Physiology and Biochemistry of Prokaryotes (4th Edition) PDF

The Physiology and Biochemistry of Prokaryotes FOURTH EDITION David White Indiana University James Drummond Indiana University Clay Fuqua Indiana University New York Oxford OXFORD UNIVERSITY PRESS Oxford University Press, Inc., publishes works that further Oxford U...

The Physiology and Biochemistry of Prokaryotes FOURTH EDITION David White Indiana University James Drummond Indiana University Clay Fuqua Indiana University New York Oxford OXFORD UNIVERSITY PRESS Oxford University Press, Inc., publishes works that further Oxford University’s objective of excellence in research, scholarship, and education. Oxford New York Auckland Cape Town Dar es Salaam Hong Kong Karachi Kuala Lumpur Madrid Melbourne Mexico City Nairobi New Delhi Shanghai Taipei Toronto With offices in Argentina Austria Brazil Chile Czech Republic France Greece Guatemala Hungary Italy Japan Poland Portugal Singapore South Korea Switzerland Thailand Turkey Ukraine Vietnam Copyright © 2012, 2007, 2000, 1995 by Oxford University Press, Inc. For titles covered by Section 112 of the US Higher Education Opportunity Act, please visit www.oup.com/us/he for the latest information about pricing and alternate formats. Published by Oxford University Press, Inc. 198 Madison Avenue, New York, New York 10016 http://www.oup.com Oxford is a registered trademark of Oxford University Press All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of Oxford University Press. Library of Congress Cataloging-in-Publication Data White, David, 1936- The physiology and biochemistry of prokaryotes / by David White, James T. Drummond, Clay Fuqua. — 4th ed. p. ; cm. Includes bibliographical references and index. ISBN 978-0-19-539304-0 (alk. paper) I. Drummond, James T. II. Fuqua, Clay. III. Title. [DNLM: 1. Bacteria—metabolism. 2. Archaea—physiology. 3. Prokaryotic Cells—physiology. QW 52] 571.2’93—dc23 2011037790 Printing number: 9 8 7 6 5 4 3 2 1 Printed in the United States of America on acid-free paper  PREFACE Introduction physiology of specific groups of prokaryotes is The fourth edition of The Physiology and emphasized. This pattern of organization lends Biochemistry of Prokaryotes, designed for use itself to the elucidation of general principles of in advanced undergraduate and beginning physiology, metabolism, responses to environ- graduate-level biology courses, provides the mental challenges, and cellular/multicellular most current, authoritative, and relevant presen- development. tation of prokaryotic physiology and biochem- Topics include cellular structure and func- istry. It presents microbial metabolism in the tion, growth and cell division, chromosome context of the chemical and physical problems replication and partitioning of chromosomes, that cells must solve in order to grow. The text membrane bioenergetics and the proton poten- is organized by topic rather than by organism, tial, electron transport , photosynthesis, the reg- therefore helping students understand the gen- ulation of metabolic pathways, bioenergetics in eral principles of physiology and metabolism. the cytosol, central metabolic pathways, RNA This new edition builds in comprehensive cov- and protein synthesis, cell wall and capsule erage of energetics. It also adds broad coverage biosynthesis, inorganic metabolism, C1 metab- of molecular machinery, applied throughout the olism, fermentations, responses to environmen- text to help create a unifying narrative across tal stress, solute transport, protein transport biological principles. Also added is broader cov- and secretion, responses to environmental cues, erage of chromosomes, macromolecular synthe- chemotaxis, photoresponses, aerotaxis, micro- sis, biofilms, and cell–cell communications. bial biofilms, cell–cell communication mecha- The prokaryotes are a diverse assemblage of nisms, and bacterial development. organisms that consists of the Bacteria (also called eubacteria) and the Archaea (also called archae- Distinctive Features bacteria). This text provides an updated descrip- Topical organization fosters understanding tion of the major aspects of the prokaryotes, such of the general principles and concepts of the as cell structure, biochemistry, bacterial devel- biochemistry and physiology of prokaryotes, opment, adaptation to environmental changes, in addition to providing detailed data and con- and signaling interactions between the cells that clusions about specific groups of prokaryotes. occur in bacterial populations such as those living End-of-chapter summaries and study ques- in biofilms. The text highlights signaling mecha- tions help students synthesize material and nisms that allow individual bacterial cells to sense prepare for examinations. and respond to the environment, and also to sig- An extensive references and notes section nal each other so that they can respond as a coop- is available in the chapters to aid further erating population of organisms. research and to provide access to the data that supports the conclusions made in the text. Organization Boxed sections call out topics of special The organization of the text is according to interest, adding historical information about topics rather than organisms, although the earlier discoveries, covering experiments xv xvi preface that established the central tenets of micro- Acknowledgments biology and biochemistry, or exploring in We would like to express gratitude to the many detail especially engaging or important top- individuals named below, and those who remain ics discussed in the text. anonymous, who have read sample chapters and have made helpful suggestions for the fourth New to the Fourth Edition edition. New coauthors: David White is joined by Jennifer Anthony, University of the Sciences in two colleagues from Indiana University: Jim Philadelphia Drummond adds expertise on biological Theodore C. Crusberg, Worcester Polytechnic macromolecules, while Clay Fuqua contrib- Institute utes authority on microbial interaction. Both John E. Gustafson, New Mexico State University are well-known authorities in their respec- Shannon Hinsa-Leasure, Grinnell College tive fields, as well as experienced educators: Adam J. Houlihan, Wagner College New chapters: The fourth edition adds a Carol R. Lauzon, California State University– more detailed account of chromosome rep- East Bay lication, protein and RNA synthesis, and a Paul W. Lepp, Minot State University more complete description of the biology of Robert J. C. McLean, Texas State University– biofilms and of intercellular communication San Marcos between bacteria: Tina Salmassi, California State University–Los Chapter 11, RNA and Protein Synthesis Angeles Chapter 21, Microbial Biofilms—Structured Kathleen Scott, University of South Florida Multicellular Assemblies Timothy Secott, Minnesota State University Chapter 22, Cell–Cell Communication Louis Sherman, Purdue University Mechanisms Teri Shors, University of Wisconsin–Oshkosh All chapters from the previous edition have been Om V. Singh, University of Pittsburgh thoroughly reviewed and revised to incorporate John G. Steiert, Missouri State University the most recent research. Ann M. Stevens, Virginia Tech Sonia M. Tiquia, University of Michigan– New themes: Two new themes have been Dearborn incorporated across the text. A comprehen- Thomas M Walter, Purdue University sive coverage of energetics adds another per- Hwan Youn, California State University–Fresno spective to the physiological and biochemical topics covered. A thorough incorporation of We also thank the reviewers of the third edition: molecular machinery helps create a unifying Carl Bauer, Yves Brun, Jim Drummond, Martin narrative across biological principles. Dworkin, Pat Foster, Heidi Kaplan, Larry Shimkets, and Ashley Williams, as well as the A note on chemical notation of acidic and basic many individuals who helped by reviewing groups: Most of the carboxyl groups are drawn the first two editions. Thanks also to the team as nonionized and the primary amino groups as at Oxford University Press, including Jason nonprotonated. However, at physiological pH Noe, senior editor for the life sciences; Katie these groups are ionized and protonated, respec- Naughton and Caitlin Kleinschmidt, editorial tively. The names of the organic acids indicate assistants; Jason Kramer, marketing manager; that they are ionized (e.g., acetate rather than Frank Mortimer; director of marketing; Patrick acetic acid). Lynch, editorial director; and John Challice, vice president and publisher. The excellent efforts of Ancillary Materials the Oxford University Press production team Instructor’s Resource Companion Website: are gratefully acknowledged: David Bradley, www.oup.com/us/white. All images are production editor; Steven Cestaro, production available to instructors for classroom director; Lisa Grzan, production team leader; use on a password-protected instructor’s Betty Lew, art director; and Brenda Griffing, website. Please contact your Oxford sales copy editor. Much of this edition, like the first representative for access. three editions, was illustrated by Eric J. White.  BRIEF CONTENTS Boxed Material xiii Preface xv Symbols xvii Conversion Factors, Equations, and Units of Energy xix Definitions xxi Chapter 1. Structure and Function 1 Chapter 2. Growth and Cell Division 55 Chapter 3. Chromosome Replication and Partitioning of Chromosomes 77 Chapter 4. Membrane Bioenergetics: The Proton Potential 111 Chapter 5. Electron Transport 146 Chapter 6. Photosynthesis 175 Chapter 7. The Regulation of Metabolic Pathways 199 Chapter 8. Bioenergetics in the Cytosol 207 Chapter 9. Central Metabolic Pathways 222 Chapter 10. Metabolism of Lipids, Nucleotides, Amino Acids, and Hydrocarbons 255 Chapter 11. RNA and Protein Synthesis 281 Chapter 12. Cell Wall and Capsule Biosynthesis 316 Chapter 13. Inorganic Metabolism 335 Chapter 14. C1 Metabolism 358 Chapter 15. Fermentations 383 Chapter 16. Responses to Environmental Stress 403 Chapter 17. Solute Transport 432 v vi brief contents Chapter 18. Protein Transport and Secretion 452 Chapter 19. Responses to Environmental Cues 482 Chapter 20. Chemotaxis, Photoresponses, Aerotaxis 534 Chapter 21. Microbial Biofilms—Structured Multicellular Assemblies 551 Chapter 22. Cell–Cell Communication Mechanisms 566 Chapter 23. Bacterial Development 587 Index 613  CONTENTS Boxed Material xiii Preface xv Symbols xvii Conversion Factors, Equations, and Units of Energy xix Definitions xxi Chapter 1. Structure and Function 1 1.1 Phylogeny 3 1.2 Cell Structure 6 1.3 Summary 44 Study Questions 46 Reference and Notes 47 Chapter 2. Growth and Cell Division 55 2.1 Measurement of Growth 55 2.2 Growth Physiology 57 2.3 Growth Yields 65 2.4 Growth Kinetics 66 2.5 Steady State Growth and Continuous Growth 68 2.6 Cell Division 69 2.7 Summary 71 Study Questions 72 References and Notes 72 Chapter 3. Chromosome Replication and Partitioning of Chromosomes 77 3.1 DNA Replication, Chromosome Separation, and Chromosome Partitioning 77 3.2 Summary 103 Study Questions 104 References and Notes 104 Chapter 4. Membrane Bioenergetics: The Proton Potential 111 4.1 The Chemiosmotic Theory 111 4.2 Electrochemical Energy 112 4.3 The Contributions of the DΨ and the DpH to the Overall Dp in Neutrophiles, Acidophiles, and Alkaliphiles 118 4.4 Ionophores 119 4.5 Measurement of the Dp 120 4.6 Use of the Dp to Do Work 122 4.7 Exergonic Reactions That Generate a Dp 124 4.8 Other Mechanisms for Creating a DΨ or a Dp 129 vii viii contents 4.9 Halorhodopsin, a Light-Driven Chloride Pump 137 4.10 The Dp and ATP Synthesis in Alkaliphiles 138 4.11 Summary 139 Study Questions 140 References and Notes 141 Chapter 5. Electron Transport 146 5.1 Aerobic and Anaerobic Respiration 147 5.2 The Electron Carriers 147 5.3 Organization of the Electron Carriers in Mitochondria 152 5.4 Organization of the Electron Carriers in Bacteria 152 5.5 Coupling Sites 156 5.6 How a Proton Potential Might Be Created at the Coupling Sites: Q Loops, Q Cycles, and Proton Pumps 159 5.7 Patterns of Electron Flow in Individual Bacterial Species 163 5.8 Summary 170 Study Questions 171 References and Notes 171 Chapter 6. Photosynthesis 175 6.1 The Phototrophic Prokaryotes 175 6.2 The Purple Photosynthetic Bacteria 178 6.3 The Green Sulfur Bacteria (Chlorobiaceae) 183 6.4 Cyanobacteria and Chloroplasts 184 6.5 Efficiency of Photosynthesis 186 6.6 Photosynthetic Pigments 187 6.7 The Transfer of Energy from the Light-Harvesting Pigments to the Reaction Center 193 6.8 The Structure of Photosynthetic Membranes in Bacteria 194 6.9 Summary 194 Study Questions 196 References and Notes 196 Chapter 7. The Regulation of Metabolic Pathways 199 7.1 Patterns of Regulation of Metabolic Pathways 199 7.2 Kinetics of Regulatory and Nonregulatory Enzymes 201 7.3 Conformational Changes in Regulatory Enzymes 204 7.4 Regulation by Covalent Modification 204 7.5 Summary 205 Study Questions 205 References and Notes 206 Chapter 8. Bioenergetics in the Cytosol 207 8.1 High-Energy Molecules and Group Transfer Potential 207 8.2 The Central Role of Group Transfer Reactions in Biosynthesis 213 8.3 ATP Synthesis by Substrate-Level Phosphorylation 215 8.4 Summary 220 Study Questions 221 References and Notes 221 Chapter 9. Central Metabolic Pathways 222 9.1 Glycolysis 224 9.2 The Fate of NADH 230 9.3 Why Write NAD+ instead of NAD, and NADH instead of NADH2? 230 9.4 A Modified EMP Pathway in the Hyperthermophilic Archaeon Pyrococcus furiosus 231 9.5 The Pentose Phosphate Pathway 231  contents ix 9.6 The Entner–Doudoroff Pathway 236 9.7 The Oxidation of Pyruvate to Acetyl–CoA: The Pyruvate Dehydrogenase Reaction 238 9.8 The Citric Acid Cycle 241 9.9 Carboxylations That Replenish Oxaloacetate: The Pyruvate and Phosphoenolpyruvate Carboxylases 245 9.10 Modification of the Citric Acid Cycle into a Reductive (Incomplete) Cycle during Fermentative Growth 246 9.11 Chemistry of Some of the Reactions in the Citric Acid Cycle 247 9.12 The Glyoxylate Cycle 248 9.13 Formation of Phosphoenolpyruvate 249 9.14 Formation of Pyruvate from Malate 251 9.15 Summary of the Relationships between the Pathways 251 9.16 Summary 252 Study Questions 253 References and Notes 254 Chapter 10. Metabolism of Lipids, Nucleotides, Amino Acids, and Hydrocarbons 255 10.1 Lipids 255 10.2 Nucleotides 264 10.3 Amino Acids 267 10.4 Aliphatic Hydrocarbons 273 10.5 Summary 275 Study Questions 278 References and Notes 279 Chapter 11. RNA and Protein Synthesis 281 11.1 RNA Synthesis 282 11.2 Protein Synthesis 296 11.3 Summary 310 Study Questions 311 References and Notes 312 Chapter 12. Cell Wall and Capsule Biosynthesis 316 12.1 Peptidoglycan 316 12.2 Lipopolysaccharide 321 12.3 Extracellular Polysaccharide Synthesis and Export in Gram-Negative Bacteria 326 12.4 Levan and Dextran Synthesis 331 12.5 Glycogen Synthesis 332 12.6 Summary 332 Study Questions 332 References and Notes 333 Chapter 13. Inorganic Metabolism 335 13.1 Assimilation of Nitrate and Sulfate 335 13.2 Dissimilation of Nitrate and Sulfate 337 13.3 Nitrogen Fixation 339 13.4 Lithotrophy 344 13.5 Summary 353 Study Questions 354 Reference and Notes 355 Chapter 14. C1 Metabolism 358 14.1 Carbon Dioxide Fixation Systems 358 14.2 Growth on C1 Compounds Other than CO2: The Methylotrophs 374 14.3 Summary 378 x contents Study Questions 380 References and Notes 380 Chapter 15. Fermentations 383 15.1 Oxygen Toxicity 383 15.2 Energy Conservation by Anaerobic Bacteria 384 15.3 Electron Sinks 385 15.4 The Anaerobic Food Chain 386 15.5 How to Balance a Fermentation 387 15.6 Propionate Fermentation via the Acrylate Pathway 388 15.7 Propionate Fermentation via the Succinate–Propionate Pathway 389 15.8 Acetate Fermentation ( Acetogenesis) 391 15.9 Lactate Fermentation 392 15.10 Mixed-Acid and Butanediol Fermentation 394 15.11 Butyrate Fermentation 397 15.12 Ruminococcus albus 400 15.13 Summary 400 Study Questions 401 References and Notes 402 Chapter 16. Responses to Environmental Stress 403 16.1 Maintaining a DpH 403 16.2 Osmotic Pressure and Osmotic Potential 406 16.3 Heat-Shock Response (HSR) 412 16.4 Repairing Damaged DNA 415 16.5 The SOS Response 421 16.6 Oxidative Stress 423 16.7 Summary 425 Study Questions 427 References and Notes 427 Chapter 17. Solute Transport 432 17.1 The Use of Proteoliposomes to Study Solute Transport 432 17.2 Kinetics of Solute Uptake 433 17.3 Energy-Dependent Transport 434 17.4 How to Determine the Source of Energy for Transport 444 17.5 Drug-Export Systems 445 17.6 Bacterial Transport Systems in Summary 446 17.7 Summary 446 Study Questions 448 References and Notes 448 Chapter 18. Protein Transport and Secretion 452 18.1 The Sec System 453 18.2 The Translocation of Membrane-Bound Proteins 457 18.3 The E. coli SRP 459 18.4 Protein Translocation of Folded Proteins: The Tat System 459 18.5 Extracellular Protein Secretion 461 18.6 Folding of Periplasmic Proteins 473 18.7 Summary 474 Study Questions 474 References and Notes 475 Chapter 19. Responses to Environmental Cues 482 19.1 Introduction to Two-Component Signaling Systems 483  contents xi 19.2 Responses by Facultative Anaerobes to Anaerobiosis 488 19.3 Response to Nitrate and Nitrite: The Nar Regulatory System 494 19.4 Response to Nitrogen Supply: The Ntr Regulon 498 19.5 Response to Inorganic Phosphate Supply: The PHO Regulon 503 19.6 Effect of Oxygen and Light on the Expression of Photosynthetic Genes in the Purple Photosynthetic Bacterium Rhodobacter capsulatus 504 19.7 Response to Osmotic Pressure and Temperature: Regulation of Porin Synthesis 506 19.8 Response to Potassium Ion and External Osmolarity: Stimulation of Transcription of the kdpABC Operon by a Two-Component Regulatory System 507 19.9 Acetyl Phosphate Is a Possible Global Signal in Certain Two-component Systems 508 19.10 Response to Carbon Sources: Catabolite Repression, Inducer Expulsion, and Permease Synthesis 510 19.11 Virulence Factors: Synthesis in Response to Temperature, pH, Nutrient Osmolarity, and Quorum Sensors 515 19.12 Summary 522 Study Questions 524 References and Notes 524 Chapter 20. Chemotaxis, Photoresponses, Aerotaxis 534 20.1 Bacteria Measure Changes in Concentration over Time 534 20.2 Tumbling 535 20.3 Adaptation 536 20.4 Proteins Required for Chemotaxis 536 20.5 A Model for Chemotaxis 537 20.6 Mechanism of Repellent Action 541 20.7 Chemotaxis That Does Not Use MCPs: The Phosphotransferase System Is Involved in Chemotaxis toward PTS Sugars 541 20.8 Chemotaxis That Is Not Identical with the Model Proposed for the Enteric Bacteria 541 20.9 Photoresponses 543 20.10 Halobacteria 544 20.11 Photosynthetic Bacteria 544 20.12 Aerotaxis 546 20.13 Summary 546 Study Questions 546 References and Notes 547 Chapter 21. Microbial Biofilms—Structured Multicellular Assemblies 551 21.1 Bacterial Multicellular Structures 551 21.2 Prevalence and Importance of Biofilms 552 21.3 Properties of Biofilms 554 21.4 Progression of Biofilm Formation and Dissolution 557 21.5 Regulation of Biofilm Formation 559 21.6 Inhibition of Biofilm Formation 560 21.7 Evolutionary Processes in Biofilms 561 21.8 Summary 562 Study Questions 563 References and Notes 563 Chapter 22. Cell–Cell Communication Mechanisms 566 22.1 Diversity of Diffusible Signal Molecules Produced by Bacteria 566 22.2 Specific Signaling Systems 566 22.3 Cell–Cell Signaling That Requires Contact 581 xii contents 22.4 Summary 583 Study Questions 583 References and Notes 583 Chapter 23. Bacterial Development 587 23.1 Myxobacteria 587 23.2 Caulobacter Development: Control of DNA Replication and Cell Cycle Genes 598 23.3 Sporulation in Bacillus subtilis 601 23.4 Summary 610 Study Questions 610 References and Note 611 Index 613  1 Structure and Function The prokaryote (procaryote) domains of life are movement on solid surfaces via a form of motil- the Bacteria and the Archaea. Prokaryotes are ity called twitching, for gliding motility among defined as organisms that have no membrane- the myxobaceria, and for mating. Flagella (sing. bound nucleus. (The prefix pro, borrowed from flagellum) are used by single cells to swim in liq- Greek pro, means earlier than or before; kary- uid; they are also used in swarming, a form of ote, borrowed from Greek káryon, means kernel group swimming on moist solid surfaces. or nut.) Besides lacking a nucleus, prokaryotes In addition, it turns out that the poles of are devoid of organelles such as mitochondria, nonspherical bacteria cells are physiologically chloroplasts, and Golgi vesicles. However, as and structurally different from the rest of the you will learn from reading this chapter, their cell, and sometimes the cell poles in the same cell structure is far from simple and reflects the cell differ from each other. This is obvious, for evolution of prokaryotes into quite sophisti- example, when flagella or pili protrude from cated organisms that are remarkably successful one or both cell poles, or when a cell pole is dis- in inhabiting diverse ecological niches. tinguished by having a stalk, as is the case for Despite the absence of organelles comparable Caulobacter, discussed in Chapter 23. In other to those in eukaryotic cells, the activities within cases, the differences between poles are less prokaryotic cells are compartmentalized. For obvious, as shown, for example, in the discus- example, as this chapter points out, compart- sion of the polar localization of the chemotaxis mentalization occurs within multienzyme gran- proteins in Chapter 20. ules that house enzymes for specific metabolic It has become clear that prokaryotes even pathways, in intracellular membranes within have an internal protein cytoskeleton, a prop- the cytosol, within the cell membrane, within erty previously thought to be restricted to a special compartment called the periplasm eukaryotic cells. As described in this chapter in gram-negative bacteria, within the cell wall and in Chapter 2, the cytoskeleton is critical itself, and within various inclusion bodies that for determining and maintaining cell shape as house specific enzymes, storage products, or well as for cell division. For a general review of photosynthetic pigments. As this chapter also subjects covered in this chapter, the reader is points out, the prokaryotes also have special- referred to ref. 1. ized external structures called appendages. Thus, as has been pointed out many times in The most notable appendages are pili and fla- recent years, the prokaryotic cell is not simply gella. Different types of pili (sing., pilus) serve a “bag of enzymes,” in accordance with ear- different functions. Depending upon the type, lier descriptions, but rather, a sophisticated pili are used for adhesion to other cells when entity, that is dynamic both structurally and that becomes necessary for colonization, for physiologically. Furthermore, as Chapter 22 1 2 the physiology and biochemistry of prokaryotes Table 1.1 Major subdivisions of prokaryotes Bacteria and their subdivisions B. Flavobacterium group Purple bacteria (now referred to as the division or Flavobacterium, Cytophaga, Saprospira, phylum Proteobacteria) Flexibacter α subdivision Purple nonsulfur bacteria (Rhodobacter, Planctomyces and relatives Rhodopseudomonas), rhizobacteria, agrobacteria, A. Planctomyces group rickettsiae, Nitrobacter, Thiobacillus (some), Planctomyces, Pasteuria Azospirillum, Caulobacter B. Thermophiles β subdivision Isocystis pallida Rhodocyclus (some), Thiobacillus (some), Chlamydiae Alcaligenes, Bordetella, Spirillum, Nitrosovibrio, Chlamydia psittaci, C. trachomatis Neisseria γ subdivision Radio-resistant micrococci and relatives Enterics (Acinetobacter, Erwinia, Escherichia, A. Deinococcus group Klebsiella, Salmonella, Serratia, Shigella, Deinococcus radiodurans Yersinia), vibrios, fluorescent pseudomonads, B. Thermophiles purple sulfur bacteria, Legionella (some), Thermus aquaticus Azotobacter, Beggiatoa, Thiobacillus (some), Photobacterium, Xanthomonas Green nonsulfur bacteria and relatives δ subdivision A. Chloroflexus group Sulfur and sulfate reducers (Desulfovibrio), Chloroflexus, Herpetosiphon myxobacteria, bdellovibrios B. Thermomicrobium group Thermomicrobium roseum Gram-positive eubacteria A. High (G + C) species Archaea subdivisions Actinomyces, Streptomyces, Actinoplanes, Extreme halophiles Arthrobacter, Micrococcus, Bifidobacterium, Halobacterium, Halococcus morrhuae Frankia, Mycobacterium, Corynebacterium B. Low (G + C) species Methanobacter group Clostridium, Bacillus, Staphylococcus, Methanobacterium, Methanobrevibacter, Streptococcus, mycoplasmas, lactic acid bacteria Methanosphaera stadtmaniae, C. Photosynthetic species Methanothermus fervidus Heliobacterium Methanococcus group D. Species with gram-negative walls Methanococcus Megasphaera, Sporomusa “Methanosarcina” group Cyanobacteria and chloroplasts Methanosarcina barkeri, Methanococcoides Oscillatoria, Nostoc, Synechococcus, Prochloron, methylutens, Methanothrix soehngenii Anabaena, Anacystis, Calothrix Methospirillum group Spirochaetes and relatives Methanospirillum hungatei, Methanomicrobium, A. Spirochaetes Methanogenium, Methanoplanus limicola Spirochaeta, Treponema, Borrelia B. Leptospiras Thermoplasma group Leptospira, Leptonema Thermoplasma acidophilum Green sulfur bacteria Thermococcus group Chlorobium, Chloroherpeton Thermococcus celer Bacteroides; flavobacteria and relatives Extreme thermophiles A. Bacteroides group Sulfolobus, Thermoproteus tenax, Bacteroides, Fusobacterium Desulfurococcus mobilis, Pyrodictium occultum Source: Hodgson, D. A. 1989. Bacterial diversity: the range of interesting things that bacteria do, pp. 4–22. In: Genetics of Bacterial Diversity. D. A. Hopwood and K. F. Chater (Eds.). Academic Press, London. explains, depending upon the growth condi- the investigator to experimentally learn how tions, prokaryotes are capable of living either the organisms are temporally and spatially as single cells, when suspended in liquid, or as organized so that myriad activities can take interacting cells in multicellular populations at place within the cells and between the cells to physical interfaces, such as solid surfaces. The enable survival in the face of environmental study of prokaryotes presents a challenge to challenges.  structure and function 3 1.1 Phylogeny Korarchaeota, has been found in hot environ- Figure 1.1 shows a current phylogeny of life- ments such as hot springs, and much less is forms based upon a comparison of ribosomal known about it, or about archaea belonging to RNA (rRNA) nucleotide sequences. For a more the newly discovered phylum Nanoarchaeota, complete explanation of Fig. 1.1 and how it which has been cultivated from a submarine hot was derived, see Box 1.1. Notice that there are vent. three lines (domains) of evolutionary descent— Bacteria, Eucarya, and Archaea—that diverged Phenotypes in the distant past from a common ancestor.2-4 For a comprehensive description of the Archaea, (The term archaeon may be used to describe the reader is referred to ref. 5. For a review of the particular Archaea.) Archaea differ from bac- cell surface structures of the Archaea, see ref. 6 teria in ribosomal RNA nucleotide sequences, and also Box 1.6. Archaea are found not only in cell chemistry, and in certain physiological in extreme environments such as hot springs, aspects, described in Sections 1.1.1 and 1.2. and alkaline and acid waters, but also in non- Table 1.1 lists examples of prokaryotes in extreme environments that exist in the oceans, the different subdivisions within the domains lakes, soil, sewage, swamps, and the animal Bacteria and Archaea. Notice that the gram- intestinal tract. They are thus widespread in the positive bacteria are a tight grouping. Although environment. there is no single grouping of gram-negative From a morphological point of view (size bacteria, most of the well-known gram-negative and shape), Archaea are similar to the typical bacteria are in the Proteobacteria division. Bacteria lineage. However, they form a group of organisms phylogenetically distinct from 1.1.1 Archaea both bacteria and eukaryotes. The Archaea that Phylogenetic lineages have been best studied commonly manifest one The two major archaeal phyla are the Euryar- of three phenotypes: methanogenic, extremely chaeota and the Crenarchaeota. A third phylum, halophilic, and extremely thermophilic. Fig. 1.1 Phylogenetic relationships among life-forms based upon rRNA sequences. The line lengths are pro- portional to the evolutionary differences. The position of the root in the tree is approximate. The “purple bacteria” are now referred to as the phylum Proteobacteria and, as summarized in Table 1.1, comprise a wide variety of gram-negative organisms including phototrophic, chemoheterotrophic, and chemolithotrophic bacteria. Source: Woese, C. R., and N. R. Pace. 1993. Probing RNA structure, function, and history by com- parative analysis. The RNA World. Cold Spring Harbor Press, Cold Spring Harbor, NY. 4 the physiology and biochemistry of prokaryotes The methanogenic archaea (Euryarchaeota), Their highly unusual metabolism is explained also referred to as methanogens, produce meth- in Chapter 14. Methanogens are obligate anaer- ane. This ability is important for the organisms’ obes that grow in environments such as anaero- survival, because they derive energy from the bic groundwaters, swamps, and sewage, as well process. The methanogens produce methane by as part of the digestive tract (rumen) of animals reducing carbon dioxide to methane or by con- such as cattle and sheep, where they produce verting acetate to carbon dioxide and methane. methane. BOX 1.1 PHYLOGENY The evolutionary relationships among all can explain the differences in nucleotide living organisms have been deduced by sequences. The simplest tree is the one that comparing the ribosomal RNA sequences requires the smallest number of nucleotide of modern organisms. If the structures of changes to evolve the collection of extant the ribosomal RNA molecules from differ- sequences from a postulated ancestral ent living organisms are sufficiently con- sequence. served in certain segments of the molecule, It has been pointed out that it is diffi- conserved sequences and secondary struc- cult to assess the validity of a phylogenetic tures can be aligned to permit comparison tree. There are several reasons for this. For of the differences in base sequences between example, none of the methods accounts for RNA molecules. Researchers analyzed the the possibility that multiple changes may number of positions that differ between have taken place at any single position. pairs of sequences, as well as other features This shortcoming is likely to result in an (e.g., which positions vary, the number of underestimate of the distances. changes that have been made in going from Underestimation of the distances, in one sequence to another). The number of turn, might bring two distantly related lin- nucleotide differences between homolo- eages much closer, and in fact might make gous sequences is used to calculate the evo- them appear to be specifically related to lutionary distance between the organisms each other when they are not. Other ambi- and to construct a phylogenetic tree. Most guities in phylogenetic trees might arise if recently published phylogenetic trees are different positions in the sequence align- based upon 16S rRNA sequences. ments have changed at very different rates. There are two major ways in which phy- Furthermore, the branching pattern itself logenetic trees are constructed from the can differ if a different algorithm is used, nucleotide differences. In the evolutionary even with the same sequences. The out- distance method, the number of nucleotide come is that there exist a variety of phyloge- differences is used as a measure of the evo- netic trees, and no single published tree has lutionary distance between the organisms. been accepted as perfectly representing the The second method, the maximum par- 4 billion years or so of bacterial evolution. simony method, is more complicated. It Despite these problems, there is consistency takes into account not only the nucleotide in the trees that are based upon 16S rRNA differences but also the positions at which sequences, and it is believed that most of the the differences occur and the nature of the phylogenetic trees derived from sequenc- differences. Parsimony means “less is bet- ing bacterial 16S rRNA are at least plau- ter” or “stinginess,” going to extreme, for sible. However, the assumption remains the sake of economy; the method attempts unproven that the relationships between to find the simplest evolutionary tree that the 16S rRNAs represent the phylogenies  structure and function 5 of the organisms rather than simply the M. Dworkin, W. Harder, and K.-H. Schleifer phylogeny of a given 16S rRNA. Because of (Eds.). Springer-Verlag, Berlin. this, it is imperative that the relationships 2. Stackebrandt, E. 1991. Unifying phylogeny between the different organisms be tested and phenotypic diversity, pp. 19–47. In: The by means of other characters (e.g., other Prokaryotes, Vol. I. A, Balows, H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer appropriate molecules besides the 16S (Eds.). Springer-Verlag, Berlin. rRNA, and various phenotypic character- 3. Felsenstein, J. 1982. Numerical methods istics of the organisms). See refs 1–4. for inferring evolutionary trees. Q. Rev. Biol. 57:379–404. REFERENCES 4. Boucher, Y., C. J. Douady, R. T. Papke, D. A. Walsh, M. E. R. Boudreau, C. I. Nesbo, R. 1. Woese, C. R. 1991. Prokaryotic systemat- J. Case, and W. F. Doolittle. 2003. Lateral gene ics: the evolution of a science, pp. 3–11. In: The transfer and the origins of prokaryotic groups. Prokaryotes, Vol. I. A, Balows, H. G. Trüper, Annu. Rev. Gen. 37:283–328. The extremely halophilic archaea (also in acceptor to oxidize hydrogen gas. These include the kingdom Euryarchaeota) require very Thermoproteus, Pyrobaculum, Pyrodictium, high sodium chloride concentrations (at least and Archaeoglobus. [Pyrobaculum and 3–5 M) for growth. They grow in salt lakes Pyrodictium use elemental sulfur (S°) as an elec- and solar evaporation ponds. The halophilic tron acceptor during autotrophic growth, i.e., archaea have light-driven proton and chloride growth on CO2 as the carbon source, whereas pumps called bacteriorhodopsin and halorho- Archaeglobus uses S2O32−.] Archaea belong- dopsin, respectively, Bacteriorhodopsin creates ing to the genus Pyrodictium have the highest an electrochemical proton gradient that is used growth temperature known, with the ability to to drive ATP synthesis, and halorhodopsin is grow at 110 °C. A few of the sulfur-oxidizing used to accumulate chloride intracellularly to archaea are acidophiles, growing in hot sulfuric maintain osmotic stability. These pumps are acid at pH values as low as 1.0. They are called described in more detail in Chapter 4 (Sections thermoacidophiles, indicating that they grow 4.8.4 and 4.9). optimally in hot acid. For example, Sulfolobus The extremely thermophilic archaea grow grows at pH values of 1 to 5 and at tempera- in thermophilic environments (generally tures up to 90 °C in hot sulfur springs, where it 55–100 °C).7 Some of these have an optimal oxidizes H2S (hydrogen sulfide) or So to H2SO4 growth temperature near the boiling point (sulfuric acid). Although most of the extreme of water! Many use inorganic sulfur either thermophiles are obligately sulfur dependent, as an electron donor or as an electron accep- some are facultative. For example, Sulfolobus tor in energy-yielding oxidation–reduction can be grown heterotrophically on organic (redox) reactions. [The pathways for sulfate carbon and O2 as well as autotrophically on reduction and sulfur oxidation are described H2S or S°, O2, and CO2. Interestingly, some of in Sections 13.2.2 and 13.4.1 (see subsection the sulfur-dependent archaea have metabolic entitled Sulfur-oxidizing prokaryotes), respec- pathways for sugar degradation not found tively.] Some of these archaea, which are also among the Bacteria. These are mentioned in called sulfur dependent, oxidize inorganic sul- Section 9.4. fur compounds such as elemental sulfur and Recently, a new genus of thermoacido- sulfide, by using oxygen as the electron accep- philic archaea was discovered.8 Picrophilus, a tor, deriving ATP from the process. Examples member of the kingdom Euryarchaeota, order include Sulfolobus and Acidianus. Other Thermoplasmales, is an obligately aerobic, extreme thermophiles are anaerobes that use heterotrophic archaeon that grows at tempera- elemental sulfur or thiosulfate as the electron tures between 45 and 65 °C at a pH of 0 with 6 the physiology and biochemistry of prokaryotes an optimum pH of 0.7. It was isolated from tor, EF-2, that can be ADP-ribosylated by hot geothermally heated acidic solfataras9 in diphtheria toxin. In contrast, bacteria use Japan. Despite the sulfur content of the habitat, formylmethionine to initiate protein synthe- Picrophilus is not sulfur dependent. sis, and their EF-2 is not sensitive to diphthe- ria toxin. However, the archaeal ribosomes Comparison of domains Archaea, are similar to bacterial ribosomes in having Bacteria, and Eucarya an overall size described as 70S; that is, they Bacteria can be distinguished from Archaea on sediment in a centrifugal field of a velocity of the basis of the following structural and physi- 70 svedberg units. ological differences. 6. The methanogenic archaea have several coenzymes that are unique to Archaea. The 1. Whereas the lipids in the membranes of bac- coenzymes are used in the pathway for the teria and eukaryotes are fatty acids, which reduction of carbon dioxide to methane and are esterlinked to glycerol (see later: Figs. in the synthesis of acetyl–CoA from H2 and 1.16 and 10.5), the archaeal lipids are meth- CO2. These coenzymes and their biochemi- yl-branched, isoprenoid alcohols, ether- cal roles are described in Section 14.1.5. linked to glycerol (Fig. 1.18).10 Archaeal membranes are discussed in Section 1.2.5, In addition to the differences just listed, there and the biosynthesis of archaeal lipids is dis- are several similarities between archaeal and cussed in Section 10.1.3. eukaryotic RNA polymerase, and archaeal and 2. Archaea lack peptidoglycan, a universal eukaryotic ribosomes (Table 1.2). component of bacterial cell walls. The cell walls of some archaea contain pseudopepti- 1.2 Cell Structure doglycan, a component absent from bacte- There are well-known differences between rial cell walls (Section 1.2.3). Archaea and Bacteria with respect to cell walls, 3. Archaea contain histones that resemble cell membranes, and flagella, and these will be eukaryal histones and bind archaeal DNA into compact structures resembling eukaryal pointed out. The structure and function of nucleosomes.11,12 the major cell components will be described, beginning with cellular appendages (pili and 4. The archaeal RNA polymerase differs from flagella) and working our way into the interior bacterial RNA polymerase by having 8 to of the cell. 10 subunits, rather than 4 subunits, and by not being sensitive to the antibiotic rifampi- cin. The difference in sensitivity to rifampi- 1.2.1 Appendages cin reflects differences in the proteins of the Numerous appendages, each designed for a spe- RNA polymerase. In fact, the archaeal RNA cific task, can extend from bacterial surfaces. polymerase resembles the eukaryotic RNA We shall describe two classes: flagella and pili. polymerase, which also has many subunits The flagella are used by single cells for swim- (10–12) and is not sensitive to rifampicin. ming in liquid and swarming, a type of group 5. Some protein components of the archaeal swimming on moist solid surfaces. Depending protein synthesis machinery differ from upon the type, the pili (sometimes called fim- those found in the bacteria. Archaeal ribo- briae) are used for adhesion to surfaces, includ- somes are not sensitive to certain inhibitors ing adhesion to the surfaces of animal cells; in of bacterial ribosomes (e.g., erythromycin, certain cases, described in Box 1.2, they serve streptomycin, chloramphenicol, and tetracy- for movement on solid surfaces (type IV pili), cline). These differences in sensitivity to anti- and for conjugal transfer or mating (sex pili). biotics reflect differences in the ribosomal proteins. In this respect, archaeal ribosomes Flagella resemble cytosolic ribosomes from eukary- For a review of all known prokaryotic motil- otic cells. Other resemblances to eukaryotic ity structures, see ref. 13. For a review of bac- ribosomes are the use of methionine rather terial flagella, see refs. 14 and 15. Swimming than formylmethionine to initiate protein bacteria have one or more flagella, which are synthesis, and a translation elongation fac- organelles of locomotion that protrude from  structure and function 7 Table 1.2 Comparison between Bacteria, Archaea, and Eucarya Characteristic Bacteria Archaea Eucarya Peptidoglycan Yes No No Lipids Ester linked Ether linked Ester linked Ribosomes 70S 70S 80S Initiator tRNA Formylmethionine Methionine Methionine Introns in tRNA No Yes Yes Ribosomes sensitive to No Yes Yes diphtheria toxin RNA polymerase One (5 subunits) Several (8–12 Three (12–14 subunits each) subunits each) Ribosomes sensitive to chloramphenicol, streptomycin, kanamycin Yes No No the cell surface. Flagella allow single cells to coli and S. typhimurium. Archaeal flagella are swim in liquid and can also be used for swarm- somewhat different, as will be described. ing on a solid surface. Swarming, a means of group swimming by which bacterial colonies 2. General structure can spread in limiting liquid, is discussed later The proteins are named after genes found in in this section. Escherichia coli and Salmonella typhimurium.17 Mutations in the mot (motility) genes result in 1. An overview paralyzed flagella, and as we shall see later, the The bacterial flagellum is a stiff, helical fila- Mot proteins provide the torque that causes ment approximately 20 nm in diameter that the flagellum to rotate. The flagellum consists rotates like a screw-type propeller; it can be of a basal body, a hook, a filament, a motor, a either a left-handed helix or a right-handed switch, an export apparatus, capping proteins, helix, depending upon the species. The bac- and junction proteins (typically referred to as terial flagellum is unrelated to the eukary- fla, fli, flg, flh, and flb). We discuss some of these otic flagellum in composition, structure, and now. mechanism of action. (For a description of eukaryotic flagella and cilia, see note 16 in The basal body. Examine Fig. 1.2. At the base the section References and Notes, at the end of the flagellum there is a basal body embedded of the chapter.) The word flagellum, which in the membrane. In gram-negative bacteria, the means whip in Latin, was first used to describe basal body consists of three stacked rings (C, the bacterial filament in 1852. The bacterial M, and S rings) and a central rod. The M and S organelle, however, is more like a stiff propel- rings are actually joined as a single ring called ler than like a whip, which is a flexible rod. the MS ring, made from different domains of When the bacterial flagellum rotates, a helical the FliF protein. This scheme is supported by wave travels from the proximal to the distal electron microscopic evidence. The term “MS” end (outward from the cell), and as a con- indicates the ring’s location: membrane and sequence the cell is pushed forward as illus- supramembranous. The P ring (FlgI protein) trated later (see Fig. 20.2). The flagellum is a and the L ring (FlgH protein) are also named very complex machine driven by a tiny rotat- according to their location: peptidoglycan and ing motor embedded in the membrane. Both lipopolysaccharide. The L ring in S. typhimu- its structure and the mechanism of its motility rium has been shown to be a lipoprotein.18 will be described. The flagella that are studied Presumably the lipid portion helps anchor the in most detail are those of Escherichia coli and protein in the lipid regions of the outer enve- Salmonella typhimurium, and we will begin lope. A central rod, made from the FlgB, FliC, with a discussion of their flagella. The flagella and FlgF proteins, passes through the rings and of other bacteria, except for the spirochaetes, leads to the hook portion (H) of the flagellum are similar in general structure to those of E. on the outside of the cell. The outermost two 8 the physiology and biochemistry of prokaryotes BOX 1.2 NONFLAGELLAR MOTILITY Many bacteria do not have flagella and yet short, intermittent jerks (hence the name are capable of motility. Depending upon the “twitching”) of the cells. bacterium, the motility may take place on a solid surface (twitching or gliding) or in liq- uid (swimming). The mechanistic bases for Pore complexes, slime extrusion, these movements differ and are related to and gliding motility specific cell structure features present in the particular cell. See refs. 1–4. Gliding motility by filamentous cyanobac- teria is apparently due to the secretion of slime through pores near the septa that Type IV pili, twitching motility, separate the cells in the filament The model and gliding motility proposes that as a consequence of the slime secretion the filament of cells is pushed for- Type IV pili are fibrillar protein append- ward through the so-called junction pores. ages located at either pole of certain gram-negative pathogenic bacteria, such as Pseudomonas aeruginosa, Bacteroides Gliding in the green fluorescent ureolyticus, Legionella pneumophila, bacteria (GFP) group Neisseria meningitidis, Ralstonia solan- acearum, and Vibrio cholerae, that infect Bacteria in the GFP group glide rapidly on animals, plants, and fungi. They are also solid surfaces. Some species actually rotate found in many nonpathogenic gram- as they glide. The cells suspended in liquid negative bacteria such as Myxococcus can propel adsorbed latex beads in multiple xanthus, which is a social bacterium that paths around the cell, indicating movement constructs multicellular fruiting bodies by cell surface molecules, perhaps poly- (discussed in Chapter 23), and in the uni- mers or fibers, to which the beads attach. cellular cyanobacterium Synechocystis. Perhaps the same molecules are part of the Type IV pili comprise the mechanosystem machinery that propels the cells when they for twitching motility, which is a form of are on a solid surface. gliding that takes place on moist surfaces. Twitching motility in M. xanthus is called REFERENCES social motility (S-motility). It is a form of smooth motility of cells gliding as groups. 1. McBride, M. J. 2001. Bacterial gliding motil- ity: multiple mechanisms for cell movement over Surfaces upon which twitching motility surfaces. Annu. Rev. Microbiol. 55:49–75. can occur include agar, epithelial tissue, plant tissue, and various other surfaces 2. Mattick, J. S. 2002. Type IV and twitching. Annu. Rev. Microbiol. 56:289–314. such as glass, plastics, and metal. Motility on such surfaces is important for rapid 3. Semmler, A. B., C. B. Whitchurch, and J. S. Mattick. 1999. A re-examination of twitch- colonization, for the formation of bio- ing in Pseudomonas aeruginosa. Microbiology films (discussed in Chapter 21), and for the 145:2863–2873. building of fruiting bodies by myxobacte- 4. McBride, M. J., T. F. Braun, and J. L. Brust. ria (Chapter 23). 2003. Flavobacterium johnsoniae GldH is a Twitching motility in P. aeruginosa and lipoprotein that is required for gliding and chitin several other bacteria is characterized by utilization. J. Bacteriol. 185:6648  structure and function 9 C ring/switch Fig. 1.2 Bacterial flagellum in a gram-negative envelope (based on Salmonella). The basal body itself, to which the hook–flagellum assembly is attached, is 22.5 nm × 24 nm in size and is composed of four rings, L, P, S, and M, connected by a central rod. The M ring is embedded in the cell membrane and the S ring appears to lie on the surface of the membrane. The S and M rings are actually one ring, the MS ring. The P ring may be in the peptidoglycan layer, and the L ring seems to be in the outer membrane. The P and L rings may act as bush- ings that allow the central rod to turn. Gram-positive bacteria have similar flagella but lack the L and P rings. The MotA and MotB proteins form complexes (Mot) that couple the influx of protons to the rotation of the rotor. The rotor consists of the MS ring with the FliG protein attached to its cytoplasmic surface and the C ring attached to the cytoplasmic surface of the MS ring. The switch complex consists of three peripheral membrane proteins, FliG (also part of the rotor), FliM, and FliN, which probably are closely apposed to the cytoplasmic face of the M ring. Not shown are hook accessory or adaptor proteins (HAP1 and HAP3) between the hook and filament, and a protein cap (HAP2) on the end of the filament. The flagellum is assembled from the proxi- mal to the distal end, with the filament being assembled last. It appears that the HAP1 and HAP3 proteins are required for the proper assembly of the filament onto the hook. Abbreviations: OM, outer membrane; pg, peptidoglycan; CM, cell membrane; R, central rod; MOT, MotA and MotB; H, hook; F, filament; L, L-ring; P, P-ring. rings (P and L) may act as bushings, allowing and surround the MS ring. The number of such the central rod to rotate in the peptidoglycan particles in various bacteria has been reported to and outer membrane. Gram-positive bacteria be 12 to 16. Such arrays of particles surround- do not have P and L rings. The basal body trans- ing the MS ring were seen in electron micro- mits torque to the hook and filament, causing graphs of freeze-fractured cell membranes, but them to rotate. 14 not when either MotA or MotB was missing.20 It has been suggested that the large periplasmic The motor. One of the fascinating components domain of MotB is attached noncovalently to of the flagellar apparatus is the tiny motor, the peptidoglycan, explaining why the MotA/ approximately 50 nm in diameter, that lies at MotB complex does not rotate when the motor the base of the flagellum and causes it to rotate. turns. For a review of the flagellar motor, see ref. 19. In agreement with the conclusion that MotA The motor consists of two parts: a nonrotating and MotB are part of the motor, mutations in part called the stator (made of the Mot pro- the motA and motB genes result in paralyzed − teins) and a rotating part called the rotor, which flagella (Mot phenotype). The MotA/MotB includes the FliG proteins that transmit torque complexes are believed to conduct protons from to the MS ring. outside the cell to inside, across the membrane, The stator consists of two different proteins, and to use this proton movement to provide the MotA and MotB, indicated as Mot in Fig. 1.2. torque to rotate the rotor. Extreme alkaliphiles These exist as particles of multiple complexes, and some marine bacteria use a sodium ion cur- (MotA)4(MotB)2, that span the cell membrane rent. This is reviewed in ref. 21. (As discussed in 10 the physiology and biochemistry of prokaryotes Chapter 4, bacteria use a proton current across chemotaxis (Chapter. 20), binds to FliM. (For the cell membrane to do other kinds of work, more information about these proteins, includ- e.g., ATP synthesis, in addition to rotating fla- ing their function, see note 28.) gella.) For an explanation of the conclusion that The hook and the HAP proteins. The central MotA and MotB form a complex, see note 22. flagellar rod is attached to an external curved Membrane vesicles prepared from strains syn- flexible hook made of multiple copies of a spe- thesizing wild-type MotA were more permeable cial protein called the hook protein, FlgE, the to protons than were vesicles prepared from product of the flgE gene. There are also two strains synthesizing mutant MotA, indicating hook-associated proteins: HAP1, also called that MotA is likely to be part of a proton chan- FlgK (the flgK gene), and HAP3, also called nel. 23 Some of the evidence in support of the FlgL (the flgL gene). These proteins are nec- conclusion that the Mot proteins form a com- essary to form the junction between the hook plex that is a transmembrane proton channel is and the filament. Mutants that lack these HAPs in ref. 24. secrete flagellin into the medium. According to How does passage of protons through the the model, the hook subunits, consisting of the Mot complex generate torque? One model pro- FlgE protein, fill the C ring and then are trans- poses that as protons pass through the MotA/ ferred en masse to the growing hook through MotB complexes, conformational changes the export apparatus, described later, which is occur in the complexes that drive stepwise rota- in the middle of the C ring. (See later subsec- tion of the attached rotor. For a model of how tion entitled The export apparatus for flagellar this might occur, consult ref. 25. components.) What is the rotor? Sometimes the C ring is referred to as the rotor, but others consider the The filament and the capping proteins. Attached rotor to be the MS and C rings together. (See to the hook is a rigid, hollow, helical filament note 26 for references.) An essential rotor com- that, along with the hook, protrudes from the ponent is FliG. FliG interacts with the Mot pro- cell. When it rotates, it acts as a propeller and teins and transmits torque generated by the Mot pushes the cell forward. The protein in the fila- complex to rotate the rotor. The FliG proteins ment is called flagellin (which in Escherichia and are part of the C ring and attach the C ring to the Salmonella is known as FliC) and is present in MS ring. As explained next, the C ring functions thousands of copies. Flagellin is not identical in as a switch that reverses the direction of rota- all bacteria. For example, the protein can vary tion of the rotor. in size from 20 kDa to 65 kDa depending upon the bacterial species. Furthermore, although The switch. In certain bacteria, such as E. coli there is homology between the C-terminal and and S. typhimurium, the motor spontaneously N-terminal ends of most flagellins, the central changes its direction of rotation periodically. part can vary considerably and is distinguished The frequency of switching is influenced by immunologically in different bacteria. In some attractants and repellents that the bacterium cases, there is no homology at all. For example, might encounter, and as such, is important for nucleotide-derived amino acid sequences for chemotaxis, described in Chapter 20. Mutations the flagellins from Sinorhizobium meliloti show that cause failure to change flagellar rotation almost no relationship to flagellins from E. coli, map in three genes, fliG, fliM, and fliN, which S. typhimurium, or Bacillus subtilis, but are code for the complex of switch proteins (FliG, 60% similar to the N and C termini of flagellin FliM, and FliN).27 It appears that the three from Caulobacter crescentus.29 Finally, a third switch proteins form a complex of peripheral HAP, HAP2, also called FliD (the fliD gene), membrane proteins closely associated with the caps the flagellar filament. cytoplasmic side of the MS ring. In particular, FliG seems to be bound to the MS ring itself; it 3. Brief summary of assembly is also bound to FliM and FliN. The latter two of the flagellum proteins form the cup-shaped C ring, located The flagellum and its associated components directly beneath the basal body attached to the are assembled in a precise order, beginning with MS ring. The regulator protein CheY, which is the components closest to the cell membrane. important in switching flagellar rotation during The first components assembled, probably in a  structure and function 11 coordinated fashion, are the MS and C rings. How can growth at the tip of the filament This step is followed by construction of the be demonstrated? Growth at the tip has been transport apparatus for export of the remain- visualized by the use of fluorophenylanine, der of the flagellar components through a chan- a phenylalanine analogue, or radioisotopes. nel in the center of the MS ring. Then the rod Incorporation of fluorophenylalanine by is assembled, followed by the hook. The hook Salmonella resulted in curly flagella that had is not completed until the P and L rings have only half the normal wavelength. When the been assembled. When the hook is complete, analogue was introduced to bacteria that had the flagellin monomers are exported and the partially synthesized flagella, the completed fla- filament is assembled. MotA and MotB are gella were normal at the ends next to the cell assembled late in the assembly process, to coin- and curly at the distal ends, implying that the cide with the appearance of the filament. As fluorophenylalanine was incorporated at the described next, the timing of assembly of the tips during flagellar growth.32 When Bacillus components is at least partially controlled at the flagella were sheared off the cells and allowed level of the transcription for the genes encoding to regenerate for 40 min before the addition of these components. radioactive amino acid ([3H]leucine), radioau- tography showed that all the radioactivity was 4. Brief summary of gene expression control at the distal region of the completed flagella.33 assembly regulating the timing and assembly The flagellin monomers are presumed to be of flagellum and its associated components transported through the central channel in the As we have seen, the flagellum and its com- filament to the tip, where they are assembled. ponents are assembled in a precise order. For more information about the biosynthesis of Interestingly, the genes for the flagellum and flagella, consult the review by Macnab.14 its associated components are expressed in the order that the gene products are used. How is The export apparatus for flagellar components. this done? In E. coli and Salmonella the genes are In the center of the C ring on the cytoplasmic in three groups that are sequentially expressed. side is a knob composed of several Flh and Fli The groups are denoted as belonging to class 1, proteins (FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, 2, or 3. The genes in class 1 (flhDC) comprise FliR). The ATPase FliI is negatively regulated an operon that encodes transcriptional activa- by FliH, and its activity is required for flagellar tors of the class 2 genes. Class 2 gene products assembly. (See note 34.) FliI and FliH are solu- comprise the basal body and hook, as well as a ble proteins, as is a chaperone protein, FliJ, and sigma factor (FliA) required for transcription of the others are integral membrane proteins. The class 3 genes. Class 3 genes are required for the knob is the transport apparatus used for export synthesis of the flagellin monomers (FliC) and of all the flagellar components (basal body, rod, the torque-generating unit (MotA and MotB). hook, filament) through a central channel in the MS ring, to be assembled beyond the cytoplas- 5. Growth of the flagellum mic membrane. One of the exported proteins Although one might expect new flagellin sub- is a muramidase called FlgJ, and it presumably units to be added to the growing filament at the functions to allow the nascent rod to penetrate base next to the cell surface, this is not the case. the peptidoglycan. The transport system for the The flagellin subunits actually travel through a bulk of flagellar materials is a flagellum-specific hollow core in the basal body, hook, and fila- type III export system. (See the description of ment and are added at the distal tip. The cap- type III export systems in Chapter 18) The P and ping protein (HAP2) is important in this regard L rings are assembled using the Sec system of because it prevents the flagellin monomers from transport, described in Section 18.1. leaking out into the medium. Somehow, the cap- ping protein must be able to move out from the 6. Differences in flagellar structure growing filament to allow extension of the fila- Although the basic structure of flagella is simi- ment, neither becoming detached nor allowing lar in all bacteria thus far studied, there are flagellin subunits to leak out into the medium. important species-dependent differences. For Models by which this may be done have been example, some bacteria have sheathed flagella, suggested.30, 31 whereas others do not. In some species (e.g., 12 the physiology and biochemistry of prokaryotes Vibrio cholerae) the sheath contains lipopoly- brane, and closely surrounded by an outer lipid saccharide and appears to be an extension of the bilayer membrane. The spirochaete flagella are outer membrane.35 In spirochaetes the sheath is called axial filaments. For a review, see ref. 40. made of protein. (See later.) For a list of diseases caused by spirochaetes, see Bacteria also vary with respect to the number note 41. of different flagellins in the filaments. Depending Most spirochaete cells are helically coiled and upon the species, there may be only one type have two or more flagella (some have 30 or 40 or of flagellin, or two or more different flagellins more) inserted subterminally near each cell pole in the same filament. For example, E. coli has and extending along the cell body. Some species one flagellin; Bacillus pumilis has two differ- have a flat meandering waveform, rather than ent flagellins, and C. crescentus has three and being helically coiled. The number of flagella S. meliloti has four.36–38 The data for B. pumilis inserted at opposite poles is the same. The fla- and C. crescentus support the conclusion that gella are usually more than half the length of the the different flagellins reside in the same fila- cell and overlap in the middle. The spirochaete ment. It has been proposed that the filaments of flagellum is often surrounded by a proteina- S. meliloti are composed of heterodimers of the ceous sheath. Borrelia burgdorferi, the spiro- two different flagellins. The flagella of many bac- chaete that causes Lyme disease, is somewhat teria that have been studied (e.g., E. coli) show different. Its axial filaments are not surrounded a smooth surface under electron microscopy by proteinaceous sheaths. Furthermore, this and are called plain filaments. However certain organism has a planar waveform shape rather bacteria (e.g., Rhizobium lupini, S. meliloti) than the corkscrew type.42 The rotation of the have “complex” filaments with obvious helical rigid periplasmic flagella between the outer patterns of ridges and grooves on the surface. membrane sheath and the cell cylinder is thought Flagella with plain filaments rotate either clock- to move the cell by propagating a helical wave wise or counterclockwise, whereas flagella with backward down the length of the highly flexible complex filaments rotate only clockwise, with cell cylinder, propelling the cell forward, which intermittent stops or slowing of rotation. It is allows the cells to “corkscrew” through viscous thought that because complex filaments are media, such as mud, sediments, and connective brittle, they are more rigid than plain filaments, tissue in animals. (For a more detailed model, hence are better suited for propelling bacteria see note 43.) There are five antigenically related in viscous media such as the gelatinous matrix flagellins in the axial filaments, but it is not through which R. lupini and S. meliloti must known whether individual filaments contain swim to infect root hairs of leguminous plants.39 more than one type of flagellin.44 Despite these A discussion of the role of bacterial flagella in differences, the spirochaete flagella and those pathogenicity can be found in the review by found in other bacteria are structurally similar Moens and Vanderleyden.39 insofar as they have a basal body composed of a Although E. coli and S. typhimurium have series of rings surrounding a central rod, plus a three rings (MS, P, and L) through which the cen- hook and a filament. tral rod passes, other bacteria may have fewer, or more. For example, gram-positive bacteria 7. Site of insertion of flagella and the lack the outer two rings (P and L). Additional number of flagella structural elements of unknown function (e.g., The site of insertion of the flagellum and the additional rings or arrays of particles surround- number of flagella vary with the bacterium. ing the basal body) have been observed in cer- Some rod-shaped or curved cells have flagella tain bacteria. that protrude from one or both of the cell poles. A bacterium with a single, polar flagellum is said Spirochaete flagella. A major difference between to be monotrichous. Bacteria with a bundle of spirochaete flagella and those found in other flagella at a single pole are lophotrichous, from bacteria is that the flagella in spirochaetes do the Greek words lophos (crest or tuft) and trichos not protrude from the cell; rather, they are in (hair). Bacteria with flagella at both poles are the periplasm, wrapped around the length of said to be bipolar. They may have either single the protoplasmic cylinder next to the cell mem- or bundled flagella at the poles. Amphitrichous  structure and function 13 refers to flagella at both poles. The prefix amphi, more than 100 years ago (1885). Now it is rec- in Greek, means on both sides.) ognized to be a widespread phenomenon among Some bacteria (e.g., spirochaetes) have sub- many bacterial genera, both gram-positive and polar flagella, which are inserted near but not gram-negative. exactly at the cell poles, and some curved bacte- The cells in swarming populations are called ria (e.g., Vibrio) have a single, medial flagellum. swarmer cells, and they are often morphologi- If the flagella are arranged laterally all around cally different from swimmer cells grown in the cell (e.g., as in Escherichia and Salmonella) liquid. The morphological changes that occur they are said to be peritrichous. (The prefix when swimming cells from a liquid culture are peri, in Greek, means around.) Peritrichous inoculated onto an agar plate and convert to flagella coalesce into a trailing bundle during swarmer cells can be more or less pronounced swimming. depending upon the bacterium and the concen- tration of the swarming agar. In general, cells 8. Role of flagella in tactic responses and that convert from swimming cells to swarming in virulence cells become nonseptate filaments, multinucle- Many swimming bacteria are capable of tactic oid, and hyperflagellated with lateral flagella. responses: that is, they swim toward environ- (Bacillus subtilis swarmer cells differ less dra- ments more favorable with respect to nutrient, matically from the nonswarmer cells in being light, and electron acceptors, and away from only slightly larger and having only two nuclei.) toxic or less favorable environments. These The lateral flagella are critical because to migrate swimming responses occur because some bac- as populations of cells, the cells physically inter- teria can sense environmental signals, transfer act with each other via the lateral flagella. these signals to the flagellum motor, and modify Swarming can be facilitated by the produc- the rotation of their flagella to swim in a particu- tion of a surfactant that reduces surface tension lar direction. How this occurs for bacteria of dif- at the aqueous periphery of the colony on hydro- ferent types is discussed in detail in Chapter 20, philic surfaces such as agar, allowing the colony which describes chemotaxis. Flagella can also to expand. The surfactant is one of the compo- make important contributions to the virulence nents in the extracellular slime produced by the of pathogenic bacteria.45 For example, it has bacteria. For example, Serratia marcescens pro- been suggested that the ability of spirochaetes duces a cyclic lipopeptide (3-hydroxydecanoic (spiral-shaped bacteria) such as Treponema pal- acid attached to five amino acids), and B. sub- lidum, the causative agent of syphilis, to swim in tilis produces a lipopeptide surfactant called a corkscrew fashion through viscous liquid aids surfactin.48,49 Isolated swarmer cells rarely in their dissemination (e.g., through connec- move. One of the differences between isolated tive tissue or the junctions between endothelial cells and cells in a group is that the cells in the cells). group are encased in much more slime than is 9. Flagella and swarming found around single cells. It could be that wet- The swarming of flagella is a type of social ting agents in the slime hydrate the external swimming in which cells move on solid surfaces medium sufficiently for the flagella to rotate, in groups (called rafts) of physically interact- allowing swarming to take place on the agar. ing cells. (For a review of the different motility To demonstrate swarming on agar, one systems that allow bacteria to move on solid inoculates petri plates contai