Starvation in Bacteria Starvation in Bacteria

Edited by Staffan Kjelleberg University 0/ Göteborg Göteborg, Sweden and University 0/ New South Wales Sydney, Australia

SPRINGER SCIENCE+BUSINESS MEDIA, LLC Library of Congress Cataloging-In-Publication Data

Starvation 1n bacteria / edited by Staffan Kjeiieberg. p. cm. Includes bibliographical references and Index.

1. M1crob1al netaboHsn. 2. Starvation. I. Kjeileberg, Staffan. QR88.S7 1993 589.9" 0133—dc 20 93-21918 CIP

This limited facsimile edition has been issued for the purpose of keeping this title available to the scientific community.

10 98765432

ISBN 978-0-306-44430-2 ISBN 978-1-4899-2439-1 (eBook) DOI 10.1007/978-1-4899-2439-1

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Preface

Concerted efforts to study starvation and survival of nondifferentiating vegeta• tive heterotrophic bacteria have been made with various degrees of intensity, in different bacteria and contexts, over more than the last 30 years. As with bacterial growth in natural ecosystem conditions, these research efforts have been intermittent, with rather long periods of limited or no production in between. While several important and well-received reviews and proceedings on the topic of this monograph have been published during the last three to four decades, the last few years have seen a marked increase in reviews on starvation survival in non-spore-forming bacteria. This increase reflects a realization that the biology of bacteria in natural conditions is generally not that of logarithmic growth and that we have very limited information on the physiology of the energy- and nutrient-limited phases of the life cyde of the bacterial cello The growing interest in nongrowing bacteria also sterns from the more recent advances on the molecular basis of the starvation-induced nongrowing bacterial cello The identification of starvation-specific gene and protein re• sponders in Escherichia coli as weIl as other bacterial species has provided molecular handles for our attempts to decipher the "differentiation-like" responses and programs that nondifferentiating bacteria exhibit on nutrient• limited growth arrest. Severallaboratories have contributed greatly to the progress made in life• after-log research. Of these it is pertinent to mention the strong pioneering work of Richard Morita and his collaborators, which predominantly studied marine Vibrio species, and Abdul Matin and co-workers, who in aseries of important publications introduced physiological and molecular aspects of starvation survival and the starvation-induced program in E. coli. Recently, the field has benefited greatly from the elegant and innovative series of studies reported by the research laboratories of Roberto Kolter and Regine Hengge• Aronis. The detailed analysis of global control systems (for the regulation above the operon level) by, primarily, Fred Neidhardt and colleagues has been of prime importance for our understanding of the starvation-induced defense systems exhibited by prokaryotes.

v vi Preface

This monograph provides an up-to-date presentation of the means by which traditionally nondifferentiating bacteria adapt to starvation conditions. The genetic program and physiological features of adaptation to starvation by different bacteria are explored. This book also addresses prevailing ecosystem conditions that lead to intermittent growth or long-tenn starvation in bacteria. It is suggested that an improved understanding of starvation survival and nongrowth biology is an essential goal in microbiology, with far-reaching implications in bacterial physiology and ecology, as weIl as in applied bacteriol• ogy and biotechnology. Public health microbiology and environmental bio• technology are areas that greatly benefit from the advances recently made in research programs that deal with starvation in bacteria. This monograph serves as an overview and introduction also for those interested in further exploring such applications. 1 wish to thank the authors for providing their excellent contributions, Plenum Senior Editor Mary Phillips Born and other Plenum staff members for constructive support throughout the various stages of preparing this mono• graph, and Kevin MarshalI for proposing that it is timely to publish a book on starvation in bacteria.

Staffan Kjelleberg Göteborg anti Sydney Contributors

MartaAlmir6n, Department ofMicrobiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

R. T. Bell, Institute of Limnology, Uppsala University, S-751 22 Uppsala, Sweden

Thomas Egli, Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600 Dübendorf, Switzerland

Klas Flärdh, Department of General and Marine Microbiology, University of Göteborg, S-413 19 Göteborg, Sweden lohn W. Foster, Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, Alabama 36688

Regine Hengge-Aronis, Department of Biology, University of Konstanz, 7750 Konstanz, Germany

Louise Holmquist, Department of General and Marine Microbiology, Univer• sity of Göteborg, S-413 19 Göteborg, Sweden

Asa louper-laan, Department of General and Marine Microbiology, University of Göteborg, S-413 19 Göteborg, Sweden

Staffan Kjelleberg, Department of General and Marine Microbiology, Uni ver• sity of Göteborg, S-413 19 Göteborg, Sweden. Present address: School of Microbiology and Immunology, University of New South Wales, Ken• sington, New South Wales 2033, Australia

Roberto Kolter, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

C. G. Kurland, Department of Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden

vii viii Contributors

c. Anthony Mason, Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600 Dübendorf, Switzerland

Riitta Mikkola, Department of Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden

D. J. W Moriarty, Department of Marine Microbiology, University of Gothen• burg, Gothenburg, Sweden. Present address: Department of Chemical Engineering, University of Queensland, St. Lucia, Queensland 4067, Aus• tralia

Richard Y. Morita, Department of Microbiology, College of Science and College of Oceanography, Oregon State University, Corvallis, Oregon 97330-3804

Thomas Nyström, Department ofMicrobiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620. Present address: Department of General and Marine Microbiology, University of Göteborg, S-413 19 Götcborg. Sweden

James D. Oliver, Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223

Jörgen Östling, Department of General and Marine Microbiology, University of Göteborg, S-413 19 Göteborg, Sweden

Deborah A. Siegele, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115. Present address: Department of Biology, Texas A&M University, College Station, Texas 77843

Michael P. Spector, Department of Biomedical Sciences, College of Allied Health, and Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, Alabama 36688

Björn Svenblad, Department of General and Marine Microbiology, University of Göteborg, S-413 19 Göteborg, Sweden

J. D. van Eisas, Institute for Soil Fertility Research, 6700AA Wageningen, The Netherlands

L. S. van Overbeek, Institute for Soil Fertility Research, 6700AA Wageningen, The Netherlands Contents

Chapter 1 Bioavailability of Energy and the Starvation State Richard Y. Morita

1. Introduetion...... 1 2. Survival of Baeteria in Natural and Artifieial Eeosystems 2 3. Organic Matter in Oligotrophie Systems...... 2 3.1. Soil ...... 3 3.2. Aquatic Systems...... 3 4. Bioavailability of Organie Matter in Oligotrophie Environments for Heterotrophie Baeteria ...... 4 4.1. Energy for Growth and/ or Reproduetion ...... 4 4.2. Bioavailability of Substrates and Generation Times 4 4.3. Syntrophy and Baeterial Aetivity ...... 7 4.4. Oligotrophie Bacteria ...... 8 5. The Starvation Proeess ...... 8 5.1. Patterns of Starvation ...... 10 5.2. Cell Size of Baeteria in Oligotrophie Environments and in Starvation Microeosms ...... 12 5.3. Cell Membrane Changes ...... 13 5.4. ATP and Adenylate Energy Charge ...... 14 5.5. Protein, DNA, and RNA ...... 14 5.6. Chemotaxis...... 16 5.7. Resistance of Starved Cells to Various Environmental Faetors ...... 16 5.8. Recovery from Starvation ...... 16 6. Conclusions...... 17 References ...... 18 ix x Contents

Chapter 2 Bacterial Growth and Starvation in Aquatic Environments D. 1. W. Moriarty and R. T. Bell

1. Introduction...... 25 2. Effects of Starvation on Natural Populations...... 27 2.1. Specific Growth Rates and Productivity ...... 27 2.2. Factors Controlling Bacterial Growth ...... 28 2.3. Bacterial Cell Sizes in Natural Environments...... 31 2.4. Effects of Nitrogen and Phosphorus ...... 32 3. Marine Environments ...... 34 3.1. Pelagic Bacterial Growth ...... 34 3.2. Bacterial Growth and Survival in Sediments ...... 37 4. Freshwater Environments ...... 40 4.1. Seasonal Dynamics of Bacterioplankton in Lakes 40 4.2. The Microbial Landscape in Lakes-Importance of Microenvironments ...... 44 4.3. Bacterial Productivity in the Surface Sediment of Lakes ...... 46 5. Concluding Comment ...... 47 References ...... 48

Chapter 3 Bacterial Responses to SoU Stimuli J. D. van Elsas and L. S. van Overbeek

1. Introduction...... 55 2. The Soil Environment...... 56 2.1. Observations on Bacterial Populations in Soil ..... 56 3. Stimuli in the Soil Environment ...... 64 3.1. Responses to Stimuli of Bulk Soil ...... 66 3.2. Responses to Stimuli of Rhizosphere Soil ...... 70 4. Concluding Remarks ...... 73 References ...... 74 Contents xi

Chapter 4 Dynamics 01 Microbial Growth in the Decelerating and Stationary Phase 01 Batch Culture C. Anthony Mason and Thomas Egli

1. Introduction ...... 81 2. Nutrient Limitation and Nutrient Starvation ...... 83 3. Growth under Nutrient-Sufficient Conditions ...... 84 4. Effects of Exhaustion of Different Nutrients on Batch Growth Curve ...... 84 4.1. Carbon ...... 84 4.2. Nitrogen ...... 85 4.3. Phosphate ...... 85 4.4. Other Nutrients ...... 88 5. Effect of Nutrient Exhaustion in Batch Culture on Physiological Parameters ...... 90 5.1. Cell Number and Morphology ...... 90 5.2. Product Formation and Utilization ...... 91 5.3. Accumulation of Storage Compounds ...... 92 5.4. Conte nt of DNA ...... 92 5.5. Content of RNA ...... 93 5.6. Proteins ...... 94 5.7. Cell Envelope ...... 95 6. Concluding Remarks ...... 96 References ...... 98

Chapter 5 Starvation and Recovery 01 Vibrio Jörgen Östling, Louise Holmquist, Klas Flärdh, Björn Svenblad, Äsa Jouper-Jaan, and Staffan Kjelleberg

1. Introduction...... 103 2. Vibrios in the Environment ...... 104 3. Adaptation to Multiple-Nutrient Limitation ...... 106 3.1. Morphology and Physiology ...... 106 xii Contents

3.2. Sequential Expression of Starvation-Induced Proteins ...... 108 4. Responses to Starvation for Individual Nutrients ...... 109 4.1. Viability ...... 109 4.2. Morphology ...... 109 4.3. RNA and Protein Synthesis ...... 111 4.4. Overlap in Protein Synthesis during Starvation for Different Individual Nutrients ...... 111 4.5. Stress Resistance ...... 111 5. Carbon Starvation ...... 112 5.1. Physiology of Carbon-Starved Cells ...... 112 5.2. Physiological Basis for Stress Resistance of Starved Cells ...... 115 5.3. Carbon Starvation Responders ...... 116 6. The Diversity of Starvation Responses in the Genus Vibrio 120 7. Appendix...... 122 References ...... 123

Chapter 6 Global Systems Approach to the Physiology of the Starved Cell Thomas Nyström

1. Introduction ...... 129 2. Protein Expression during Starvation-Induced Growth Arrest ...... " 131 2.1. Physiology of the Starved Cell ...... 131 2.2. Sequential Expression of Starvation-Inducible Proteins ...... 131 3. Overlap between Starvation Stimulons ...... 134 3.1. Unique, General, and Universal Starvation Proteins 134 3.2. Identity of Some General Starvation Proteins ..... 135 3.3. Possible Regulation of General Starvation and Stress Proteins ...... 136 4. Role of Global Regulatory Networks in Starvation-Survival 141 4.1. Stringent Control ...... 141 4.2. The Heat-Shock Response...... 143 Contents xiii

5. Protein Expression during Recovery from Starvation . . . .. 144 6. Perspectives...... 144 References ...... 146

Chapter 7 Approaches to the Study of Survival and Death in Stationary-Phase Escherichia coli Deborah A. Siegele, Marta Almir6n, and Roberto Kolter

1. Introduction...... 151 2. Terminology ...... 152 3. Culture Conditions ...... 152 4. Methods to Distinguish Viable and Nonviable Cells ..... 153 4.1. By Plating ...... 153 4.2. By Microscopy ...... 153 4.3. By Centrifugation ...... 154 5. Stationary-Phase Survival Studies with ZK126 ...... 154 6. Survival Genes ...... 159 7. Reverse Genetics to Study Stationary Phase Gene Expression ...... 163 8. Appendixes ...... 164 8. 1. Culture Media ...... 164 8.2. AO Staining ...... 165 8.3. Renografin-76 Equilibrium Density Gradients ..... 166 8.4. Notes about Reverse Genetics ...... 166 References ...... 167

Chapter 8 The Role of rpoS in Early Stationary Phase Gene Regulation in Escherichia coli K12 Regine Hengge-Aronis

1. Introduction...... 171 2. Identification of rpoS as a Central Regulatory Gene for Early Stationary-Phase Gene Expression ...... 172 xiv Contents

3. The Role of rpoS in Stationary-Phase Stress Resistance ...... 174 3.1. Thermotolerance...... 175 3.2. Oxidative Stress Resistance ...... 176 3.3. Other Stationary-Phase Stress Resistances ...... 177 3.4. Implications for the Functions of the Structural Genes Involved in Multiple Stationary-Phase Stress Resistance ...... 178 4. rpoS-Regulated Genes and Their Cellular Functions ..... 179 4.1. The Morphogene bolA and Stationary-Phase Cell Morphology ...... 180 4.2. xtha, katE, dps and Hydrogen Peroxide Resistance ...... 181 4.3. The Role of otsA, otsB, and treA in Trehalose Metabolism, Osmoprotection, and Thermotolerance ...... 182 4.4. gIgS and Glycogen Synthesis ...... 183 4.5. csi-5 (osmY): A Periplasmic Protein of Unknown Function ...... 184 4.6. The Lipoprotein Encoded by osmB ...... 185 4.7. appY: A Secondary Regulator Involved in the Control of Acid Phosphatase (appA) and a Third Cytochrome Oxidase (cyxAB) ...... 185 4.8. mcc-Directed Synthesis of Microcin C7 ...... 186 4.9. Genes Involved in Pathogenesis (csgA, spv) ..... 186 4.10. rpoS-dependent Genes of Unknown Function (csiD, csiE, pex) ...... 187 5. The Mechanisms of Gene Regulation by rpoS ...... 187 5.1. rpoS Encodes a a 7o-like Sigma Factor (aS) ...... 187 5.2. What is the Consensus Promoter Recognized byaS •...... •...... •...... •...... 188 5.3. Regulatory Subfamilies of rpoS-Controlled Genes ...... 191 5.4. Multiple Promoters in the Control Region of rpoS-Dependent Genes ...... 192 6. Perspectives...... 193 References ...... 194 Contents xv

Chapter 9 Starvation-Stress Response (SSR) of Salmonella typhimurium: Gene Expression and Survival during Nutrient Starvation Michael P. Spector and lohn W. Foster

1. Introduction ...... 201 2. Alterations of Cellular Constituents during Nutrient Starvation ·in Salmonella ...... 202 3. Two-Dimensional Gel Electrophoretic Analysis of the Starvation-Stress Response of Salmonella ...... 203 4. Genetic Analysis of the Starvation-Stress Response of Salmonella ...... 208 4.1. Identification of Starvation-Regulated Loci Using Mud-Iac Gene Fusions ...... 208 4.2. Additional Aspects of the Physiological Regulation of sti Gene Expression ...... 209 4.3. Temporal Expression of Starvation-Regulated lac Gene Fusions during Carbon Starvation ...... 209 4.4. Starvation-Survival in Salmonella typhimurium .... 210 4.5. Negative Regulation of Starvation-Survival Loci by the cAMP Receptor Protein (CRP) in cAMP- Dependent and -Independent Manners ...... 214 4.6. Stringent Control and Regulation of Starvation- Survival Loci ...... 215 4.7. P-Starvation-Induction of Starvation-Survival Loci is Independent of re IA and phoP ...... 218 5. Comparison of S. typhimurium Sti Proteins with Pex Proteins of E. coli and the Sti Proteins of Marine Vibrio . 218 References ...... 221

Chapter 10 The Impact of Nutritional State on the Microevolution of Ribosomes C. G. Kurland and Riitta Mikkola

1. Introduction...... 225 2. The Variability of Natural Isolates ...... 226 xvi Contenls

3. Ribosome Mutants ...... 228 4. Ribosomes of Natural Isolates ...... 230 5. Drift...... 232 6. Carbon Starvation ...... 234 7. Conc1usions...... 235 References ...... 236

Chapter 11 Formation of Viable but Nonculturable Cells James D. Oliver

1. Introduction...... 239 2. General Characteristics of the VBNC State ...... 241 2.1. Loss of Plateability ...... 242 2.2. Size Reduction and Ultrastructural Changes ...... 242 2.3. Time Required to Enter the VBNC State ...... 243 2.4. Response to aReversal of the Inducing Factor .... 245 3. Methods for Determining the VBNC State ...... 246 3.1. Direct Viable Count ...... 246 3.2. Detection of Respiration ...... 247 3.3. Monoclonal Antibodies ...... 248 3.4. Cellular Integrity and Staining of Nuc1eic Acid with Acridine Orange or DAPI ...... 248 3.5. Loss of Radiolabel ...... 249 3.6. Intracellular ATP Levels ...... 250 3.7. Flow Cytometry ...... 250 4. Bacteria Reported to Enter the VBNC State ...... 250 5. Factors Inducing the Nonculturable Response...... 252 5.1. Temperature ...... 252 5.2. Physiological Age of the Culture ...... 253 5.3. Salt Levels ...... 253 5.4. Nutrient Levels...... 254 5.5. Light...... 254 5.6. Aeration ...... 255 5.7. Cell Washing ...... 255 Contents xvii

6. Physiological and Biochemical Changes in VBNC Cells 255 6.1. Macromolecular Synthesis ...... 255 6.2. Respiration ...... 257 6.3. Peptidoglycan...... 258 6.4. Plasmids ...... 259 6.5. Chromosomal DNA ...... 259 6.6. Lipids ...... 260 6.7. Capsule...... 261 7. Resuscitation from the VBNC State ...... 261 7.1. In Vitro Studies ...... 261 7.2. Do Nonculturable Cells of Pathogenic Bacteria Retain Virulence? ...... 263 8. Relationship between the VBNC and Starvation States ... 265 9. Conc1usions...... 267 References ...... 268

Index...... 273