 1881.

Out of the hills of Habersham,
Down the valleys of Hall,
I hurry amain to reach the plain,
Run the rapid and leap the fall,
Split at the rock and together again,
Accept my bed, or narrow or wide,
And flee from folly on every side
With a lover’s pain to attain the plain
Far from the hills of Habersham,
Far from the valleys of Hall.
All down the hills of Habersham,
All through the valleys of Hall,
The rushes cried Abide, abide,
The willful waterweeds held me thrall,
The laving laurel turned my tide,
The ferns and the fondling grass said Stay,
The dewberry dipped for to work delay,
And the little reeds sighed Abide, abide,
Here in the hills of Habersham,
Here in the valleys of Hall.
High o’er the hills of Habersham,
Veiling the valleys of Hall,
The hickory told me manifold
Fair tales of shade, the poplar tall
Wrought me her shadowy self to hold,
The chestnut, the oak, the walnut, the pine,
Overleaning, with flickering meaning and sign,
Said, Pass not, so cold, these manifold
Deep shades of the hills of Habersham,
These glades in the valleys of Hall.
And oft in the hills of Habersham,
And oft in the valleys of Hall,
The white quartz shone, and the smooth brook-stone
Did bar me of passage with friendly brawl,
And many a luminous jewel lone
—Crystals clear or acloud with mist,
Ruby garnet and amethyst—

Ruby, garnet and amethyst—
Made lures with the lights of streaming stone
In the clefts of the hills of Habersham,
In the beds of the valleys of Hall.
But oh, not the hills of Habersham,
And oh, not the valleys of Hall
Avail: I am fain for to water the plain.
Downward the voices of Duty call—
Downward, to toil and be mixed with the main,
The dry fields burn, and the mills are to turn,
And a myriad flowers mortally yearn,
And the lordly main from beyond the plain
Calls o’er the hills of Habersham,
Calls through the valleys of Hall.

NIGHT AND DAY
[5]
The innocent, sweet Day is dead.
Dark Night hath slain her in her bed.
O, Moors are as fierce to kill as to wed!
—Put out the light, said he.
A sweeter light than ever rayed
From star of heaven or eye of maid
Has vanished in the unknown Shade
—She’s dead, she’s dead, said he.
Now, in a wild, sad after-mood
The tawny Night sits still to brood
Upon the dawn-time when he wooed
—I would she lived, said he.
Star-memories of happier times,
Of loving deeds and lovers’ rhymes,
Throng forth in silvery pantomimes.
—Come back, O Day! said he.

FROM “THE MARSHES OF GLYNN”
[6]

As the marsh-hen secretly builds on the watery sod,
Behold I will build me a nest on the greatness of God:
I will fly in the greatness of God as the marsh-hen flies
In the freedom that fills all the space ’twixt the marsh and the skies:
By so many roots as the marsh-grass sends in the sod
I will heartily lay me a-hold on the greatness of God:
Oh, like to the greatness of God is the greatness within
The range of the marshes, the liberal marshes of Glynn.
And the sea lends large, as the marsh: lo, out of his plenty the sea
Pours fast: full soon the time of the flood-tide must be:
Look how the grace of the sea doth go
About and about through the intricate channels that flow
Here and there,
Everywhere,
Till his waters have flooded the uttermost creeks and the low-lying
lanes,
And the marsh is meshed with a million veins,
That like as with rosy and silvery essences flow
In the rose-and-silver evening glow.
Farewell, my lord Sun!
The creeks overflow; a thousand rivulets run
’Twixt the roots of the sod; the blades of the marsh-grass stir;
Passeth a hurrying sound of wings that westward whirr;
Passeth, and all is still; and the currents cease to run;
And the sea and the marsh are one.
How still the plains of the waters be!
The tide in his ecstasy.
The tide is at his highest height:
And it is night.
And now from the Vast of the Lord will the waters of sleep
Roll in on the souls of men,
But who will reveal to our waking ken
The forms that swim and the shapes that creep
Under the waters of sleep?
And I would I could know what swimmeth below when the tide
comes in

comes in
On the length and breadth of the marvellous marshes of Glynn.

Charles Edward Carryl
Charles Edward Carryl, father of Guy Wetmore Carryl (see page 142), was born
in New York City, December 30, 1842. He was an officer and director in various
railroads but found leisure to write two of the few worthy rivals of the immortal
Alice in Wonderland. These two, Davy and the Goblin (1884), which has gone
through twenty printings, and The Admiral’s Caravan (1891), contain many lively
and diverting ballads as well as inspired nonsense verses in the manner of his
model who, in spite of the slight difference in spelling, was also a Carroll.
C. E. Carryl lived the greater part of his life in New York, but on retiring from
business, removed to Boston and lived there until his death, which occurred in the
summer of 1920.

“Canary-birds feed on sugar and seed,
Parrots have crackers to crunch;
And as for the poodles, they tell me the noodles
Have chickens and cream for their lunch.
But there’s never a question
About MY digestion—
Anything does for me!
“Cats, you’re aware, can repose in a chair,
Chickens can roost upon rails;
Puppies are able to sleep in a stable,
And oysters can slumber in pails.
But no one supposes
A poor Camel dozes—
Any place does for me!
“Lambs are enclosed where it’s never exposed,
Coops are constructed for hens;
Kittens are treated to houses well heated,
And pigs are protected by pens.
But a Camel comes handy
Wherever it’s sandy—
Anywhere does for me!
“People would laugh if you rode a giraffe,
Or mounted the back of an ox;
It’s nobody’s habit to ride on a rabbit,
Or try to bestraddle a fox.
But as for a Camel, he’s
Ridden by families—
Any load does for me!
“A snake is as round as a hole in the ground;
Weasels are wavy and sleek;
And no alligator could ever be straighter
Than lizards that live in a creek.
But a Camel’s all lumpy
And bumpy and humpy—
Any shape does for me!”

The night was thick and hazy
When the “Piccadilly Daisy”
Carried down the crew and captain in the sea;
And I think the water drowned ’em;
For they never, never found ’em
And I know they didn’t come ashore with me.
Oh! ’twas very sad and lonely
When I found myself the only
Population on this cultivated shore;
But I’ve made a little tavern
In a rocky little cavern,
And I sit and watch for people at the door.
I spent no time in looking
For a girl to do my cooking,
As I’m quite a clever hand at making stews;
But I had that fellow Friday,
Just to keep the tavern tidy,
And to put a Sunday polish on my shoes.
I have a little garden
That I’m cultivating lard in,
As the things I eat are rather tough and dry;
For I live on toasted lizards,
Prickly pears, and parrot gizzards,
And I’m really very fond of beetle-pie.
The clothes I had were furry,
And it made me fret and worry
When I found the moths were eating off the hair;
And I had to scrape and sand ’em,
And I boiled ’em and I tanned ’em,
Till I got the fine morocco suit I wear.
I sometimes seek diversion
In a family excursion
With the few domestic animals you see;
And we take along a carrot
As refreshment for the parrot,
And a little can of jungleberry tea.

Then we gather as we travel,
Bits of moss and dirty gravel,
And we chip off little specimens of stone;
And we carry home as prizes
Funny bugs, of handy sizes,
Just to give the day a scientific tone.
If the roads are wet and muddy
We remain at home and study,—
For the Goat is very clever at a sum,—
And the Dog, instead of fighting,
Studies ornamental writing,
While the Cat is taking lessons on the drum.
We retire at eleven,
And we rise again at seven;
And I wish to call attention, as I close,
To the fact that all the scholars
Are correct about their collars,
And particular in turning out their toes.

James Whitcomb Riley
James Whitcomb Riley, who was possibly the most widely read native poet of his
day, was born October 7, 1849, in Greenfield, Indiana, a small town twenty miles
from Indianapolis, where he spent his later years. Contrary to the popular belief,
Riley was not, as many have gathered from his bucolic dialect poems, a struggling
child of the soil; his father was a lawyer in comfortable circumstances and Riley
was not only given a good education but was prepared for the law. His
temperament, however, craved something more adventurous. At eighteen he shut
the heavy pages of Blackstone, slipped out of the office and joined a traveling
troupe of actors who sold patent medicines during the intermissions. Riley’s
functions were varied: he beat the bass-drum, painted their flaring banners, wrote
local versions of old songs, coached the actors and, when occasion arose, took part
in the performance himself.
Even before this time, Riley had begun to send verses to the newspapers, frank
experiments, bits of homely sentiment, simple snatches and elaborate hoaxes—the
poem “Leonainie,” published over the initials “E. A. P.,” being accepted in many
quarters as a newly discovered poem by Poe. In 1882, when he was on the staff of
the Indianapolis Journal, he began the series of dialect poems which he claimed
were by a rude and unlettered farmer, one “Benj. F. Johnson, of Boone, the
Hoosier poet”—printing long extracts from “Boone’s” ungrammatical and badly-
spelt letters to prove his find. A collection of these rustic verses appeared, in 1883,
as The Ole Swimmin’ Hole; and Riley leaped into widespread popularity.
Other collections followed rapidly: Afterwhiles (1887), Old-Fashioned Roses
(1888), Pipes o’ Pan at Zekesbury (1889), Rhymes of Childhood (1890). All met an
instant response; Riley endeared himself, by his homely idiom and his childlike
ingenuity, to a countryful of readers, adolescent and adult.
But Riley’s simplicity is not always as artless as it seems. Time and again, one
can see him trading wantonly on the emotions of his unsophisticated readers; he
sees them about to smile—and broadens the point of his joke; he observes them on
the point of tears—and pulls out the sobbing tremolo stop. In many respects, he is
patently the most artificial of those poets who claim to give us the stuff of the soil.
He is the poet of obtrusive sentiment rather than of quiet convictions; of lulling
assurance, of philosophies that never disturb his readers, of sweet truisms rather
than searching truths.

That work of his which may endure, will survive because of the personal flavor
that Riley often fused into it. Such poems as “When the Frost is on the Punkin,”
“The Raggedy Man,” “Our Hired Girl” are a part of American folk literature; “Little
Orphant Annie” is read wherever there is a schoolhouse or, for that matter, a
nursery. In 1912 the schools throughout the country observed his birthday.
Riley died in his little house in Lockerbie Street, Indianapolis, July 22, 1916.

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