Chapter 1 the microbial world partial
- 1. 1 Dr Bilal Houshaymi Chapter 1 The Microbial World And You bhoushaymi@gmail.com
- 2. 2 topic % of final grade Exams I 25% Exams II 25% Final Exam 40% Participation and project 10% GRADING: EXAMS: The questions will be based primarily on lectures, however, some material from the assigned reading (and not necessarily covered in class) may appear on exams. Questions will be multiple choice, true/false, or short answer, essay. Make up exams will be given orally and could have an increased degree of difficulty. •Please ask questions. Class participation may help you. Please share comments or news •You will be responsible for all material covered. •Lectures will focus on clarifying the most important concepts. •You should take notes during class. •Exams will focus mainly on the most important concepts.
- 3. 3 Microbes in Our Lives • Microorganisms are organisms that are too small to be seen with the unaided eye. • The group includes: Bacteria, viruses, fungi, (yeasts and molds), protozoa, and also some algae. The relative size and the nature of these are shown in the Table below. However there are a few exceptions, for example, the fruiting bodies of many fungi such as mushrooms are frequently visible to the naked eye; equally, some algae can grow to meter in length. • Most of these microorganisms are single- celled. Viruses are not even cells, just genetic material surrounded by a protein coat, and are incapable of independent existence. • Are microorganisms the same as germs? Microorganisms are the scientific name for what most people refer to as “Germ”. The term microorganism has a neutral connotation, whereas germ has a negative connotation and generally refers to something capable of causing disease. Microbe Approximate range of sizes Nature of cell Chapter of book Viruses 0.01 - 0.25 µm Acellular 13 Bacteria 0.1 – 10 µm Prokaryote 11 Fungi 2µm - >1m Eukaryote 12 Protozoa 2-1000 µm Eukaryote 12 Algae 1 µm – several meters Eukaryote 12
- 4. 4 Microorganisms: Figure 1.1 a. Bacteria, b. fungus, c. amoeba, d, alga, e, human immunodeficiency virus.
- 5. 5 Indeed, well over 99% of microorganisms contributes to the quality of life: for example: • Microorganisms that help to maintain the balance of chemical elements in natural environment by recycling C, N, S, and phosphorous, and other elements. • Microorganisms that form the foundation of many food chains; produce fermented foods such as vinegar, cheese, yogurt, pickles and bread. • Microorganisms that produce industrial chemicals such as ethyl alcohol, organic acids, enzymes, acetone and many drugs. • Microorganisms that help to break down the remains of all the dies (microorganism of the soil). • Human and many animals depend on the microorganisms in their intestines for digestion and the synthesis of some vitamins that their bodies require, including some B vitamins for metabolism and vitamins K for blood clotting. Are all microorganisms involved in infectious disease?
- 6. 6 • A minority of microorganisms (1%) cause disease in human body. These organisms are overwhelm human system either by sheer force of numbers, or they produce powerful toxins that interfere with body systems. Example, the bacteria that cause botulism and tetanus produce toxins that affect the flow of nerve impulses. • Viruses inflict damage by replicating within tissue cells, thereby causing tissue degeneration. These conditions and many others may lead to infectious disease. In which way are microorganisms detrimental to health? Do microorganisms perform photosynthesis? • Photosynthesis is the chemical process in which energy from the sun is used in the synthesis of carbon-containing compounds such as carbohydrates, which maintain the energy as chemical energy. • We generally consider photosynthesis to be the domain of green plants, but certain species of microorganisms perform photosynthesis. – Such organisms as cyanobacteria (formerly called blue green algae) have the enzyme systems for photosynthesis. As a result of the process they contribute much of oxygen to the environment. – Single celled (unicellular) algae also perform photosynthesis and manufacture the carbohydrates used as energy sources by other organisms. In this way, microorganisms benefit all living things.
- 7. 7 Naming and Classifying Microorganisms NAMING OF MICOROGNISMS • Carolus Linnaeus, a Swedish botanist, developed a hierarchal system of naming the organisms (the system of scientific nomenclature). • Each organism has two names: the genus and specific epithet (species). • Both names are italicized or underlined. • The genus is capitalized and the specific epithet (species) is lower case. • May be descriptive or honor a scientist. Staphylococcus aureus – Staphylo-, describes the clustered arrangement of the cells; coccus, they are shaped like sphere; aureus, the golden color of the colonies. (found on skin) Escherichia coli – Escherichia, honors the discoverer, Theodor Eshcerich, where are its specific epithet, coli, describes the bacterium’s habitat, the large intestine or colon. • After the first use, scientific names may be abbreviated with the first letter of the genus and the specific epithet: For example: Staphylococcus aureus and Esherichia coli are found in the human body. S. aureus is on skin and E. coli, in the large intestine.
- 8. 8 In 1978 Carl Woese devised a system of classification based on the cellular organization of organisms. • It groups all organisms in three domains: – Bacteria (cell walls contain peptidoglycan) – Archaea (Cell walls, if present, lack peptidoglycan) – Eukarya which include: – Protists (slime molds, protozoa, and algae) – Fungi (yeast, mold, and mushrooms) – Plants (flowering plants, etc) – Animal (worms, insects, sponges, vertebrates, etc) CLASSIFYING OF MICOROGNISM The science of classifying and naming organisms called Taxonomy.
- 9. 9 Kingdoms Species no. • Bacteria and Archae 4,800 • Protista 150,000 • Fungi 81,000 • Plantae 270,000 • Animalia 1,000,000 Total 1,501,800 >30 million species thought to exist
- 10. 10 • Prokaryotes • Peptidoglycan cell walls (largely composed of carbohydrates and protein complex) • Binary fission (reproduce by dividing into two equal cells) • For energy, use organic chemicals, inorganic chemicals, or photosynthesis Bacteria • Prokaryotes • Lack peptidoglycan • Live in extreme environments and are divided into three main groups: – Methanogens (produce methane) – Extreme halophiles (live in extremely salt environment) – Extreme thermophiles (live in hot sulfurous water). • Not known to cause disease in human. Archaea
- 11. 11 • Eukaryotes • Chitin cell walls • Use organic chemicals for energy (so they obtain nourishment by absorbing solutions of organic material from their environment, whether soil, seawater, freshwater, or an animal or plant host. • Reproduce sexually or asexually • Unicellular or multicellular – Molds and mushrooms are multicellular consisting of masses of mycelia, which are composed of filaments called hyphae – Yeasts are unicellular Fungi • Eukaryotes • Unicellular • Absorb or ingest organic chemicals • May be motile via pseudopods (ex. Amoebas), cilia, or flagella • Reproduce sexually or asexually Protozoa
- 12. 12 • Eukaryotes • Cellulose cell walls • Use photosynthesis for energy • Are abundant in fresh and salt water, soil. • Produce oxygen and organic compounds which are utilized by other organisms. Thus, they play an important role in the balance of nature. Algae • Acellular • Consist of DNA or RNA core • Core is surrounded by a protein coat • Sometime the coat may be enclosed by an additional layer, a lipid membrane called an envelope • Viruses are replicated only when they are in a living host cell Viruses
- 13. 13 • Eukaryote • Multicellular animals • Most of these animals belong to the two phyla: – Parasitic flatworms – Parasitic round worms • These worms are commonly called helminths Multicellular Animal Parasites The tapeworm found in the intestine of dogs, wolves, and foxes.
- 14. 14 A Brief History of Microbiology • Ancestors of bacteria were the first life on Earth. • The science of microbiology did not start until the invention of microscope in the mid 16th century. • The first microbes were observed in 1664 by Robert Hooke who described the fruiting structures of molds. He was the first to report that living things were composed of little boxes or cells. • 1684 - Antoni van Leeuwenhoek built his own microscope and was the first person to observe live microorganisms, he called them “animalcules.” Among his descriptions were those of protozoa, fungi, and various kinds of bacteria. • In 1858, Rudolf Virchow said cells arise from preexisting cells. • Cell Theory. All living things are composed of cells and come from preexisting cells
- 15. 15 • Scientists debated the theory of spontaneous generation . The theory stated that living organisms arise from nonliving matter. According to spontaneous generation, a “vital force’ Forms life. • In 1765 a scientist named Lazzaro Spellanzani disputed the theory by showing that boiled beef broth (nutrient fluid), would not give rise to microscopic forms of life after being sealed in a flask. Spellanzani’s observations were criticized on the grounds that there was not enough oxygen in the sealed flasks to support microbial life, when Laurent Lavoisier showed the importance of oxygen to life. • In 1858 a scientist called Rudolf Virchow challenged spontaneous generation with the concept of biogenesis: living organisms arise from preexisting life. • In 1865 Louis Pasteur dispelled the myth of Spontaneous Generation with a series of experiments showing that microorganisms are present in air and can contaminate sterile solutions, but air itself does not create microbes. Refer to Fig. 1.3 in the text (SLIDE 17). The Debate Over the Theory of Spontaneous Generation
- 17. 17 Pasteur’s Scientific Method 1. State the problem. 1. Food allowed to stand for some time spoiled. 2. Form a hypothesis after gathering all information on the topic. 2. Observed that structures in the air that resembled microorganisms found in putrefied materials. Hypothesis: Microorganisms found in putrefying food originate from microbes in the air - foods protected from the airbourne microbes should not putrefy. 3. Devise experiments to test the validity of the hypothesis Slide 18 4. Observe results of the experiment Slide 18 5. Interpret the data Slide 18 6. Draw conclusions 6. Food allowed to stand for some time spoiled is due to microorganisms. Microorganisms in these products can be killed by heating Some advancements from these studies and conclusions: 1867—pasteurization – heating food to 55-60oC for a short time. 7. Always carefully design, document and write results of the experiment!!!
- 18. 18 Louis Pasteur refutes the theory of spontaneous generation 3. Devise experiments to test the validity of the hypothesis 4. Observe results of the experiment 5. Interpret the data
- 19. 19 The Golden Age of Microbiology • 1857-1914 • Beginning with Pasteur’s work, discoveries included the relationship between microbes and disease, immunity, and antimicrobial drugs. Also improved the techniques for performing microscopy and culturing microorganisms, and developed vaccines and surgical techniques. Some of the events that occurred: Fermentation • One of the Key steps that established the relationship between microbes and disease, when Pasteur was asked by French merchants to found out why wine and beer become soured, to develop a method that would prevent the spoilage when those beverages were shipped long distances. • Many scientist believed that air converted the sugars in these fluids into alcohol. • Pasteur showed that microorganisms (called yeast) are responsible for fermentation. Fermentation is the conversation of sugar to alcohol, in the absence of air, to make beer and wine. • Different microorganisms called bacteria is responsible for spoilage or souring. • In the presence of air, bacteria use alcohol and produce acetic acid which spoil wine by turning it to vinegar (acetic acid). Fermentation and Pasteurization (1860s)
- 20. 20 Pasteurization • Pasteur demonstrated that these spoilage bacteria could be killed by heat that was not hot enough to evaporate the alcohol in wine. This application of a high heat for a short time is called pasteurization.
- 21. 21 • 1835: Agostino Bassi showed a silkworm disease was caused by a fungus. • 1865: Pasteur believed that another silkworm disease was caused by a protozoan. • 1840s: Ignaz Semmelwise advocated hand washing to prevent transmission of childbirth fever from one obstetrical patient to another. • 1860s: Joseph Lister used a chemical disinfectant to prevent surgical wound infections after looking at Pasteur’s work showing microbes are in the air, can spoil food, and cause animal diseases. • 1876: Robert Koch provided proof that a bacterium causes anthrax and provided the experimental steps. These steps are known today as Koch’s postulates, used to prove that a specific microbe causes a specific disease. The Germ Theory of Disease postulated by Pasteur (microorganisms might cause disease)
- 22. 22 1873-76 Robert Koch (Koch’s Postulates) devises a set of principles for describing the cause and effect relationship between a given microorganism and a specific disease. - Suspected causative agent must be found in every case of the disease and be absent from healthy hosts. - Agent must be isolated and grown outside the host. - When agent in introduced into a healthy, susceptible host, the host must get the disease. - Same agent must be reisolated from diseased experimental host
- 23. 23 • Developed by Robert Koch. – In order to link a specific microbe to a specific process (ex. disease), the organism must first be isolated in pure culture. – Bacterial colonies on solid media can arise from a single bacterial cell and have different characteristic (for example, shape and color, etc.). If the medium is liquid then it is difficult to separate the various species. Solid media (AGAR) 1884 - Walter Hesse first used agar (is a solidifying agent). • Agar is composed of a polysaccharide derived from red algae. • Agar liquifies at 55°C and solidified at lower temperature. Petri dish 1887 - Richard Petri modified Koch’s flat plate technique and designed the Petri dish. Advantages: • can be sterilized separately from the medium (ex. nutrient agar) • cover: prevents contamination • colonies formed on the surface are fully exposed to air and easily manipulated • currently made of glass which is sterilized by dry heat or made of plastic which is sterilized by a gas sterilant. Development of methods for isolating pure cultures on solid media
- 24. 24 • 1796: Edward Jenner inoculated a person with cow-pox virus. • The virus swept through Europe killing 1000’s also wiped out 90% of Nature Americans. • A milkmaid informed Jenner that she could not get smallpox because she had a much milder disease called cow-pox. He inoculate a healthy child with cowpox. The child get mildly sick but recover and never again contracted cowpox or small pox. The process was called vaccination. • The protection from disease provided by vaccination is called immunity. The smallpox is caused by an orthopoxvirus . The virus is transmitted by the respiratory route, it infect many internal organs before their eventual movement into bloodstream leads to infection of the skin and the production of more recognized symptoms such as lesions in the skin. Vaccination
- 25. 25 • After the relationship between microorganisms and disease was established, treatment was required. • Treatment using chemical substances is called chemotherapy. • Chemotherapeutic agents used to treat infectious disease are synthetic drugs. • Chemicals produced naturally by bacteria and fungi that inhibit or kill other microbes are called antibiotics. Examples: – Quinine which obtained from the bark of Cinchona tree was long used to treat malaria (antimalarial agents effective against intraerythrocytic Plasmodium vivax). – synthetic arsenic drug, salvarsan, to treat syphilis (developed by Paul Ehrlich in 1910). Treponema pallidium – Sulfonamides were synthesized (1930s) The Birth of Modern Chemotherapy: Dreams of a “magic bullets”
- 26. 26 • 1928: Alexander Fleming discovered the first antibiotic. • He observed a mold that could inhibit the growth of bacterium. The mold was then identified as Penicillium notatum (produced by a fungus). Fleming named the mold’s active inhibitor penicillin, that killed S. aureus. • In 1940s Penicillin was finally tested clinically and mass produced. The Birth of Modern Chemotherapy
- 27. 27 New braches of microbiology were developed including: – Bacteriology is the study of bacteria. – Mycology is the study of fungi. – Parasitology is the study of protozoa and parasitic worms. – Virology is the study of viruses. • Recombinant DNA Technology (or genetic engineering): using microorganisms that can be genetically engineered to manufacture large amount of human hormones and other urgently needed medical substances. Recombinant DNA technology or genetic engineering involves microbial genetics and molecular biology. • Recent advances in genomics, the study of an organism’s genes, have provided new tools for classifying microorganisms such as bacteria and fungi according to their genetic relationships with other bacteria, fungi, protozoa. Modern Developments in Microbiology
- 28. 28 Chapter 3 Observing Microorganisms Through a Microscope
- 29. 29 Microscopy: The Instruments Units of Measurement • 1 µm = 10-6 m = 10-3 mm • 1 nm = 10-9 m = 10-6 mm • 1000 nm = 1 µm • 0.001 µm = 1 nm
- 30. 30 Types of Microscope • Brightfield Microscope: (called light microscope used for teaching laboratories) Dark objects are visible against a bright background. – To observe various stained specimens and to count microbes. It shows various structures and the outline of the transparent pellicle (external covering). • Darkfield microscopy: Light objects are visible against a dark background. – To examine living microorganisms that are invisible in bright field microscope, do not stain easily, or are distorted by staining. • Phase-contrast microscopy: – To facilitate detailed examination of the internal structures of living specimens.
- 31. 31 • Differential Interference Contrast Microscopy (similar to phase-contrast microscopy) – To provide three dimensional images.. • Fluorescence Microscopy – Uses UV light. – The principle use of fluorescence microscopy is a diagnostic technique called the fluorescence antibody technique. – To rapidly detect and identify microbes in tissue of clinical specimens. • Confocal Microscopy – Uses a laser light.
- 32. 32 Electron Microscopy (Uses electrons instead of light). There are two types: A. Transmission Electron Microscopy (TEM) – To examine viruses or the internal ultrastructure in thin sections of the cell. (usually magnified 10,000-100,000x). B. Scanning Electron Microscopy (SEM) - To study the surface features of cells and viruses (usually magnified 1000-10,000x) Scanned-Probe Microscopy There are two types: A. Scanning Tunneling Microscopy (STM) • Uses thin metal probe that scan an image. • Provide very detailed views of molecules inside cells. B. Atomic Force Microscopy • uses a metal and diamond probe inserted into the specimen. • Provides images of biological molecules in nearly atomic details. Produce three dimensional image.
- 33. 33 Preparation of Specimens for Light Microscopy (any kind of microscope that uses visible light to observe specimens) • Because the cytoplasm of bacteria, fungi, protozoa and other microorganism is transparent, it would be very difficult to observe these microorganisms without the benefit of staining. Staining simply means coloring the microorganism with a dye that emphasizes certain structures. • Prior to staining, a thin film of a solution of microbes placed on a slide. This film which called a smear allowed to dry. • The smear is usually fixed: – to attach the microbes to the slide, – to kill the microbes that may be alive, – to prepare microbes for staining by making the cell wall and membrane more permeable to the dye. This can be done by passing the slide through the flame of a Bunsen burner several times or by covering the slide with methyl alcohol for 1 minute. Stain is applied and then washed off the slide to be ready for microscopic examination. Note: Heat fixation, a process by which heat is briefly applied to the slide to bind microorganisms that may be alive.
- 34. 34 Stains • Are salts composed of a positive and negative ion. The part of the dye molecule which is responsible for the color of the dye is called Chromophore. • Staining that carry a positive charges are called a basic dye, the chromophore is a cation. Examples are methylene blue, crytal violet, safranin, and malachite green. • Staining that carry a negative charges are called a acidic dye, the chromophore is an anion. Examples are eosin, acid fuchsin, nigrosin. • Acidic dyes are not attracted to most types of bacteria because the dye’s negative ions are repelled by the negatively charged bacterial surface, so the stain colors the background instead. Staining the background instead of the cell is called negative staining. It is valuable in the observation of overall cell shapes, sizes, and capsule because the cells are made highly visible against a contrasting dark background.
- 35. 35 To apply acidic or basic dyes, microbiologist use three kinds of staining techniques: Simple, differential, and special. Simple Stains (single step) – The purpose of a simple stain is to highlight the entire microorganism so that cellular shape and basic structures are visible. – Use of a single basic dye such as methylene blue, crystal violet, safranin. – The stain is applied to the fixed smear for a certain length of time and then washed off, and the slide is dried and examined. In this way staining thus takes place. Note: – Negative staining is a single step procedure. – A chemical called mordant may be used to hold the stain or coat a structure (such as flagellum) to make thicker and easier to see after it is stained with a dye. Differential Stains (Multiple-step) The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain. The Gram stain classifies bacteria into gram-positive and gram-negative. The composition of the cell wall is the base.
- 36. 36 Differential Stains: Gram Stain Color of Gram +ve cells Color of Gram –ve cells Primary stain: Crystal violet Purple Purple Mordant: Iodine Purple Purple Decolorizing agent: Alcohol-acetone Purple Colorless Counterstain: Safranin Purple Red The gram stain is regarded as differential stain because it separates, differentiates, bacteria into two separate groups depending on how they react to the procedure. The simple stain procedure, by contrast is not a differential technique because it does not divide bacteria into groups. Fig: Gram staining. The rods and cocci (purple) are Gram +ve, and the vibros (pink) are Gram –ve.
- 37. 37 • The Gram stain classifies bacteria into gram-positive and gram- negative. – Gram-positive bacteria are sensitive to penicillin and are producers of exotoxins. They are susceptible to phenol disinfectants and include organisms such as Bacillus anthrax, Staphylococcus aureus, Streptococcus pyogenes, Clostridium tetani, and Corynebacterium diphtheriae. – The Gram-negative bacteria are usually sensitive to the tetracycline antibiotics and to aminoglycode antibiotics such as gentamicin, neomycin, and kanamycin. Also, they produce endotoxins. They are susceptible to chlorine, iodine, and detergent disinfectants and include such organisms as Salmonella typhi, Shigella sonnei, Bordetella pertussis, and Yersinia pestis.
- 38. 38 • This Acid-Fast stain binds only to bacteria that have a waxy material in their cell wall. • The technique separates species of Mycobacterium from other bacteria, including the two important pathogens M. tuberculosis, the causative agent of tuberculosis, and M. leprae, the causative agent of leprosy. • In the Acid-Fast staining procedure: All bacteria receive the first stain in the procedure, a red stain called carbolfuchsin (the slide is gently heated for several minutes). The slide is cooled and washed with water. When Acid-alcohol is added (decolorizer), all bacteria except Mycobacterium species lose the stain and become transparent. Differential Stains: Acid-Fast Stain - Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. - Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained with a different color basic stain such as methylene blue to see them. Non–acid- fast cells appear blue.
- 39. 39 Special stains are used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal presence of capsules. • Negative staining is useful for capsules. Procedure: mix bacteria in a solution containing India Ink or nigrosin to provide a dark background and then stain with safranin. Capsule do not accept most dyes and thus appear as halos surrounding each stained bacterial cell. • Heat is required to drive a stain into endospores. Procedure: Malachite green, is used as a primary stain. Heat to steaming for 5 min. Wash with water for about 30 seconds. Apply safranin (counterstain). The endospores appear green within red or pink cells. • Flagella staining requires a mordant to make the flagella wide enough to see. Special Stains
- 40. 40 Chapter 4 Functional Anatomy of Prokaryotic and Eukaryotic Cells
- 41. 41 Comparing Prokaryotic and Eukaryotic Cells: Overview – Prokaryote comes from the Greek words for prenucleus. – Eukaryote comes from the Greek words for true nucleus. Prokaryotes and Eukaryotes are chemically similar, in the sense that they both contain nucleic acids, proteins, lipids, and carbohydrates. They use the same kinds of chemical reactions to metabolize food and build protein. • One circular chromosome, not in a membrane • No histones • No organelles • Peptidoglycan cell walls • Binary fission Prokaryote Eukaryote • Paired chromosomes, in nuclear membrane • Histones • Organelles • Polysaccharide cell walls • Mitotic spindle
- 42. 42 The structure of bacterium The major features of Eukaryotic cells
- 43. 43 The Prokaryotic cell • Average size: 0.2-2.0 µm in diameter and from 2- 8 µm in length • Basic shapes: Spherical coccus Rod-shaped bacillus Spiral
- 44. 44 • Unusual shapes – Star-shaped cells ( genus Stella: a recently described genus of Gram –ve bacteria found in fresh water, sewage and soil) – Rectangular cells (genus Haloarcula, a genus of halophilic archae) • Most bacteria are monomorphic which means that they maintain a single shape. • A few bacteria (such as Rhizobuim: a genus of Gram –ve bacteria found in soil and Corynebacterium a genus of Gram +ve bacteria found in vegetable and soil) are pleomorphic which means that they can have many shapes, not just one.
- 45. 45 Arrangements Chains: streptobacilli Pairs: diplococci, Pairs: diplobacilli Clusters: staphylococci Chains: streptococci
- 47. 47 Structure external to the cell wall Glycocalyx Glycocalyx (meaning sugar coat) is general term used for substances that surrounded cells. • Certain species of bacteria are able to form a sticky, gelatinous layer of polysaccharides and proteins known as capsule. A capsule is neatly organized and can find in pathogenic species such as S. pneumoniae. • If the substance is less, more flowering or unorganized, and loosely attached to the cell wall, the Glycocalyx is described as a slime layer.
- 48. 48 Flagella Some prokaryotic cells have flagella, which are long filamentous appendages that propel bacteria. • Made of chains of flagellin. • They are long and thin and cannot be seen by the light microscope unless stained. • Attached to a protein hook • Anchored to the cell wall and membrane by the basal body. • Bacterial cells have four arrangements of flagella: – Monotrichous: single flagellum – Amphitrichous: have a flgellum at each end of the cell. – Lophotrichous: have multiple flagella at the ends of the cells. – Peritrichous: have flagella distributed over the entire body of the cell.
- 49. 49 Bacterial cells can alter the speed and directions of rotation of flagella and thus are capable of various patterns of motility, the ability of an organism to move by itself. • The advantage of motility is that it enables a bacterium to move toward a favorable environment or away from an adverse one. • The movement of bacterium toward or away from a particular stimulus is called taxis. Such movement include: – chemical (chemotaxis) – light (photoctaxis). • Rotate flagella to run or tumble • The flagellar protein called H antigens is useful for distinguishing among serovars (serotypes), or variations within species, of gram-negative bacteria. (e.g., there are at least 50 different H antigen for E. coli ). Motile Cells
- 50. 50 Figure 4.9 Flagella and bacterial motility: A bacterium running and tumbling.
- 51. 51 • Spirochetes are a group of bacteria that have unique structure and motility. • One of the best studied spirochetes is Treponema pallidium (Gram –ve), the causative agent of syphilis. • The movement is by axial filaments or endoflagella, bundles of fibrils that arise at ends of the cell beneath outer sheath and spiral around the cell. • Axial filaments anchored at one end of a cell, have a structure similar to that of flagella. • Rotation causes cell to move Axial Filaments
- 52. 52 Fambriae and Pili Many gram-negative bacteria contain short, hairlike appendages that are shorter, straighter, and thinner than flagella and are used for attachment rather than for motility. • These structure consists of protein called pilin. • They are divided into two types: fimbriae (few to several hundreds per cell) and pili (one to two per cell). – Fimbriae are distributed over the entire surface of the cell and allow attachment for example, the fimbriae of Neisseria gonorrhoeae, the causative agent of gonorrhea, help the microbe colonize mucous membrane, so the bacteria cause the disease. – Pili are used to transfer DNA from one cell to another, that is why sometimes they are called sex pili.
- 53. 53 Cell Wall • Outside cell membrane; not an organelle • Maintains cell shape; • Prevents osmotic lysis (cell bursting) • It contributes to the ability of some species to cause disease • It is the site of action of some antibiotics. • The components of the cell wall is used to differentiate major types of bacteria.
- 54. 54 Peptidoglycan • Peptidoglycan (murein) – (in bacteria only); – Polysaccharides containing N acetylglucosamine & N acetylmuramic acid linked by numerous chain of amino acid (polypeptides: tetrapeptide side chain and peptide cross chain). Antibiotic (penicillin interference)
- 56. 56 Gram-positive cell walls • The cell wall of Gram-positive bacteria consisting of many layers of peptidoglycan forming a thick, rigid structure. • Teichoic acids (consists primarily of an alcohol and phosphate) – Lipoteichoic acid which spans the peptidoglycan layer and is linked to the cytoplasmic membrane. – Wall teichoic acid, which is linked to the peptidoglycan layer. • Teichoic acids – may bind and regulate the movement of cations into and out of the cell. – Play a role in cell growth, preventing extensive wall breakdown and possible cell lysis. – Provide much of wall’s antigenic specificity and thus make it possible to identify bacteria by certain laboratory test. Thick peptidoglycan layer and teichoic acids ( act as attachment sites for viruses such as bacteriophages) Gram stain: purple; so it retains crystal violet color( primary stain)
- 57. 57 Gram-negative cell walls • The cell wall of Gram-negative bacteria consists of a thin peptidoglycan (one or a few layers) and an outer membrane. The peptidoglycan is bonded to a lipoprotein and is in periplasm (fluid- filled space contains a high concentration of degradative enzymes and transport proteins). • No teichoic acids • More susceptible to mechanical breakage. The outer membrane: Lipopolysaccharides (LPS), lipoproteins, phospholipids. • Protect the cell from phagocytes, antibiotics, detergents, digestive enzymes and certain dye. • Dose not provide a barrier to all substance. • Part of the permeability of the outer membrane is due to Porins (proteins) that form channels through membrane to permit the passage of molecules (nucleotides, disaccharides, peptides, amino acids, iron, vitamin). Porins - proteins that allow small molecules to cross membrane.
- 58. 58 LPS provides 2 important characteristics: Polysaccharides portion is composed of sugars called, O polysaccharides, that function as antigens and are useful for distinguishing species. e.g., pathogen E. coli O157:H7. (comparable to teichoic acid in G+ve bacteria). Lipid portion called lipid A, called endotoxin, and is toxic when in host’s blood stream or gastrointestinal track. It causes fever.
- 59. 59 The mechanism is based on differences in the structure of the cell walls of gram-positive and gram-negative bacteria. • Crystal violet (CV) stains both type of cells and the cells becomes purple. • When iodine added, it forms large crystals with the dye that are too large to escape through the cell wall. • Gram-positive – Alcohol dehydrates peptidoglycan – CV-I crystals do not leave • Gram-negative – Alcohol dissolves outer membrane and leaves holes in peptidoglycan – CV-I washes out • Because gram-ve bacteria are colorless after the alcohol wash, the addition of safranin turns the cell pink. Gram Stain Mechanism
- 60. 60 Atypical Cell Walls • Mycoplasmas (prokaryotes) – Lack cell walls – Sterols in plasma membrane which are thought to help protect them from lysis (rupture). • Archaea – Wall-less, or – Walls of pseudomurein (lack NAM and D amino acids)
- 61. 61 Damage to Cell Walls • Lysozyme digests disaccharide in peptidoglycan. This enzyme occurs naturally in some eukaryotic cells and is a constituent of tears, mucus, and saliva. – Lyzozyme can completely destroy the cell wall of gram +ve cell. If lysis do not occur and the cellular contents remained intact, this wall-less cell is called protoplast (spherical and still capable of carrying on metabolism). – Spheroplast is a wall-less gram -ve cell. – Protoplasts and spheroplasts are susceptible to osmotic lysis. • Some genera can loose its cell wall and swell into irregular shapes are called L forms. They may form spontaneously or develop in response to Penicillin which inhibits peptide bridges in peptidoglycan (so inhibit cell wall formation), or lyzozyme.
- 62. 62 Structures Internal to the cell wall • Cytoplasmic or Plasma membrane • This thin barrier, 8 nm thick, controls traffic into and out of the cell. • Like other membranes, the plasma membrane is selectively permeable, allowing some molecules and ions to pass through the membrane, but others prevented from passing through it. • The main macromolecules in membranes are lipids and proteins, but include some carbohydrates and sterols, such as cholesterol. Bacteria have no sterols , except Mycoplasmas - no cell wall and have cholesterol to add rigidity.
- 63. 63 • The most abundant lipids are phospholipids. The phospholipid molecules are arranged in two parallel rows, called phospholipid bilayer. • Each phospholipid molecule contains a polar head, composed of phosphate group and glycerol that is hydrophilic (water loving) and soluble in water, and non polar tails, composed of fatty acids that are hydrophobic (water fearing) and insoluble in water. • Phospholipids and most other membrane constituents are amphipathic molecules. – Amphipathic molecules have both hydrophobic (water fearing) regions and hydrophilic (water loving) regions.
- 64. 64 •Proteins: carriers, channels pores, enzymes •Proteins determine most of the membrane’s specific functions. There are two populations of membrane proteins. Peripheral proteins are not embedded in the lipid bilayer at all. Instead, they are loosely bounded to the surface of the protein, often connected to the other population of membrane proteins. They may function: − as enzymes that catalyze chemical reactions, − as a “scaffold” for support, − and as a mediators of changes in membrane shape during movement. Integral proteins: some penetrate the membrane completely, and are called a transmembrane protein. Some integral proteins are channels that have a pore through which substances enter and exist the cell. The phospholipids and proteins in membranes create a unique physical environment, described by the fluid mosaic model.
- 65. 65 Inclusions With the cytoplasm of prokaryotic cells are several kind s of reserve deposits, known as inclusions. Some inclusions are common to a wide variety of bacteria, whereas others are limited to a small number of species and therefore serve as basis for identification. Cells may accumulate certain nutrients when they are plentiful and use them when the environment is deficient. Metachromatic granules (volutin) Corynebacterium diphtheriae Phosphate reserves Polysaccharide granules Energy reserves Lipid inclusions Energy reserves Sulfur granules Genus Thiobacillus Energy reserves Carboxysomes Ribulose 1,5-diphosphate carboxylase for CO2 fixation Gas vacuoles Found in many aquatic prokaryotes. Rows of several individual gas vesicles, which are hollow cylinders covered by protein. Magnetosomes By several Gram –ve Iron oxide used as magnet, (destroys H2O2)
- 66. 66 Internal Structures in Eukaryotic cells Ribosomes – free in cytoplasm or attached to ER • Endoplasmic Reticulum – Smooth : no ribosomes; makes lipids & membranes – Rough : ribosomes; makes proteins for use outside of cell • Lysosomes: – digestive enzymes • Peroxisomes: – organelle – converts hydrogen peroxide to water + oxygen • Vacuoles: – stores materials – starch, glycogen, fat • Cytoskeleton: – protein fibers to give support, add rigidity, shape to cell External structure: Flagella and Cilia • Few and long projections called Flagella. • Numerous and short projections called cilia. Cell Wall and Glycocalyx • Most cells have cell walls (Algae, fungi, plant) • Some cells, the plasma membrane is covered by glycocalyx.
- 67. 67 Fig 12.8 Two ways in which a sorting signal can be built into a protein. A. The signal resides in a single discrete stretch of amino acid sequence, called a signal sequence, that is exposed in the folded protein. Signal sequence often occur at the end of the polypeptide chain, but the can also located internally. B. A signal patch can be formed by the juxtaposition of amino acid.
- 68. 68 Fig 12.26 Protein import by mitochondria. The N-terminal signal sequence of the precursor protein is recognized by receptors of the TOM complex. The protein is thought to be translocated across both mitochondrial membranes at or near special contact sites. The signal sequences is cleaved off by a signal peptidase in the matrix to form the mature protein. The free signal sequence is rapidly degraded.
- 69. 69 Fig 12.7 Vesicle budding and fusion during vesicular transport. In this process, soluble components (red spots) are transferred from lumen to lumen. The membrane is also transferred and the original orientation of both proteins and lipids in the donor-compartment membrane is preserved in the target-compartment membrane.
- 70. 70 Fig 12.6 Simplified “roadmap” of protein traffic. Proteins can move from one compartment to another by gated transport, transmembrane protein, or vesicular transport. The signals that direct a given protein’s movement through the system, and thereby determine its eventual location in the cell, are contained in each proteins amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol or on a ribosome of the ER and terminates when the final destination is reached. At each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. In principle, a signal could be required for either retention in or exit from a compartment.
- 71. 71 Endospores • When essential nutrients are depleted, certain gram +ve cells, form specialized resting cells called endospores. • Bacillus, Clostridium • Resistant to heat, radiation, acids, drying, chemicals • The process of endospore formation within a vegetative cell takes several hours (8 h) and is known as sporulation. • Germination: Return to vegetative state. Germination occurs, under favorable conditions, in a matter of minutes. • How long can spores survive? It has been reported that 250 million year old spores have been revived These spores were preserved in salt crystals of Permian age.
- 72. 72 • In spore formation, the DNA of the cell and a small amount of cytoplasm gather at one region of the cell. See slide 70. • Depending on the species, the endospore might be located terminally (at one end) such as clostridium species, or centrally inside the vegetative cell such as Bacillus. • Endospores can survive in boiling water for several hours or more whereas most vegetative cells can be killed at temperature 70ºC.
- 73. 73 Sporulation Initiated when nutrients limiting ~200 genes involved 8 h for entire process
- 76. 76 1. Synthesis of ribosomal RNA and ribosomes: Protein synthesis takes place in ribosomes. 1. Each cell contains thousands of ribosomes. 2. Consist of two subunits (large and small) in prokaryotes and eukaryotes, in combination with ribosomal proteins. 3. E. coli 70S model: (nt: nucleotide) • 50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins • 30S subunit = 16S (1,542 nt) + 20 proteins 4. Mammalian 80S model: • 60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins • 40S subunit = 18S (1,900 nt) + 35 proteins
- 77. 77 tRNA required for the ribosome to translate the mRNA.
- 78. 78