Thermus Species BIOTECHNOLOGY HANDBOOKS Series Editors: Tony Atkinson and Roger F. Sherwood Centre for Applied Microbiology and Research Division of Biotechnology Salisbury, Wiltshire, England Volume 1 PENICILLIUM AND ACREMONIUM Edited by John F. Peberdy Volume 2 BACILLUS Edited by Colin R. Harwood Volume 3 CLOSTRIDIA Edited by Nigel P. Minton and David]. Clarke Volume 4 SACCHAROMYCES Edited by Michael F. Tuite and Stephen G. Oliver Volume 5 METHANE AND METHANOL UTILIZERS Edited by]. Colin Murrell and Howard Dalton Volume 6 PHOTOSYNTHETIC PROKARYOTES Edited by Nicolas H. Mann and Noel G. Carr Volume 7 ASPERGILLUS Edited by]. E. Smith Volume 8 SULFATE-REDUCING BACTERIA Edited by Larry L. Barton Volume 9 THERMUS SPECIES Edited by Richard Sharp and Ralph Williams A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher. Thermus Species Edited by Richard Sharp The Centre for Applied Microbiology and Research Porton Down, Salisbury, England and Ralph Williams Queen Mary and Westfield College London, England Springer Science+Business Media, LLC Llbrary of Congress Cataloglng-ln-Publlcatlon Data Thermus species I edited by Richard Sharp and Ralph Williams. p. cm. -- (Biotechnology handbooks ; v. 9) Includes bibliographical references and index. ISBN 978-1-4613-5741-4 ISBN 978-1-4615-1831-0 (eBook) DOI 10.1007/978-1-4615-1831-0 1. Thermophil ic bacteria. 1. Sharp. Richard (Richard J.) II. Wi 11 iams. R. A. D. (Ralph Anthony David). 1938- III. Series. OR84.8.T46 1995 589.9·2--dc20 95-12394 CIP ISBN 978-1-4613-5741-4 © 1995 Springer Science+Business Media New York Originally published by Plenum Press in 1995 Softcover reprint of the hardcover Ist edition 1995 10987654321 AII rights reserved 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 permis sion from the Publisher Contributors Gudni A. Alfredsson • University of Iceland, Institute of Biology, Mi crobiology Laboratory, Armuli lA, IS-I08 Reykjavik, Iceland Peter L. Bergquist • Bacterial Genetics and Microbiology, School of Biological Sciences, University of Auckland, New Zealand Doug Cossar • Cangene Corporation, 3403 American Drive, Missis sauga, Ontario, Canada, L4V IT4 Milton S. da Costa • Departmento de Zoologia, Universidade de Coimbra, 3049 Coimbra Cedex, Portugal Melanie L. Duffield • Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom Jakob K. Kristjansson • University of Iceland, Institute of Biology, Microbiology Laboratory, ArmUli lA, IS-I08 Reykjavik, Iceland Hugh W. Morgan • Thermophile Research Unit, University of Wai kato, Private Bag 3105, Hamilton, New Zealand Tairo Oshima • Department of Life Science, Tokyo Institute of Tech nology, Nagatsuta, Yokohama, Japan Neil D. H. Raven • Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OjG, United Kingdom Richard Sharp • Centre for Applied Microbiology and Research, Por ton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom Ralph Williams • Queen Mary & Westfield College, Biochemistry De partment, Faculty of Basic Medical Sciences, Mile End Road, London E 14 NS, United Kingdom v Preface There is considerable interest in thermophile microorganisms, in their environments, their ability to survive at temperatures which normally denature proteins, but more importantly, as a valuable resource for bio technology. The first reported isolation of Thermus by Tom Brock was in 1969. This initiated the present era of thermophilic research with the realization that where liquid water is available, there may be no limits to the temper ature at which microorganisms can grow. Considerable research into the ecology, physiology, metabolism, and thermostable enzymes of thermo philes has led to their evaluation for a range of industrial and commercial processes. The past fifteen years have been an explosive period of dis covery of many new genera and species, including the descriptions of a new fundamental kingdom-the Archaea. Much of the current research has been focused on the Archaea; but it is significant that during this period, the original type strain YT-l of Thermus aquaticus described by Brock has provided a major step forward in molecular biology. DNA polymerase from strain YT-I has proved to be the major success in the commercialization of enzymes from thermophilic microorganisms to date. The ease with which Thermus strains can be handled in laboratories without specialized equipment, together with the large investment in de scribing their structure, metabolism, and genetics, should ensure a con tinuing effort in Thermus research. This book brings together many groups that have isolated and con tributed to our knowledge of Thermus. It covers aspects of ecology and isolation, taxonomy, physiology, molecular biology and genetics, cell struc ture and biotechnology. This volume aims primarily at established re searchers in universities, research institutes, and industry, but it introduc es this important group of thermophilic bacteria to undergraduates and postgraduates who are interested in thermophilic microorganisms as po tential biotechnological tools and as fascinating laboratory curiosities. Richard Sharp Ralph Williams VB Contents Chapter 1 The Taxonomy and Identification of Thermus ............... . Ralph Williams and Richard Sharp I. Introduction and General Properties of Thermus ............ 1 2. Cell Wall Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.1. Peptidoglycan..................................... 4 3. Pigments and Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1. Pigments.......................................... 4 3.2. Menaquinones..................................... 5 3.3. Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.4. Complex Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4. Phenotypic Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.1. Physiological and Biochemical Tests. . . . . . . . . . . . . . . . . . 6 4.2. Numerical Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5. Nucleic Acid Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 5.1. DNA Base Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 5.2. DNA:DNA Homology............................... 22 5.3. Ribosomal RNA Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . 24 6. The Species of Thermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 6.1. The Yellow-Pigmented and Colorless Species. . . . . . . . . . 34 6.2. The Taxonomic Position of Thermus ruber. . . . . . . . . . . . . . 39 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Chapter 2 Ecology, Distribution, and Isolation of Thermus . . . . . . . . . . . . . . 43 Gudni A. Alfredsson and Jakob K. Kristjansson 1. Ecology and Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 1.1. Characteristics of the Thermal Environment. . . . . . . . . . . 43 IX x CONTENTS 1.2. Different Thermal Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . 44 1.3. Habitats of Thermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 1.4. Sources of Organic Compounds for Thermus. . . . . . . . . . . . 46 1.5. Thermus and Oligotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 1.6. Effects of Some Environmental Factors . . . . . . . . . . . . . . . . 50 1.7. Rotund Bodies and Their Possible Function. . . . . . . . . . . . 54 1.8. Distribution of Thermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2. Isolation of Thermus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.1. Introduction....................................... 56 2.2. Physiological and Nutritional Characteristics. . . . . . . . . . . . 56 2.3. Sampling and Enumeration. . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.4. Culture Media and Isolation .......... . . . . . . . . . . . . . . . 57 2.5. Growth Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Chapter 3 Physiology and Metabolism of Thermus . . . . . . . . . . . . . . . . . . . . . . 67 Richard Sharp, Doug Cossar, and Ralph Williams 1. Enzymes of the Main Metabolic Pathways. . . . . . . . . . . . . . . . . . . 67 1.1. Glycolysis and Gluconeogenesis. . . . . . . . . . . . . . . . . . . . . . . 67 1.2. The Tricarboxylic Acid and Glyoxylate Cycles. . . . . . . . . . 69 1.3. Amino Acid Metabolism ............................. 70 2. Utilization of Substrates for Growth and Energy. . . . . . . . . . . . . 71 2.1. Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 2.2. Carbohydrates...................................... 72 2.3. Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3. Energetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.1. Electron Acceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.2. The Electron Transport Chain. . . . . . . . . . . . . . . . . . . . . . . . 73 3.3. ATP Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 3.4. Defenses against Oxygen Toxicity. . . . . . . . . . . . . . . . . . . . . 74 4. Microbial Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 4.1. Medium Development and Growth Requirements. . ... . . 75 4.2. Growth Kinetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 4.3. Growth and Pigmentation. . . ... .. .. . . . .. . . . . . . . . . . . . . 81 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 CONTENTS xi Chapter 4 Enzymes of Thermus and Their Properties. . . . . . . . . . . . . . . . . . . 93 Melanie L. Duffield and Doug Cossar 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 2. Thermal Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 2.1. Proposed Mechanisms of Thermal Stability . . . . . . . . . . . 9S 2.2. Examples of Thermal Stability. . .. . .. . .. . . . . . . . . . . . . 101 3. Oxidoreductases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 3.1. Lactate Dehydrogenase ............. . . . . . . . . . . . . . . . 101 3.2. Malate Dehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 3.3. NADP-Dependent Isocitrate Dehydrogenase. . . . . . . . . . 104 3.4. 3-Isopropylmalate Dehydrogenase. . . . . . . . . . . . . . . . . . . 105 3.5 Ferredoxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 3.6 Glyceraldehyde-3-Phosphate Dehydrogenase. . . . . . . . . . 107 3.7. L-Alanine Dehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . lOS 3.S. NADH Dehydrogenase. . .. . . . . . . . . . . . .. . . . . . .. . . . .. lOS 3.9. NADH Oxidase.. . . . . . ... . .. . . . . . . . . . . . . .... . . . . . . 109 3.10. Catalase.......................................... 110 3.11. Superoxide Dismutase ............................. 110 3.12. Cytochromes...................................... 111 4. Transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.1. tRNA Methyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.2. Phosphofructokinase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.3. Phosphoglycerokinase...... . . . . . . . . . . . . . . . . . . . . . . . . 114 4.4. RNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 4.5. DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5. Hydrolases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5.1. Proteases..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5.2. Carbohydrases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.3. Alkaline Phosphatase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.4. Ribonucleases...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 5.5. EFTu............................................ 120 5.6. Asparaginase..................................... 121 5.7. Inorganic Pyrophosphatase. . . . . . . . . . . . . . . . . . . . . . . . . 122 5.S. ATPase.......................................... 122 5.9. DNA Endonucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 6. Lyases. . . . . . . . .. . .... . . . . . . .. . . .. . . . . . .. . . . . . . .. . . . . . . . 124 6.1. Anthranilate Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 6.2. Enolase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 xii CONTENTS 6.3. Fumarase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 6.4. Tryptophan Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7. Isomerases ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7.1. D-Xylose Isomerase. .... . . . . . . . . . . . . . . . . . . . . . . . ... . . 126 8. Ligases. . . . . . . .. . . . . . . . .. . . .. . . . . . . . . . .. . . . . . . . . . . . . . . .. 127 8.1. Aminoacyl tRNA Synthetase. . . . . . . . . . . . . . . . . . . . . . . . .. 127 8.2. Succinyl-CoA Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 8.3. DNA Ligase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 9. Miscellaneous. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 9.1. Chaperonins....................................... 131 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Chapter 5 The Cell Walls and Lipids of Thermus . . . . . . . . . . . . . . . . . . . . . . . 143 Milton S. da Costa 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 2. Cell Wall Structure and Composition. . . . . . . . . . . . . . . . . . . . . . . 143 3. Lipid Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 3.1. Respiratory Quinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 3.2. Polar Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 145 3.3. Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 3.4. Carotenoids........................................ 150 4. Temperature-Induced Alterations in Lipids. . . . . . . . . . . . . . . . . 151 4.1. Polar Lipids. . . . . . . . . . . . . . . . . . . . .. . . . .. . . . .. . .. . . . . . 151 4.2. Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 5. Final Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Chapter 6 Genetics of Thermus: Plasmids, Bacteriophage, Potential Vectors, Gene Transfer Systems . . . . . . . . . . . . . . . . . . . . 157 Neil D. H. Raven 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 2. Thermus Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158