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2. 2. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 MICROBIAL BIOTECHNOLOGY Knowledge in microbiology is growing exponentially through the determination of genomic sequences of hundreds of microorganisms and the invention of new technologies, such as genomics, transcriptomics, and proteomics, to deal with this avalanche of information. These genomic data are now exploited in thousands of applications, rang- ing from medicine, agriculture, organic chemistry, public health, and biomass conversion,tobiomining.MicrobialBiotechnologyfocusesonusesofmajorsoci- etalimportance,enablinganin-depthanalysisofthesecriticallyimportantappli- cations. Some, such as wastewater treatment, have changed only modestly over time; others, such as directed molecular evolution, or green chemistry, are as current as todays headlines. Thisfullyrevisedsecondeditionprovidesanexcitinginterdisciplinaryjourney through the rapidly changing landscape of discovery in microbial biotechnology. An ideal text for courses in applied microbiology and biotechnology, this book will also serve as an invaluable overview of recent advances in this eld for pro- fessional life scientists and for the diverse community of other professionals with interests in biotechnology. Alexander N. Glazer is a biochemist and molecular biologist and has been on the faculty of the University of California since 1964. He is a Professor of the Graduate School in the Department of Molecular and Cell Biology at the University of Cali- fornia, Berkeley. Dr. Glazer is a member of the National Academy of Sciences and a Fellow of the American Academy of Arts and Sciences, the American Academy of Microbiology, the American Association for the Advancement of Science, and the California Academy of Sciences. He was twice the recipient of a Guggenheim Fellowship. He was the recipient of the Botanical Society of America Darbaker Prize, 1980 and the National Academy of Sciences Scientic Reviewing Prize, 1991, a lecturer of the Foundation for Microbiology, 199698; and a National GuestLecturer,NewZealandInstituteofChemistry,1999.Dr.Glazerhasauthored over 250 research papers and reviews. He is a co-inventor on more than 40 U.S. patents.Since1996,hehasservedasamemberoftheEditorialAffairsCommittee of Annual Reviews, Inc. Hiroshi Nikaido is a biochemist and microbiologist. He received his M.D. from Keio University in Japan in 1955 and became a faculty member at Harvard Med- ical School in 1963, before moving to University of California in 1969. He is a Professor of Biochemistry and Molecular Biology in the Department of Molecu- larandCellBiologyattheUniversityofCalifornia,Berkeley.Dr.NikaidoisaFellow of the American Academy of Arts and Sciences and the American Academy of Microbiology. He was the recipient of a Guggenheim Fellowship, NIH Senior International Fellowship, Paul Ehrlich prize (1969), Hoechst-Roussel Award of American Society for Microbiology (1984), and Freedom-to-Discover Award for Distinguished Research in Infectious Diseases from Bristol-Myers Squibb (2004). He was an Editor of Journal of Bacteriology from 1998 to 2002. Dr. Nikaido has authored nearly 300 research papers and reviews. i
3. 3. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 MICROBIAL BIOTECHNOLOGY Knowledge in microbiology is growing exponentially through the determination of genomic sequences of hundreds of microorganisms and the invention of new technologies, such as genomics, transcriptomics, and proteomics, to deal with this avalanche of information. These genomic data are now exploited in thousands of applications, rang- ing from medicine, agriculture, organic chemistry, public health, and biomass conversion,tobiomining.MicrobialBiotechnologyfocusesonusesofmajorsoci- etalimportance,enablinganin-depthanalysisofthesecriticallyimportantappli- cations. Some, such as wastewater treatment, have changed only modestly over time; others, such as directed molecular evolution, or green chemistry, are as current as todays headlines. Thisfullyrevisedsecondeditionprovidesanexcitinginterdisciplinaryjourney through the rapidly changing landscape of discovery in microbial biotechnology. An ideal text for courses in applied microbiology and biotechnology, this book will also serve as an invaluable overview of recent advances in this eld for pro- fessional life scientists and for the diverse community of other professionals with interests in biotechnology. Alexander N. Glazer is a biochemist and molecular biologist and has been on the faculty of the University of California since 1964. He is a Professor of the Graduate School in the Department of Molecular and Cell Biology at the University of Cali- fornia, Berkeley. Dr. Glazer is a member of the National Academy of Sciences and a Fellow of the American Academy of Arts and Sciences, the American Academy of Microbiology, the American Association for the Advancement of Science, and the California Academy of Sciences. He was twice the recipient of a Guggenheim Fellowship. He was the recipient of the Botanical Society of America Darbaker Prize, 1980 and the National Academy of Sciences Scientic Reviewing Prize, 1991, a lecturer of the Foundation for Microbiology, 199698; and a National GuestLecturer,NewZealandInstituteofChemistry,1999.Dr.Glazerhasauthored over 250 research papers and reviews. He is a co-inventor on more than 40 U.S. patents.Since1996,hehasservedasamemberoftheEditorialAffairsCommittee of Annual Reviews, Inc. Hiroshi Nikaido is a biochemist and microbiologist. He received his M.D. from Keio University in Japan in 1955 and became a faculty member at Harvard Med- ical School in 1963, before moving to University of California in 1969. He is a Professor of Biochemistry and Molecular Biology in the Department of Molecu- larandCellBiologyattheUniversityofCalifornia,Berkeley.Dr.NikaidoisaFellow of the American Academy of Arts and Sciences and the American Academy of Microbiology. He was the recipient of a Guggenheim Fellowship, NIH Senior International Fellowship, Paul Ehrlich prize (1969), Hoechst-Roussel Award of American Society for Microbiology (1984), and Freedom-to-Discover Award for Distinguished Research in Infectious Diseases from Bristol-Myers Squibb (2004). He was an Editor of Journal of Bacteriology from 1998 to 2002. Dr. Nikaido has authored nearly 300 research papers and reviews. i
4. 4. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 1 10 9 12 11 8 7 6 5 43 2 13 14 15 1 4 5 3 2 MOLDS 1 Penicillium chrysogenum 2 Monascus purpurea 3 Penicillium notatum 4 Aspergillus niger 5 Aspergillus oryzae YEASTS 1 Saccharomyces cerevisiae 2 Candida utilis 3 Aureobasidium pullulans 4 Trichosporon cutaneum 5 Saccharomycopsis capsularis 6 Saccharomycopsis lipolytica 7 Hanseniaspora guilliermondii 8 Hansenula capsulata 9 Saccharomyces carlsbergensis 10 Saccharomyces rouxii 11 Rhodotorula rubra 12 Phaffia rhodozyma 13 Cryptococcus laurentii 14 Metschnikowia pulcherrima 15 Rhodotorula pallida Cultures of molds and yeasts on nutrient agar in glass Petri dishes. From H. Phaff, Indus- trial microorganisms, Scientific American, September 1981. Copyright 1981 by Scientific American, Inc. All rights reserved. ii
5. 5. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 MICROBIAL BIOTECHNOLOGY Fundamentals of Applied Microbiology, Second Edition Alexander N. Glazer University of California, Berkeley Hiroshi Nikaido University of California, Berkeley iii
6. 6. CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, So Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 8RU, UK First published in print format ISBN-13 978-0-521-84210-5 ISBN-13 978-0-511-34136-6 Alexander N. Glazer and Hiroshi Nikaido 2007 2007 Information on this title: www.cambridge.org/9780521842105 This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. ISBN-10 0-511-34136-9 ISBN-10 0-521-84210-7 Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Published in the United States of America by Cambridge University Press, New York www.cambridge.org hardback eBook (EBL) eBook (EBL) hardback
7. 7. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 We dedicate this book to Eva and Kishiko, for the gift of years of support, tolerance, and patience. v
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9. 9. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Contents in Brief Preamble page xiii Acknowledgments xvii 1 Microbial Diversity 1 2 Microbial Biotechnology: Scope, Techniques, Examples 45 3 Production of Proteins in Bacteria and Yeast 90 4 The World of Omics: Genomics, Transcriptomics, Proteomics, and Metabolomics 147 5 Recombinant and Synthetic Vaccines 169 6 PlantMicrobe Interactions 203 7 Bacillus thuringiensis (Bt) Toxins: Microbial Insecticides 234 8 Microbial Polysaccharides and Polyesters 267 9 Primary Metabolites: Organic Acids and Amino Acids 299 10 Secondary Metabolites: Antibiotics and More 324 11 Biocatalysis in Organic Chemistry 398 12 Biomass 430 13 Ethanol 458 14 Environmental Applications 487 Index 541 Advances of particular relevance and importance will be posted periodically on the website www.cambridge.org/glazer. vii
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11. 11. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Contents Preamble page xiii Acknowledgments xvii 1 Microbial Diversity 1 Prokaryotes and Eukaryotes 2 The Importance of the Identication and Classication of Microorganisms 10 Plasmids and the Classication of Bacteria 16 Analysis of Microbial Populations in Natural Environments 19 Taxonomic Diversity of Bacteria with Uses in Biotechnology 25 Characteristics of the Fungi 35 Classication of the Fungi 35 Culture Collections and the Preservation of Microorganisms 41 Summary 42 Selected References and Online Resources 43 2 Microbial Biotechnology: Scope, Techniques, Examples 45 Human Therapeutics 46 Agriculture 54 Food Technology 59 Single-Cell Protein 64 Environmental Applications of Microorganisms 67 Microbial Whole-Cell Bioreporters 74 Organic Chemistry 77 Summary 85 Selected References and Online Resources 86 3 Production of Proteins in Bacteria and Yeast 90 Production of Proteins in Bacteria 90 Production of Proteins in Yeast 125 Summary 143 Selected References 144 ix
12. 12. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 x Contents 4 The World of Omics: Genomics, Transcriptomics, Proteomics, and Metabolomics 147 Genomics 147 Transcriptomics 155 Proteomics 158 Metabolomics and Systems Biology 164 Summary 165 Selected References 166 5 Recombinant and Synthetic Vaccines 169 Problems with Traditional Vaccines 170 Impact of Biotechnology on Vaccine Development 172 Mechanisms for Producing Immunity 179 Improving the Effectiveness of Subunit Vaccines 184 Fragments of Antigen Subunit Used as Synthetic Peptide Vaccines 189 DNA Vaccines 193 Vaccines in Development 194 Summary 199 Selected References 200 6 PlantMicrobe Interactions 203 Use of Symbionts 204 Production of Transgenic Plants 210 Summary 230 Selected References 231 7 Bacillus thuringiensis (Bt) Toxins: Microbial Insecticides 234 Bacillus thuringiensis 235 Insect-Resistant Transgenic Crops 250 Benet and Risk Assessment of Bt Crops 259 Summary 263 Selected References and On-Line Resources 264 8 Microbial Polysaccharides and Polyesters 267 Polysaccharides 268 Xanthan Gum 272 Polyesters 281 Summary 295 References 296 9 Primary Metabolites: Organic Acids and Amino Acids 299 Citric Acid 299 Amino Acid: l-Glutamate 301 Amino Acids Other Than Glutamate 308 Amino Acid Production with Enzymes 320 Summary 322 Selected References 322
13. 13. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Contents xi 10 Secondary Metabolites: Antibiotics and More 324 Activities of Secondary Metabolites 325 Primary Goals of Antibiotic Research 338 Development of Aminoglycosides 339 Development of the -Lactams 352 Production of Antibiotics 369 Problem of Antibiotic Resistance 382 Summary 393 Selected References 394 11 Biocatalysis in Organic Chemistry 398 Microbial Transformation of Steroids and Sterols 400 Asymmetric Catalysis in the Pharmaceutical and Agrochemical Industries 402 Microbial Diversity: A Vast Reservoir of Distinctive Enzymes 406 High-Throughput Screening of Environmental DNA for Natural Enzyme Variants with Desired Catalytic Properties: An Example 407 Approaches to Optimization of the Best Available Natural Enzyme Variants 409 Rational Methods of Protein Engineering 416 Large-Scale Biocatalytic Processes 418 Summary 426 References 427 12 Biomass 430 Major Components of Plant Biomass 432 Degradation of Lignocellulose by Fungi and Bacteria 441 Degradation of Lignin 444 Degradation of Cellulose 448 Degradation of Hemicelluloses 453 The Promise of Enzymatic Lignocellulose Biodegradation 454 Summary 455 References and Online Resources 456 13 Ethanol 458 Stage I: From Feedstocks to Fermentable Sugars 461 Stage II: From Sugars to Alcohol 463 Simultaneous Saccharication and Fermentation: Stages I and II Combined 479 Prospects of Fuel Ethanol from Biomass 483 Summary 483 References and Online Resources 484 14 Environmental Applications 487 Degradative Capabilities of Microorganisms and Origins of Organic Compounds 487
14. 14. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 xii Contents Wastewater Treatment 490 Microbiological Degradation of Xenobiotics 500 Microorganisms in Mineral Recovery 527 Microorganisms in the Removal of Heavy Metals from Aqueous Efuent 532 Summary 536 References 538 Index 541
15. 15. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Preamble Il ny a pas des sciences appliquees . . . mais il ya des applications de la sci- ence. (There are no applied sciences . . . but there are the applications of science.) Louis Pasteur Microorganisms are the most versatile and adaptable forms of life on Earth, and they have existed here for some 3.5 billion years. Indeed, for the rst 2 billion years of their existence, prokaryotes alone ruled the biosphere, col- onizing every accessible ecological niche, from glacial ice to the hydrother- mal vents of the deep-sea bottoms. As these early prokaryotes evolved, they developed the major metabolic pathways characteristic of all living organ- isms today, as well as various other metabolic processes, such as nitrogen xation, still restricted to prokaryotes alone. Over their long period of global dominance, prokaryotes also changed the earth, transforming its anaero- bic atmosphere to one rich in oxygen and generating massive amounts of organic compounds. Eventually, they created an environment suited to the maintenance of more complex forms of life. Today, the biochemistry and physiology of bacteria and other micro- organisms provide a living record of several billion years worth of genetic responses to an ever-changing world. At the same time, their physiologic and metabolic versatility and their ability to survive in small niches cause them to be much less affected by the changes in the biosphere than are larger, more complex forms of life. Thus, it is likely that representatives of most of the microbial species that existed before humans are still here to be explored. Such an exploration is by no means a purely academic pursuit. The many thousandsofmicroorganismsalreadyavailableinpurecultureandthethou- sands of others yet to be cultured or discovered represent a large fraction of the total gene pool of the living world, and this tremendous genetic diver- sity is the raw material of genetic engineering, the direct manipulation of the heritable characteristics of living organisms. Biologists are now able to greatly accelerate the acquisition of desired traits in an organism by directly modifying its genetic makeup through the manipulation of its DNA, rather thanthroughthetraditionalmethodsofbreedingandselectionatthelevelof xiii
16. 16. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 xiv Preamble the whole organism. The various techniques of manipulation summarized under the rubric of recombinant DNA technology can take the form of removing genes, adding genes from a different organism, modifying genetic control mechanisms, and introducing synthetic DNA, sometimes enabling a cell to perform functions that are totally new to the living world. In these ways, new stable heritable traits have by now been introduced into all forms of life. One result has been a signicant enhancement of the already consid- erablepracticalvalueofappliedmicrobiology.Appliedmicrobiologycoversa broad spectrum of activities, contributing to medicine, agriculture, green chemistry, exploitation of sources of renewable energy, wastewater treat- ment, and bioremediation, to name but a few. The ability to manipulate the genetic makeup of organisms has led to explosive progress in all areas of this eld. The purpose of this book is to provide a rigorous, unied treatment of all facets of microbial biotechnology, freely crossing the boundaries of for- mal disciplines in order to do so: microbiology supplies the raw materials; genomics, transcriptomics, and proteomics provide the blueprints; bio- chemistry, chemistry, and process engineering provide the tools; and many otherscienticeldsserveasimportantreservoirsofinformation.Moreover, unlike a textbook of biochemistry, microbiology, molecular biology, organic chemistry, or some other vast basic eld, which must concentrate solely on teaching general principles and patterns in order to provide an overview, this one will continually emphasize the importance of diversity and unique- ness. In applied microbiology, one is frequently likely to seek the unusual: a producer of a novel antibiotic, a parasitic organism that specically infects a particularly widespread and noxious pest, a hyperthermophilic bacterium thatmightserveasasourceofenzymesactiveabove100 C.Insum,thisbook examines the fundamental principles and facts that underlie current prac- tical applications of bacteria, fungi, and other microorganisms; describes those applications; and examines future prospects for related technologies. The stage on which microbial biotechnology performs today is vastly different from that portrayed in the rst edition of this book, published 12 years ago. The second edition has been extensively rewritten to incorporate the avalanche of new knowledge. What are some of the most inuential of these recent advances? Hundreds of prokaryotic and fungal genomes have been fully sequenced, andpartialgenomicinformationisavailableformanymoreorganismsavail- able in pure culture. The understanding of the phylogenetic and evolutionary relationships among microorganisms now rests on the objective foundation provided by this large body of sequence data. These data have also revealed the mosaic and dynamic aspects of microbial genomes. Environmental DNA libraries offer a glimpse of the immensity and func- tionaldiversityofthemicrobialworldandproviderapidaccesstogenesfrom tens of thousands of yet-uncultured microorganisms.
17. 17. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Preamble xv Extensive databases of annotated sequences along with sophisticated computational tools allow rapid access to the burgeoning body of infor- mation and reveal potential functions of new sequences. The polymerase chain reaction coupled with versatile techniques for the generation of recombinant organisms allows exploitation of sequence infor- mation to create new molecules or organisms with desired properties. Genomics, transcriptomics, and metabolomics use powerful new tech- niques to map how complex cell functions arise from coordinated regulation of multiple genes to give rise to the interdependent pathways of metabolism and to the integration of the sensory inputs that ensure proper functioning of cells in responding to environmental change. In the past 10 years, these developments have also changed the processes used in all of the classical areas of biotechnology for instance, in the production of amino acids, antibiotics, polymers, and vaccines. The growing human population of the earth, equipped with the ability to effect massive environmental change by applying ever-increasing tech- nological sophistication, is placing huge and unsustainable demands on natural resources. Microbial biotechnology is of increasing importance in contributing to the generation of crops with resistance to particular insect pests, tolerance to herbicides, and improved ability to survive drought and high levels of salt. The urgent need to minimize the discharge of organic chemical pollutants into the environment along with the need to conserve declining reserves of petrochemicals has led to the advent of green chem- istry with attendant rapid growth in the use of biocatalysts. The future of the use of biomass as a renewable source of energy is critically dependent on progress in efcient direct microbial conversion of complex mixtures of polysaccharides to ethanol. The treatment of wastewater, a critical contri- bution of microorganisms to maintaining the life-support systems of the planet, is an important area for future innovation. The application of biotechnology to medicine, agriculture, the chemical industry, and the environment is changing all aspects of everyday life, and the pace of that change is increasing. Thus, basic understanding of the many facetsofmicrobialbiotechnologyisimportanttoscientistsandnonscientists alike. We hope that both will nd this book a useful source of information. Although a strong technical background may be necessary to assimilate the ne points described herein, we have tried to make the fundamental con- cepts and issues accessible to readers whose background in the life sciences is quite modest. The attempt is vital, for only an informed public can distin- guish desirable biotechnological options from the undesirable, those likely to succeed from those likely to result in costly failure.
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19. 19. P1: JZP 0521842107pre CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:13 Acknowledgments We are grateful to our colleagues who read various chapters, to Moira Lerner for her helpful developmental editing of three of the chapters, and to the manyscientistsandpublisherswhoallowedustoreproduceillustrationsand other material and generously provided their original images and electronic les for this purpose. We are indebted to Kirk Jensen for his interest in our plans for this book and for introducing us to Cambridge University Press. Working with the Cambridge staff has been a pleasure. Dr. Katrina Halliday provided encour- agement and steady editorial guidance from the early stages of this project through the completion of the manuscript. We are particularly grateful to Clare Georgy and Alison Evans for their careful review of the manuscript and forundertakingthearduoustaskofsecuringpermissionstoreproducemany illustrations and other material. We thank Marielle Poss for her oversight of the production process, and are grateful to Alan Gold for designing the creative and elegant layout for the book. We thank Ken Karpinski at Aptara for his oversight and meticulous attention to detail in the production of this book and his unfailing gracious help when there were snags in the process. Finally, we thank Georgette Koslovsky for her precise and thoughtful copy editing. The combined efforts of all of these individuals have contributed a great deal to the accuracy and aesthetic quality of this book. The authors are responsible for any imperfections that remain. xvii
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21. 21. P1: SPP 0521842105c01 CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:18 ONE Microbial Diversity Molecular phylogenies divide all living organisms into three domains Bac- teria (true bacteria), Archaea, and Eukarya (eukaryotes: protists, fungi, plants, animals). The place of viruses (Box 1.1) in the phylogenetic tree of life isuncertain.Inthisbook,wefocusonthecontributionsofBacteria,Archaea, and Fungi to microbial biotechnology. In so doing, we include organisms from all three domains. We also devote some attention to the uses of viruses as well as to the problems they pose in certain technological contexts. The domains of Bacteria and Archaea encompass a huge diversity of organismsthatdifferintheirsourcesofenergy,theirsourcesofcellcarbonor nitrogen, their metabolic pathways, the end products of their metabolism, and their ability to attack various naturally occurring organic compounds. Different bacteria and archaea have adapted to every available climate and microenvironment on Earth. Halophilic microorganisms grow in brine ponds encrusted with salt, thermophilic microorganisms grow on smolder- ing coal piles or in volcanic hot springs, and barophilic microorganisms live under enormous pressure in the depths of the seas. Some bacteria are symbiontsofplants;otherbacterialiveasintracellularparasitesinsidemam- malian cells or form stable consortia with other microorganisms. The seem- ingly limitless diversity of the microorganisms provides an immense pool of raw material for applied microbiology. The morphological variety of organisms classied as fungi rivals that of Viruses differ from all other organ- isms in three major respects: they contain only one kind of nucleic acid, either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); only the nucleic acid is necessary for their re- production; and they are unable to re- produce outside of a hosts living cell. Viruses are not described further in this chapter but will be encountered later in the discussion of vaccines (Chapter 5) BOX 1.1 the bacteria and archaea. Fungi are particularly effective in colonizing dry woodandareresponsibleformostofthedecompositionofplantmaterialsby secreting powerful extracellular enzymes to degrade biopolymers (proteins, polysaccharides, and lignin). They produce a huge number of small organic molecules of unusual structure, including many important antibiotics. On the other hand, fungi as a group lack some of the metabolic capabilities of the bacteria. In particular, fungi do not carry out photosynthesis or nitrogen xation and are unable to exploit the oxidation of inorganic compounds as a source of energy. Fungi are unable to use inorganic compounds other than oxygen as terminal electron acceptors in respiration. Fungi as a group are also less versatile than bacteria in the range of organic compounds they can 1
22. 22. P1: SPP 0521842105c01 CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:18 2 Microbial Diversity useassolesourcesofcellcarbon.Frequently,fungiandbacteriacomplement each others abilities in degrading complex organic materials. A consortium is a system of several organisms (frequently two) in which each organism contributes something needed by the others. Many funda- mental processes in nature are the outcome of such interactions among microorganisms inuencing the biosphere on a worldwide scale. For exam- ple, consortia of bacteria and fungi play an indispensable role in the cycling of organic matter. By decomposing the organic by-products and the remains of plants and animals, they release nutrients that sustain the growth of all living things. The top six inches of fertile soil may contain over two tons of fungi and bacteria per acre. In fact, the respiration of bacteria and fungi has been estimated to account for over 90% of the carbon dioxide production in the biosphere. Technology, too, takes advantage of the special abilities of mixed cultures of microorganisms, employing them in beverage, food, and dairy fermentations, for example, and in biotreatment processes for waste- water. Lately, the challenges posed by the need to clean up massive oil spills and to decontaminate toxic waste sites with minimum permanent damage to the environment have directed attention to the powerful degradative capabili- ties of consortia of microorganisms. Experience suggests that encouraging the growth of natural mixed microbial populations at the site of contami- nation can contribute more successfully to the degradation of undesirable organic compounds in diverse ecological settings than can the introduction of a single ingeniously engineered recombinant microorganism with new metabolic capabilities. We are still far from an adequate understanding of microbial interactions in natural environments. This chapter has a dual purpose: to provide a guide to the relative place- ments of important microorganisms on the taxonomic map of the microbial world and to explore the importance of the diversity of microorganisms to biotechnology. PROKARYOTES AND EUKARYOTES Cellular organisms fall into two classes that differ from each other in the fun- damental internal organization of their cells. The cells of eukaryotes contain a true membrane-bounded nucleus (karyon), which in turn contains a set of chromosomes that serve as the major repositories of genetic information in the cell. Eukaryotic cells also contain other membrane-bounded organelles that possess genetic information, namely mitochondria and chloroplasts. In the prokaryotes, the chromosome (nucleoid) is a closed circular DNA molecule, which lies in the cytoplasm, is not surrounded by a nuclear mem- brane, and contains all of the information necessary for the reproduction of the cell. Prokaryotes also have no other membrane-bounded organelles whatsoever.Bacteriaandarchaeaareprokaryotes,whereasfungiareeukary- otes. The choice of a fungus (such as the yeast Saccharomyces cerevisiae) or a bacterium (such as Escherichia coli) for a particular application is often
23. 23. P1: SPP 0521842105c01 CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:18 Prokaryotes and Eukaryotes 3 TABLE 1.1 A comparison of Bacterial, Archaeal, and Eukaryal cells Bacteria Archaea Eukarya STRUCTURAL FEATURES Chromosome number One One More than one Nuclear membrane Absent Absent Present Nucleolus Absent Absent Present Mitotic apparatus Absent Absent Present Microtubules Absent Absent Present Membrane lipids Glycerol diesters Glycerol diethers or glycerol tetraethers Glycerol diesters Membrane sterols Rare Rare Nearly universal Peptidoglycan Present Absent Absent GENE STRUCTURE, TRANSCRIPTION, AND TRANSLATION Introns in genes Rare Rare Common Transcription coupled with translation Yes May occur No Polygenic mRNA Yes Yes No Terminal polyadenylation of mRNA Absent Present Present Ribosome subunit sizes 30S, 50S 30S, 50S 40S, 60S (sedimentation coefficient) (cytoplasmic) Amino acid carried by initiator tRNA Formylmethionine Methionine Methionine METABOLIC PROCESSES Oxidative phosphorylation Membrane dependent Membrane dependent In mitochondria Photosynthesis Membrane dependent Membrane dependent In chloroplasts Reduced inorganic compounds as energy source May be used May be used Not used Nonglycolytic pathways for anaerobic energy generation May occur May occur Do not occur Poly--hydroxybutyrate as organic reserve material Occurs Occurs Does not occur Nitrogen fixation Occurs Occurs Does not occur OTHER PROCESSES Exo- and endocytosis Does not occur Does not occur May occur Amoeboid movement Does not occur Does not occur May occur mRNA, messenger RNA; tRNA, transfer RNA. dictated by the basic genetic, biochemical, and physiological differences between prokaryotes and eukaryotes. THE TWO GROUPS OF PROKARYOTES Among prokaryotes, a general distinction is made between the bacteria and the archaea. The evolutionary distance between the bacteria, the archaea, and the eukaryotes, estimated from the divergence in their ribosomal RNA (rRNA) sequences, is so great that it is believed that these three groups may have diverged from an ancient progenitor rather than evolving from one another. With respect to many molecular features, the archaea are almost as different from the bacteria as the latter are from eukaryotes (Table 1.1). For
24. 24. P1: SPP 0521842105c01 CUFX119/Glazer 0 521 84210 7 June 21, 2007 19:18 4 Microbial Diversity O O OH CH2OHCH2OH O COCH3 COOH O O O NHCOCH3NH CH3 CH N-Acetyl muramic acid N-Acetylglucosamine nFIGURE 1.1 Repeating unit of the polysaccharide back- bone of the peptidoglycan layer in the cell wall of bacteria. example, the cell wall structure of bacteria is based on a cross-linked poly- mer called peptidoglycan with an N-acetylglucosamineN-acetylmuramic acid repeating unit (Figure 1.1). Because of the virtually universal pres- ence of peptidoglycan in bacteria and its absence in eukaryotes, the pres- ence of muramic acid is considered a bacterial signature. The different archaea have a variety of cell wall polymers, but none of them incorporates muramic acid. The most dramatic difference between these organisms is in the nature of the glycerol lipids that make up the cytoplasmic membrane. The hydrophobic moieties in the archaea are ether-linked and branched aliphatic chains, whereas those of bacteria and eukaryotes are ester-linked straight aliphatic chains (Figure 1.2). Initially, the archaea were believed to be typical of extreme environ- ments tolerated by few bacteria and fewer eukaryotes. The archaea include three distinct kinds of microorganisms, all found in extreme environments: the methanogens, the extreme halophiles, and the thermoacidophiles. The methanogens live only in oxygen-free environments and generate methane by the reduction of carbon dioxide. The halophiles require very high concen- trations of salt to survive and are found in natural habitats such as the Great Salt Lake and the Dead Sea as well as in man-made salt evaporation ponds. Thethermoacidophilesarefoundinhotsulfurspringsattemperaturesabove 80 Cinstronglyacidicenvironments(pH