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MICROBIAL BIOTECHNOLOGY

Knowledge in microbiology is growing exponentially through the determinationof genomic sequences of hundreds of microorganisms and the invention of newtechnologies, such as genomics, transcriptomics, and proteomics, to deal withthis avalanche of information.

These genomic data are now exploited in thousands of applications, rang-ing from medicine, agriculture, organic chemistry, public health, and biomassconversion, to biomining. Microbial Biotechnology focuses on uses of major soci-etal importance, enabling an in-depth analysis of these critically important appli-cations. Some, such as wastewater treatment, have changed only modestly overtime; others, such as directed molecular evolution, or “green” chemistry, are ascurrent as today’s headlines.

This fully revised second edition provides an exciting interdisciplinary journeythrough the rapidly changing landscape of discovery in microbial biotechnology.An ideal text for courses in applied microbiology and biotechnology, this bookwill also serve as an invaluable overview of recent advances in this field for pro-fessional life scientists and for the diverse community of other professionals withinterests in biotechnology.

Alexander N. Glazer is a biochemist and molecular biologist and has been on thefaculty of the University of California since 1964. He is a Professor of the GraduateSchool 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 anda Fellow of the American Academy of Arts and Sciences, the American Academyof Microbiology, the American Association for the Advancement of Science, andthe California Academy of Sciences. He was twice the recipient of a GuggenheimFellowship. He was the recipient of the Botanical Society of America DarbakerPrize, 1980 and the National Academy of Sciences Scientific Reviewing Prize,1991, a lecturer of the Foundation for Microbiology, 1996–98; and a NationalGuest Lecturer, New Zealand Institute of Chemistry, 1999. Dr. Glazer has authoredover 250 research papers and reviews. He is a co-inventor on more than 40 U.S.patents. Since 1996, he has served as a member of the Editorial Affairs Committeeof Annual Reviews, Inc.

Hiroshi Nikaido is a biochemist and microbiologist. He received his M.D. fromKeio 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 aProfessor of Biochemistry and Molecular Biology in the Department of Molecu-lar and Cell Biology at the University of California, Berkeley. Dr. Nikaido is a Fellowof the American Academy of Arts and Sciences and the American Academy ofMicrobiology. He was the recipient of a Guggenheim Fellowship, NIH SeniorInternational Fellowship, Paul Ehrlich prize (1969), Hoechst-Roussel Award ofAmerican Society for Microbiology (1984), and Freedom-to-Discover Award forDistinguished Research in Infectious Diseases from Bristol-Myers Squibb (2004).He was an Editor of Journal of Bacteriology from 1998 to 2002. Dr. Nikaido hasauthored nearly 300 research papers and reviews.

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1

10

9

12

11

8

7

6

5

432

13

14

15

1

4

5 3

2MOLDS 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 ScientificAmerican, Inc. All rights reserved.

ii


MICROBIALBIOTECHNOLOGYFundamentals of AppliedMicrobiology, SecondEdition

Alexander N. GlazerUniversity of California, Berkeley

Hiroshi NikaidoUniversity of California, Berkeley

iii

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo

Cambridge University PressThe 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

http://www.cambridge.orghttp://www.cambridge.org/9780521842105


We dedicate this book to Eva and Kishiko,

for the gift of years of support, tolerance, and patience.

v


vi


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 Plant–Microbe 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 postedperiodically on the website www.cambridge.org/glazer.

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viii


Contents

Preamble page xiii

Acknowledgments xvii

1 Microbial Diversity 1

Prokaryotes and Eukaryotes 2

The Importance of the Identification and Classification

of Microorganisms 10

Plasmids and the Classification 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

Classification 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

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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


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


6 Plant–Microbe Interactions 203

Use of Symbionts 204

Production of Transgenic Plants 210

Summary 230


7 Bacillus thuringiensis (Bt) Toxins: Microbial Insecticides 234

Bacillus thuringiensis 235

Insect-Resistant Transgenic Crops 250

Benefit 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



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 β-Lactams 352

Production of Antibiotics 369

Problem of Antibiotic Resistance 382

Summary 393


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 Saccharification 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


xii Contents

Wastewater Treatment 490

Microbiological Degradation of Xenobiotics 500

Microorganisms in Mineral Recovery 527

Microorganisms in the Removal of Heavy Metals from

Aqueous Effluent 532

Summary 536

References 538

Index 541


Preamble

Il n’y a pas des sciences appliquées . . . mais il y’a 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 first 2billion 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, theydeveloped the major metabolic pathways characteristic of all living organ-isms today, as well as various other metabolic processes, such as nitrogenfixation, still restricted to prokaryotes alone. Over their long period of globaldominance, prokaryotes also changed the earth, transforming its anaero-bic atmosphere to one rich in oxygen and generating massive amounts oforganic compounds. Eventually, they created an environment suited to themaintenance 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 geneticresponses to an ever-changing world. At the same time, their physiologicand metabolic versatility and their ability to survive in small niches causethem to be much less affected by the changes in the biosphere than arelarger, more complex forms of life. Thus, it is likely that representatives ofmost of the microbial species that existed before humans are still here to beexplored.

Such an exploration is by no means a purely academic pursuit. The manythousands of microorganisms already available in pure culture and the thou-sands of others yet to be cultured or discovered represent a large fraction ofthe total gene pool of the living world, and this tremendous genetic diver-sity is the raw material of genetic engineering, the direct manipulation ofthe heritable characteristics of living organisms. Biologists are now able togreatly accelerate the acquisition of desired traits in an organism by directlymodifying its genetic makeup through the manipulation of its DNA, ratherthan through the traditional methods of breeding and selection at the level of

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xiv Preamble

the whole organism. The various techniques of manipulation summarizedunder the rubric of “recombinant DNA technology” can take the form ofremoving genes, adding genes from a different organism, modifying geneticcontrol mechanisms, and introducing synthetic DNA, sometimes enablinga cell to perform functions that are totally new to the living world. In theseways, new stable heritable traits have by now been introduced into all formsof life. One result has been a significant enhancement of the already consid-erable practical value of applied microbiology. Applied microbiology covers abroad 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 thegenetic makeup of organisms has led to explosive progress in all areas of thisfield.

The purpose of this book is to provide a rigorous, unified treatment ofall 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 manyother scientific fields serve as important reservoirs of information. Moreover,unlike a textbook of biochemistry, microbiology, molecular biology, organicchemistry, or some other vast basic field, which must concentrate solely onteaching 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: aproducer of a novel antibiotic, a parasitic organism that specifically infectsa particularly widespread and noxious pest, a hyperthermophilic bacteriumthat might serve as a source of enzymes active above 100◦C. In sum, this bookexamines the fundamental principles and facts that underlie current prac-tical applications of bacteria, fungi, and other microorganisms; describesthose applications; and examines future prospects for related technologies.

The stage on which microbial biotechnology performs today is vastlydifferent from that portrayed in the first edition of this book, published 12years ago. The second edition has been extensively rewritten to incorporatethe avalanche of new knowledge. What are some of the most influential ofthese recent advances?

■ Hundreds of prokaryotic and fungal genomes have been fully sequenced,and partial genomic information is available for many more organisms avail-able in pure culture.

■ The understanding of the phylogenetic and evolutionary relationshipsamong microorganisms now rests on the objective foundation provided bythis large body of sequence data. These data have also revealed the mosaicand dynamic aspects of microbial genomes.

■ Environmental DNA libraries offer a glimpse of the immensity and func-tional diversity of the microbial world and provide rapid access to genes fromtens of thousands of yet-uncultured microorganisms.


Preamble xv

■ Extensive databases of annotated sequences along with sophisticatedcomputational 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 thegeneration 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 regulationof multiple genes to give rise to the interdependent pathways of metabolismand to the integration of the sensory inputs that ensure proper functioningof cells in responding to environmental change.

■ In the past 10 years, these developments have also changed the processesused in all of the “classical” areas of biotechnology – for instance, in theproduction of amino acids, antibiotics, polymers, and vaccines.

■ The growing human population of the earth, equipped with the abilityto effect massive environmental change by applying ever-increasing tech-nological sophistication, is placing huge and unsustainable demands onnatural resources. Microbial biotechnology is of increasing importance incontributing to the generation of crops with resistance to particular insectpests, tolerance to herbicides, and improved ability to survive drought andhigh levels of salt. The urgent need to minimize the discharge of organicchemical pollutants into the environment along with the need to conservedeclining reserves of petrochemicals has led to the advent of “green” chem-istry with attendant rapid growth in the use of biocatalysts. The future ofthe use of biomass as a renewable source of energy is critically dependenton progress in efficient direct microbial conversion of complex mixtures ofpolysaccharides to ethanol. The treatment of wastewater, a critical contri-bution of microorganisms to maintaining the life-support systems of theplanet, is an important area for future innovation.

The application of biotechnology to medicine, agriculture, the chemicalindustry, and the environment is changing all aspects of everyday life, andthe pace of that change is increasing. Thus, basic understanding of the manyfacets of microbial biotechnology is important to scientists and nonscientistsalike. We hope that both will find this book a useful source of information.Although a strong technical background may be necessary to assimilate thefine points described herein, we have tried to make the fundamental con-cepts and issues accessible to readers whose background in the life sciencesis quite modest. The attempt is vital, for only an informed public can distin-guish desirable biotechnological options from the undesirable, those likelyto succeed from those likely to result in costly failure.


xvi


Acknowledgments

We are grateful to our colleagues who read various chapters, to Moira Lernerfor her helpful developmental editing of three of the chapters, and to themany scientists and publishers who allowed us to reproduce illustrations andother material and generously provided their original images and electronicfiles for this purpose.

We are indebted to Kirk Jensen for his interest in our plans for this bookand for introducing us to Cambridge University Press. Working with theCambridge staff has been a pleasure. Dr. Katrina Halliday provided encour-agement and steady editorial guidance from the early stages of this projectthrough the completion of the manuscript. We are particularly grateful toClare Georgy and Alison Evans for their careful review of the manuscript andfor undertaking the arduous task of securing permissions to reproduce manyillustrations and other material. We thank Marielle Poss for her oversightof the production process, and are grateful to Alan Gold for designing thecreative and elegant layout for the book. We thank Ken Karpinski at Aptarafor his oversight and meticulous attention to detail in the production of thisbook and his unfailing gracious help when there were snags in the process.Finally, we thank Georgette Koslovsky for her precise and thoughtful copyediting.

The combined efforts of all of these individuals have contributed a greatdeal to the accuracy and aesthetic quality of this book. The authors areresponsible for any imperfections that remain.

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ON

EMicrobial 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 lifeis uncertain. In this book, we focus on the contributions of Bacteria, Archaea,and Fungi to microbial biotechnology. In so doing, we include organismsfrom all three domains. We also devote some attention to the uses of virusesas well as to the problems they pose in certain technological contexts.

The domains of Bacteria and Archaea encompass a huge diversity oforganisms that differ in their sources of energy, their sources of cell carbon ornitrogen, 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 climateand microenvironment on Earth. Halophilic microorganisms grow in brineponds encrusted with salt, thermophilic microorganisms grow on smolder-ing coal piles or in volcanic hot springs, and barophilic microorganismslive under enormous pressure in the depths of the seas. Some bacteria aresymbionts of plants; other bacteria live as intracellular parasites inside mam-malian cells or form stable consortia with other microorganisms. The seem-ingly limitless diversity of the microorganisms provides an immense pool ofraw material for applied microbiology.

The morphological variety of organisms classified as fungi rivals that ofViruses differ from all other organ-isms in three major respects: theycontain only one kind of nucleic acid,either deoxyribonucleic acid (DNA)or ribonucleic acid (RNA); only thenucleic acid is necessary for their re-production; and they are unable to re-produce outside of a host’s living cell.Viruses are not described further inthis chapter but will be encounteredlater in the discussion of vaccines(Chapter 5)

BOX 1.1

the bacteria and archaea. Fungi are particularly effective in colonizing drywood and are responsible for most of the decomposition of plant materials bysecreting powerful extracellular enzymes to degrade biopolymers (proteins,polysaccharides, and lignin). They produce a huge number of small organicmolecules of unusual structure, including many important antibiotics. Onthe other hand, fungi as a group lack some of the metabolic capabilities ofthe bacteria. In particular, fungi do not carry out photosynthesis or nitrogenfixation and are unable to exploit the oxidation of inorganic compounds asa source of energy. Fungi are unable to use inorganic compounds other thanoxygen as terminal electron acceptors in respiration. Fungi as a group arealso less versatile than bacteria in the range of organic compounds they can

1


2 Microbial Diversity

use as sole sources of cell carbon. Frequently, fungi and bacteria complementeach other’s abilities in degrading complex organic materials.

A consortium is a system of several organisms (frequently two) in whicheach organism contributes something needed by the others. Many funda-mental processes in nature are the outcome of such interactions amongmicroorganisms influencing the biosphere on a worldwide scale. For exam-ple, consortia of bacteria and fungi play an indispensable role in the cyclingof organic matter. By decomposing the organic by-products and the remainsof plants and animals, they release nutrients that sustain the growth of allliving things. The top six inches of fertile soil may contain over two tons offungi and bacteria per acre. In fact, the respiration of bacteria and fungi hasbeen estimated to account for over 90% of the carbon dioxide productionin the biosphere. Technology, too, takes advantage of the special abilities ofmixed cultures of microorganisms, employing them in beverage, food, anddairy fermentations, for example, and in biotreatment processes for waste-water.

Lately, the challenges posed by the need to clean up massive oil spills andto decontaminate toxic waste sites with minimum permanent damage to theenvironment have directed attention to the powerful degradative capabili-ties of consortia of microorganisms. Experience suggests that encouragingthe growth of natural mixed microbial populations at the site of contami-nation can contribute more successfully to the degradation of undesirableorganic compounds in diverse ecological settings than can the introductionof a single ingeniously engineered recombinant microorganism with newmetabolic capabilities. We are still far from an adequate understanding ofmicrobial 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 microbialworld and to explore the importance of the diversity of microorganisms tobiotechnology.

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 containa true membrane-bounded nucleus (karyon), which in turn contains a set ofchromosomes that serve as the major repositories of genetic information inthe cell. Eukaryotic cells also contain other membrane-bounded organellesthat possess genetic information, namely mitochondria and chloroplasts.In the prokaryotes, the chromosome (nucleoid) is a closed circular DNAmolecule, which lies in the cytoplasm, is not surrounded by a nuclear mem-brane, and contains all of the information necessary for the reproductionof the cell. Prokaryotes also have no other membrane-bounded organelleswhatsoever. Bacteria and archaea are prokaryotes, whereas fungi are eukary-otes. The choice of a fungus (such as the yeast Saccharomyces cerevisiae) ora bacterium (such as Escherichia coli) for a particular application is often



TABLE 1.1 A comparison of Bacterial, Archaeal, and Eukaryal cells

Bacteria Archaea Eukarya

STRUCTURAL FEATURES

Chromosome number One One More than oneNuclear membrane Absent Absent PresentNucleolus Absent Absent PresentMitotic apparatus Absent Absent PresentMicrotubules Absent Absent PresentMembrane lipids Glycerol diesters Glycerol diethers or

glycerol tetraethersGlycerol diesters

Membrane sterols Rare Rare Nearly universalPeptidoglycan Present Absent Absent

GENE STRUCTURE, TRANSCRIPTION, AND TRANSLATION

Introns in genes Rare Rare CommonTranscription coupled with translation Yes May occur NoPolygenic mRNA Yes Yes NoTerminal polyadenylation of mRNA Absent Present PresentRibosome 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 mitochondriaPhotosynthesis Membrane dependent Membrane dependent In chloroplastsReduced inorganic compounds as energy

sourceMay be used May be used Not used

Nonglycolytic pathways for anaerobic energygeneration

May occur May occur Do not occur

Poly-β-hydroxybutyrate as organic reservematerial

Occurs Occurs Does not occur

Nitrogen fixation Occurs Occurs Does not occur

OTHER PROCESSES

Exo- and endocytosis Does not occur Does not occur May occurAmoeboid movement Does not occur Does not occur May occur

mRNA, messenger RNA; tRNA, transfer RNA.

dictated by the basic genetic, biochemical, and physiological differencesbetween prokaryotes and eukaryotes.

THE TWO GROUPS OF PROKARYOTES

Among prokaryotes, a general distinction is made between the bacteria andthe 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 mayhave diverged from an ancient progenitor rather than evolving from oneanother. With respect to many molecular features, the archaea are almost asdifferent from the bacteria as the latter are from eukaryotes (Table 1.1). For



OO

OH

CH2OHCH2OH

O

COCH3

COOH

OO

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 cellwall of bacteria.

example, the cell wall structure of bacteria is based on a cross-linked poly-mer called peptidoglycan with an N-acetylglucosamine–N-acetylmuramicacid 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 differentarchaea have a variety of cell wall polymers, but none of them incorporatesmuramic acid. The most dramatic difference between these organisms is inthe nature of the glycerol lipids that make up the cytoplasmic membrane.The hydrophobic moieties in the archaea are ether-linked and branchedaliphatic chains, whereas those of bacteria and eukaryotes are ester-linkedstraight 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 includethree distinct kinds of microorganisms, all found in extreme environments:the methanogens, the extreme halophiles, and the thermoacidophiles. Themethanogens live only in oxygen-free environments and generate methaneby 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 GreatSalt Lake and the Dead Sea as well as in man-made salt evaporation ponds.The thermoacidophiles are found in hot sulfur springs at temperatures above80◦C in strongly acidic environments (pH < 2). However, analyses of 16S rDNAanalyzed in environmental samples show archaea to be present in marinesediments, in coastal and open ocean waters, and in freshwater sedimentsand soils. Planktonic members of the Crenarchaeota phylum are reportedto represent about 20% of all of the bacterial and archaeal cells found inthe oceans. An archaeal symbiont, Crenarchaeum symbiosum, lives in thetissues of the marine sponge Axinella mexicana in coastal waters of about10◦C. It now appears that bacteria and archaea have many types of habitatsin common.

GRAM STAIN METHOD

The Gram stain procedure was described by the Danish physician HansChristian Gram in 1884 and has survived in virtually unmodified form. Gramworked at the morgue of the City Hospital of Berlin, where he developed amethod to detect bacteria in tissues by differential staining. In a widely used



EUBACTERIAL LIPID

ARCHAEBACTERIAL LIPIDS

Ester link

H2C O

OO

H2C

CH2

CH2 CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2 CH3

CH2

OR

C

HC O C

CH3

CH2 CH

CH

CH2CH2

CH2

CH2 CH

CH

CH2CH2

CH2CH2

CH2CH2

CH2

CH2 CH

CH

CH2CH2

CH2

CH2 CH

CH

CH2CH2

CH2CH2

CH3CH3

CH2

CH3 CH3 CH3

CH3 CH3 CH3 CH3

Ether link

H2C O

H2C OR

HC O

Diether

CH

CH2

CH2 CH

CH2CH2 CH2

CH2 CH

CH2

CH2 CH

CH2CH2 CH2

CH2 CH2CH2 CH2 CH2 CH2CH2 CH2CH2

CH CHO

CH2RO

CHCH2 CHCH2 CHCH2 CH2

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

CH

CH2

CH2 CH

CH2CH2 CH2

CH2 CH

CH2

CH2 CH

CH2CH2 CH2

CH2 CH2CH2 CH2 CH2 CH2CH2 CH2CH2

CH CH2OCHCH2 CHCH2 CHCH2 CH2

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

H2C O

H2C OR

HC O

Tetraether

FIGU RE 1.2

Membrane lipids of bacteria and eukary-otes are glycerol esters of straight-chainfatty acids such as palmitate. Archaeal mem-brane lipids are diethers or tetraethers inwhich the glycerol unit is linked by an etherlink to phytanols, branched-chain hydrocar-bons. Moreover, the configuration about thecentral carbon of the glycerol unit is D in theester-linked lipids but L in the ether-linkedlipids. R is phosphate or phosphate esters inphospholipids and sugars in glycolipids.

version of his empirical procedure, a heat-fixed tissue sample or smear ofbacteria on a glass slide is stained first with a solution of the dye crystalviolet and then with a dilute solution of iodine to form an insoluble crystalviolet-iodine complex. The preparation is then washed with either alcoholor acetone. Bacteria that are rapidly decolorized by this means are said to beGram-negative; those that remain violet are said to be Gram-positive. Theease of dye elution, and consequently the Gram staining behavior of bac-teria, correlates with the structure of the cell walls. Gram-positive bacteriahave a thick cell wall of highly cross-linked peptidoglycan, whereas Gram-negative bacteria usually have a thin peptidoglycan layer covered by an outer



GRAM-POSITIVE

Peptidoglycan

Membrane

A

Plasma membrane

Peptidoglycan

B

Plasma membrane

Peptidoglycan(inner layer)

Outer membrane

GRAM-NEGATIVE

Peptidoglycan

Membrane

Lipopolysaccharideand protein

FIGURE 1.3

Electron micrographs of bacterial cell walls.(A) Gram-positive, Arthrobacter crystallopoietes.Magnification, 126,000×. (B) Gram-negative,Leucothrix mucor. Magnification, 165,000×.[Reproduced with permission from Brock,T. D., and Madigan, M. T. (1988). Biology ofMicroorganisms, 5th Edition, Englewood Cliffs,NJ: Prentice Hall, Figure 3.22.]

membrane. The outer membrane is an asymmetric lipid bilayer membrane:a lipopolysaccharide forms the exterior layer and phospholipid forms theinner layer (Figure 1.3).

The presence of the outer membrane on Gram-negative bacteria con-fers a higher resistance to antibiotics, such as penicillin, and to degradativeenzymes, such as lysozyme. Eubacteria are almost equally divided betweenGram-positive and Gram-negative types, and the result of the Gram stainremains a valuable character in bacterial classification.

PRINCIPAL MODES OF METABOLISM

Organisms that use organic compounds as their major source of cell carbonare called heterotrophs; those that use carbon dioxide as the major sourceare called autotrophs. Organisms that use chemical bond energy for thegeneration of adenosine triphosphate (ATP) are called chemotrophs, whereasthose that use light energy for this purpose are called phototrophs. Thesedescriptions lead to the division of microorganisms into the four types listedin Table 1.2. Those chemoautotrophs that obtain energy from the oxidationof inorganic compounds are also called chemolithotrophs.



TABLE 1.2 Principal modes of metabolism

Type Prokaryotes Eukaryotes

Chemoautotrophs + noneChemoheterotrophs + + (“animals,” fungi)Photoautotrophs + + (“plants”)Photoheterotrophs + none

All organisms need energy and reducing power inorder to conduct the biosynthetic reactions requiredfor growth. In all cases, the energy-generating pro-cesses produce ATP (a molecule with high phos-phate group donor potential); reducing power is storedin nicotinamide adenine dinucleotides (NADH andNADPH; molecules with high electron donor poten-tial). Prokaryotes exhibit a wider range of energy-generating schemes than do eukaryotes. The three types of processes thatlead to the formation of ATP in prokaryotes are reviewed very briefly belowand summarized in Table 1.3.

Abstraction of Chemical Bond Energy from PreformedOrganic Compounds (Chemoheterotrophy)

Catabolic pathways are sequences of chemical reactions in which carboncompounds are degraded. The molecules are altered or broken into smallfragments, usually by reactions involving the removal of electrons (thatis, by oxidations). The enzymes that catalyze catabolic reactions are usu-ally located in the cytoplasm. There are two classes of energy-producingcatabolic pathways: fermentations and respirations.

Fermentations are catabolic pathways that operate when no exogenouselectron acceptor is present and in which the structures of carbon com-pounds are rearranged, thereby releasing free energy, which is used to makeATP. It is essential to distinguish between the biological meaning of fer-mentation as presented here and its meaning in the common parlance ofapplied microbiology. To the biotechnologist, a fermentation is any processmediated by microorganisms that involves a transformation of organic sub-stances. The rigorous, chemical definition of a fermentation is that it is aprocess in which no net oxidation–reduction occurs; the electrons of thesubstrate are distributed among the products. For example, in a lactic acidfermentation, one mole of glucose is converted to two moles of lactic acid(Figure 1.4). The process whereby some of the released free energy is con-served in activated compounds formed in the course of catabolism and thenused to generate ATP is called substrate-level phosphorylation.

Respirations are catabolic pathways by which organic compounds canbe completely oxidized to carbon dioxide (mainly via the tricarboxylic acidcycle) because an exogenous terminal electron acceptor is present. Releasedfree energy is conserved in the form of a protonic potential, or a protonmotive force, generated by the vectorial (unidirectional) translocation of pro-tons across a membrane within which components of an electron transportchain are contained. The vectorial translocation of protons is driven by thepassage of electrons along the electron transport chain to the molecule thatserves as the terminal electron acceptor. ATP is generated at the expenseof the proton gradient upon return of the protons through a transmem-brane enzyme complex, an FoF1-type adenosine triphosphatase (ATPase).This process is called oxidative phosphorylation.


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Overall equation for the fermentation reac-tion sequence, in which glucose is convertedto lactic acid (homolactic fermentation).

In aerobic respiration, molecular oxygen (O2) is utilized as the termi-nal electron acceptor. In anaerobic respiration, other oxidized substancesare used as terminal electron acceptors for electron transport chains. Suchmolecules include nitrate (NO3−), sulfur (S), sulfate (SO42−), carbonate(CO32−), ferric ion (Fe3+), and even organic compounds such as fumarateion, and trimethylamine N-oxide.

Abstraction of Chemical Bond Energy from InorganicCompounds (Chemolithotrophy)

Certain prokaryotes use reduced inorganic compounds such as hydrogen(H2), Fe2+, ammonia (NH3), nitrite (NO2−), sulfur, or hydrogen sulfide (H2S)as electron donors to specific electron transfer chains, commonly with O2as terminal electron acceptor but in some instances with CO2 or sulfate, togenerate ATP by oxidative phosphorylation.

Conversion of Light Energy to Chemical Energy (Phototrophy)

Photosynthesis is performed within membrane-bound macromolecularcomplexes containing pigments (bacteriochlorophylls, chlorophylls, caro-tenoids, bilins) that absorb light energy. The absorbed energy is conveyedto reaction centers, where it produces a charge separation in a special pairof chlorophyll (or bacteriochlorophyll) molecules. Reaction centers are spe-cialized electron transport chains. The charge separation initiates electronflow within reaction centers, and the light-energy driven electron flow gen-erates a vectorial proton gradient in a manner analogous to that describedabove for respiratory electron flow.

Some bacteria perform photosynthesis only under anaerobic conditions.This is termed anoxygenic photosynthesis. In other bacteria, photosynthesisis accompanied by the light-driven evolution of oxygen (similar to the pho-tosynthesis in chloroplasts). Such photosynthesis is termed oxygenic photo-synthesis.

Halobacteria perform a unique type of photosynthesis when the oxy-gen partial pressure is low. In the late 1960s, the cytoplasmic membraneof these organisms was found to contain an intrinsic membrane protein,bacteriorhodopsin, with a covalently attached carotenoid, retinal, as a chro-mophore. Absorption of light drives the isomerization of the retinal, afterwhich the retinal rapidly returns to its original conformation. The retinalphotocycle results in a vectorial pumping of protons by bacteriorhodopsinto the exterior of the cell with the generation of a proton motive force. ATP isgenerated at the expense of the proton gradient. Extensive screening of envi-ronmental samples shows that photosynthesis based on bacteriorhodopsinhomologs appears to be widespread in many genera of marine planktonicbacteria and most likely in bacteria in other environments as well.

Different prokaryotes use one or another of the above processes as a pre-ferred mode of energy generation. However, almost all prokaryotes are ableto switch from one form of energy production to another, depending on



the nature of the available substrates and on the environmental conditions.For example, purple nonsulfur bacteria grow on a variety of organic acidsas substrates and obtain energy from respiration when oxygen is present.However, under anaerobic conditions and in the presence of light, theseorganisms synthesize intracellular membranes that possess the complexesneeded for photosynthesis, and then they use light energy to generate ATP.Under aerobic conditions, the enteric bacterium E. coli oxidizes substratessuch as succinate and lactate and utilizes an electron transport system withubiquinone, cytochrome b, and cytochrome o as components and O2 as aterminal electron acceptor. Under anaerobic conditions, with formate asa substrate, E. coli utilizes an electron transport system with ubiquinoneand cytochrome b as components and nitrate as a terminal electron accep-tor. When E. coli is growing on oxaloacetate as a substrate under anaerobicconditions, the sequence of carriers is NADH, flavoprotein, menaquinone,and cytochrome b, and fumarate is the terminal electron acceptor. Thereare hundreds of other well-defined examples of such metabolic versatilityamong prokaryotes. This flexibility in modes of energy generation is lim-ited to the prokaryotes and gives these organisms a virtual monopoly on thecolonization of certain ecological niches.

THE IMPORTANCE OF THE IDENTIFICATION ANDCLASSIFICATION OF MICROORGANISMS

In the search for organisms to assist in a technical process or to produceunusual metabolites, each time a new organism can be placed within a well-studied genus, strong and readily testable predictions can be made con-cerning many of its genetic, biochemical, and physiological characteristics(Box 1.2).

CLASSIFICATION AND PHYLOGENY

Taxonomic systems for biological organisms are hierarchical. The most“Taxonomy (the science of classifica-tion) is often undervalued as a glori-fied form of filing – with each speciesin its folder, like a stamp in its pre-scribed place in an album; but taxon-omy is a fundamental and dynamicscience, dedicated to exploring thecauses of relationships and similari-ties among organisms. Classificationsare theories about the basis of nat-ural order, not dull catalogues com-piled only to avoid chaos.”

Source: Gould, S. J. (1989). Wonderful Life.The Burgess Shale and the Nature of History,New York: W. W. Norton & Co.

BOX 1.2

inclusive unit of classification is a kingdom (or domain), followed by phylum(or division), class, order, family, genus, species, and subspecies. By conven-tion, the scientific names of genera and species of organisms are italicized orare underlined (Table 1.4). An additional rank below the subspecies level –pathovar, serovar, or biotype – is added when it is desired to distinguish astrain by a special character that it possesses. For example, the rank of apathovar (or pathotype) is applied to an organism with pathogenic proper-ties for a certain host or hosts, as exemplified by Xanthomonas campestris pvvesicatoria, the causal agent of bacterial spot of pepper and tomato. Serovar(or serotype) refers to distinctive antigenic properties, and biovar (or bio-type) is applied to strains with special biochemical or physiological proper-ties.

In principle, any group of organisms can be classified according to any setof criteria, as long as the scheme results in reproducible identification of new


The Importance of the Identification and Classification of Microorganisms 11

TABLE 1.4 Ranking of taxonomic categories

Category Examples

Domain Archaea Bacteria FungiPhylum Crenarchaeota Proteobacteria AscomycotaClass Thermoprotei α-Proteobacteria SaccharomycetesOrder Sulfolobales Legionellales SaccharomycetalesFamily Sulfolobaceae Legionellaceae SaccharomycetaceaeGenus Sulfolobus Legionella SaccharomycesSpecies Sulfolobus acidocaldarius Legionella pneumophila Saccharomyces cerevisiae

strains. However, a classification scheme based on totally arbitrary criteriais likely to be of very limited practical use. Thus taxonomists may grouptogether apparently similar, presumably related species into a genus andpresumably related genera into a family in the hope that this classificationaccurately reflects the evolutionary or phylogenetic relationships amongvarious organisms. A hierarchical classification of this type was still beingused by the recognized authority in prokaryote taxonomy, Bergey’s Manualof Determinative Bacteriology (ninth edition), in 1994.

But how does one build such a taxonomic scheme? To classify a microor-ganism in this manner, one must first obtain a large uniform population ofindividuals, a pure culture. In the traditional methods of taxonomy, one thenexamines the organism’s phenotypic characters – that is, the properties thatresult from the expression of its genotype, which is defined as the completeset of genes that it possesses. Phenotype includes morphological character-istics such as the size and shape of individual cells and their arrangement inmulticellular clusters, the occurrence and arrangement of flagella, and thenature of membrane and cell wall layers; behavioral characteristics such asmotility and chemotactic or phototactic responses; and cultural character-istics such as colony shape and size, optimal growth temperature and pHrange, tolerance of the presence of oxygen and of high concentrations of salts,and the ability to resist adverse conditions by the formation of spores. Therange of compounds that support the growth of a given organism, the waythese compounds are degraded, and the nature of the end products (includ-ing the involvement of oxygen in the process) represent an important set ofphenotypic characters.

It is customary to examine dozens of characters; in the computer-basedmethod of numerical taxonomy, hundreds of characters may be examined.For identification of bacteria, armed with such information, one could thenconsult the ninth edition of Bergey’s Manual of Systematic Bacteriology. Theidentification of a bacterium is thus a relatively straightforward matter. How-ever, some difficulty is encountered when one wants to deduce phylogeneticrelationships between organisms on the basis of the classification schemepresented in that edition of Bergey’s Manual. A series of comments parallelto those made concerning prokaryotes can be made about the classificationof fungi.



In basing a classification scheme on phenotypic characters a taxonomistmust decide which characters are more fundamental and thus useful fordividing organisms into major groups, such as families, and which charactersare more variable and thus suitable for dividing the major groups into smallerones, such as species. In traditional taxonomy, the shape of the bacterial cell,for example, has been used for dividing bacteria into large groups. Thus ofthe lactic acid bacteria (which, as we will see later, characteristically obtainenergy by fermenting hexoses into lactic acid plus sometimes ethanol andcarbon dioxide), those with round cells and those with rod-shaped cells wereplaced in two completely different groups in the ninth edition of Bergey’sManual.

More recent quantitative information on the phylogenetic relationshipsamong organisms has become available through comparison of their DNAsequences. Because the prokaryote world is so diverse, however, this methodis only useful for comparing species of bacteria that are very closely related.Otherwise, the DNA sequences will be so dissimilar that no data of signif-icance will be obtained. Thus it was the use of rRNA sequences for com-parison, pioneered by Carl Woese in the early 1970s, that revolutionized thefield. rRNA is present and performs an identical function in every cellularorganism, and more importantly, its sequence has changed extremely slowlyduring the course of evolution. It is therefore an ideal marker for compar-ing distantly related organisms. Characteristic sequences of nucleotides, or“signature” sequences, may be conserved for a long time in a given branch ofthe phylogenetic tree and enable scientists to assign organisms on differentbranches with great confidence.

Returning to the classification of lactic acid bacteria, although the round-shaped lactic acid bacteria were placed far away from the rod-shaped onesin the 1994 Bergey’s Manual, their rRNA sequences show that many of theformer are actually very closely related to the latter.

We have now entered the era of phylogenetic systems of classification.The 2001 edition of Bergey’s Manual of Determinative Bacteriology (sec-ond edition) “follows a phylogenetic framework, based on analysis of thenucleotide sequence of the ribosomal small subunit RNA, rather than a phe-notypic structure.” We must always keep in mind the vast time scale weare dealing with when we consider the evolution of bacteria. Even bacte-ria that are thought to be closely related phylogenetically can be quite dis-tant on the evolutionary time scale, relative to the changes that have takenplace among higher organisms. Thus, if we are looking at characteristics thatchange rapidly during the course of evolution, then the phylogenetic rela-tionship may not offer much help. However, it will certainly help us in thestudy of slowly changing characters. An example is the organization and reg-ulation of biosynthetic pathways. Because the prokaryotic world is so diverse,different pathways are seen in the biosynthesis of even such common com-pounds as amino acids. The distribution and the mechanism of control ofthese pathways, which we need to know in order to use bacteria to pro-duce amino acids (see Chapter 9), clearly follow the 16S rRNA phylogeneticlines.



INFORMATION CONTENT OF 16S rRNA

The 16S rRNA is a component of the small ribosomal subunit (30S ribosomalsubunit) and is sometimes referred to as SSU rRNA. The predicted secondarystructure of 16S RNA is shown in Figure 1.5. This structure was based on theanalysis of approximately 7000 16S RNA sequences and was in about 98%accord with the crystal structure of the 16S RNA as seen in the high-resolutioncrystal structure of the 30S ribosomal subunit. Thus a common core of sec-ondary or higher order structures is preserved throughout evolution, withsome 67% of the bases involved in helix formation by intramolecular basepairing. Functional roles of the 16S RNA, conserved throughout evolution,doubtless dictate this high level of structure conservation.

Several websites provide databases of aligned 16S ribosomal DNA (rDNA)sequences (see references at the end of this chapter). Phylogenetic relation-ships are inferred from the number and character of positional differencesbetween the aligned sequences (see Box 1.3). These primary data are thensubjected to analysis by one of several tree-building algorithms. A tree isconstructed from the results of such an analysis in which the terminal nodes(the 16S rDNA sequences) represent a particular organism and the internalnodes (the inferred common ancestor 16S rDNA sequences) are connectedby branches. The branching pattern indicates the path of evolution, and thecombined lengths of the peripheral and internal branches connecting twoterminal nodes are a measure of the phylogenetic distance between two 16SrDNA sequences that serve as the surrogates for the source organisms.

On the basis of analyses of the relationships between 16S RNA genesequences, two phyla are recognized within Archaea and 23 phyla withinBacteria. The evolutionary relationships between these phyla are illustratedin Figure 1.6. The archaea cluster into two phyla, Crenarchaeota and Eur-yarcheota. The bacterial phyla cluster into three broad groups: deep-rootedbacterial groups, particularly thermophiles; the Gram-negative bacteria; andthe Gram-positive bacteria. Figure 1.7 shows the relationship between thesephyla and the major phenotypic groups of prokaryotes selected as the basisof the classification in the earlier version of Bergey’s Manual of SystematicBacteriology (ninth edition). The comparison illustrates vividly how a classi-fication based on phenotypic criteria can split into multiple groups speciesthat belong within a single phylogenetic group.

LIMITATIONS OF 16S rRNA PHYLOGENY

All biological classifications are human-imposed subdivisions upon the real-ity of the paucity of sharp discontinuities among the species in nature (Box1.4). Moreover, a classification, based on a single character even one as richin information as the 16S rRNA sequence, is bound to suffer from othershortcomings as well. This is evident from the following observations.

■ The divergence of present-day rRNA sequences allows us to establish thesuccession of common ancestral sequences. However, it does not allow adirect correlation to a time scale.


FIGURE 1.5

Predicted secondary structure of 16S rRNA. [Data from http://www.rna.icmb.utexas.edu/ and Cannone, J. J., Subramanian, S., Schnare,M. N., Collett, J. R., D’Souza, L. M., Du, Y., Feng, B., Lin, N., Madabusi, L. V., Muller, K. M., Pande, N., Shang, Z., Yu, N., and Gutell, R. R. (2002).The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, andother RNAs. BMC Bioinformatics, 3, 2; correction: BMC Bioinformatics, 3, 15.]

14



Information Content of 16S rDNA

There are 974 (63.2%) variable (informative) positions in the 16S rDNA of Bacteriaand 971 (63%) in that of Archaea. Four nucleotides may occupy a given position,and the maximum information content per position is defined by the number ofpossible character states (potential deletion or insertion is not considered).

Hence, the possible number of information bits is log2n × p, where n is thenumber of character states and p is the number of informative positions. This yields1948 bits of information for Bacteria and 1942 for Archaea. However, empiricallyit is found that the number of allowed character states varies from position toposition as follows:

Number ofnucleotidesper position Bacteria ArchaeaFour 407 (26.4%) 301 (19.5%)Three 209 (13.6%) 233 (15.2%)Two 358 (23.2%) 437 (28.3%)

Taking the above data into account, the information content is reduced to 1506bits for Bacteria and 1385 bits for Archaea.

Source: Ludwig, W., and Klenk, H-P. Overview: a phylogenetic backbone and taxonomic frame-work for prokaryotic systematics. (2001). In Bergey’s Manual of Systematic Bacteriology, 2ndEdition, Volume 1, G. M. Garrity (ed.), pp. 49–65, New York: Springer-Verlag.

BOX 1.3

■ A similarity in 16S rRNA gene se-quence between strains that exceeds97% is used to assign them to thesame genus. However, the genomesof some organisms contain multiplecopies of rRNA sequences. In cer-tain of these organisms, a signifi-cant degree of sequence divergenceexists between the multiple homol-ogous genes. For example, the acti-nomycete Thermospora bispora bis-pora contains two copies of the 16SrRNA gene on the same chromo-some within the same cell that dif-fer from each other at the sequencelevel by 6.4%. The archaeon Haloar-cula marismortui contains two rRNAoperons, which show a sequencedivergence of 5%. Such a situationposes problems for assignment of16S rRNA gene-based relationshipsfor these organisms.

■ Some organisms have identical 16S rRNA sequences but differ more at thewhole genome level than do other organisms whose rRNAs differ at severalvariable positions.

■ Sequencing of complete genomes shows that lateral gene transfer (dis-cussed later in this chapter) and recombination have played a significant rolein the evolution of prokaryote genomes. There is clear evidence in bacteriaclassified within the genera Bradyrhizobium, Mesorhizobium, and Sinorhi-zobium that distinct segments along the 16S rRNA gene sequences wereintroduced by lateral gene transfer followed by recombination (Figure 1.8).This resulted in incorrect tree topology and genus assignments and raisesthe strong possibility that other phylogenetic placements based solely on16S rRNA gene sequence divergence may need to be reassessed in the futureas more genomic information becomes available.

■ It is widely agreed that 16S rRNA phylogenetic relationships are of limitedvalue in predicting adequately the phenotypic capabilities of microorgan-isms.

DNA–DNA HYBRIDIZATION

It is now evident that there is insufficient difference between 16S rRNAsequences to distinguish between closely related species and that inter-strain DNA–DNA hybridization is the method of choice for assigning strainsto a species. This method measures levels of homology between completegenomes. The phylogenetic definition of a species by this technique is as



FIGURE 1.6

A two-dimensional projection of the phylo-genetic tree of the major prokaryotic groups.Groups that lie close to together are morelikely to have a recent common ancestrythan are those that are well separated. Thedashed lines in the time dimension belowthe plane indicate the still uncertain evo-lutionary origins of these groups. The com-putational procedure used to generate suchtwo-dimensional projections of the genomicsequence data is outlined by G. M. Garrityand J. G. Holt (2001) in Bergey’s Manual of Sys-tematic Bacteriology, 2nd Edition, Volume 1,Garrity, G. M. (ed.), pp. 119–123, New York:Springer-Verlag. (Courtesy of Peter H. A.Sneath.)

“strains with approximately 70% or greater DNA–DNA relatedness and with5◦C or less�Tm. Both values must be considered.” (Source: Wayne, L. G., et al.(1987). Report of the ad hoc committee on the reconciliation of approachesto bacterial systematics. International Journal of Systematic Bacteriology,37, 463–46). Tm is the melting temperature of the hybrid DNA duplexes asmeasured by stepwise denaturation by heating (see Figure 1.9). �Tm is thedifference in ◦C between homologous and heterologous hybrid duplexesformed under standard conditions.

PLASMIDS AND THE CLASSIFICATION OF BACTERIA

The genetic information of a bacterial cell is contained not only in the mainchromosome but also in extrachromosomal DNA elements called plasmids.Plasmids are self-replicating within a cell, and many plasmids have a blockof genes that enable them to move from one bacterial cell to another. Lossof its plasmids has no effect on the essential functions of a bacterial cell.Consequently, the cell is seen to act as host to the plasmids. Similar tobacterial chromosomes, but much smaller, plasmids are circular double-stranded DNA molecules. Plasmid DNA often replicates at a different rateand sometimes on a different schedule from those of chromosomal DNA,and cells may contain multiple copies of specific plasmids. Some plasmidsencode resistance to certain antibiotics or heavy metal ions or to ultraviolet


FIGU RE 1.7

Occurrence of major phenotypic groups within the 25 prokaryotic phyla. This figure illustrates the relationship between these phyla and the major phenotypicgroups of prokaryotes selected as the basis of the classification in the earlier version of Bergey’s Manual of Systematic Bacteriology (ninth edition). [Reproducedwith permission from Garrity, G.M., and Holt, J.G. (2001), The road map to the manual. In Bergey’s Manual of Systematic Bacteriology, 2nd Edition, Volume 1, Garrity,G.M. (ed.) p. 124, New York: Springer-Verlag.]

Prokaryotic phyla1 Group 12 Aerobic chemolithotropic bacteria and associated generaA1 Crenarcheota B12 Proteobacteria Group 13 Budding and/or appendaged bacteriaA2 Euryarcheota B13 Firmicutes Group 14 Sheathed bacteriaB1 Aquificae B14 Actinobacteria Group 15 Nonphotosynthetic, nonfruiting, gliding bacteriaB2 Thermotogae B15 Planctomycetes Group 16 Fruiting gliding bacteria: the myxobacteriaB3 Thermodesulfobacteria B16 Chlamydiae Group 17 Gram positive cocciB4 “Deinococcus-Thermus” B17 Spirochaetes Group 18 Endospore-forming Gram-positive rods and cocciB5 Chrysiogenetes B18 Fibrobacteres Group 19 Regular, nonsporulating, Gram-positive rodsB6 Chloroflexi B19 Acidobacteria Group 20 Irregular, nonsporulating, Gram-positive rodsB7 Thermomicrobia B20 Bacteroidetes Group 21 MycobacteriaB8 Nitrospirae B21 Fusobacteria Group 22 Nocardioform actinomycetesB9 Deferrobacteres B22 Verrucomicrobia Group 23 Actinomycetes with multilocular sporangiaB10 Cyanobacteria B23 Dictyoglomi Group 24 ActinoplanetesB11 Chlorobi Group 25 Streptomycetes and related genera

Group 26 MaduromycetesGroup 27 Thermomonospora and related generaGroup 28 ThermoactinomycetesGroup 29 Other actinomycete generaGroup 30 MycoplasmasGroup 31 The methanogensGroup 32 Archaeal sulfate reducersGroup 33 Extremely halophilic ArchaeaGroup 34 Archaea lacking a cell wallGroup 35 Extremely thermophilic and hyperthermophilic S-metabolizing

ArchaeaGroup 36 Hyperthermophilic non–S-metabolizing ArchaeaGroup 37 Thermophilic and hyperthermophilic bacteria

Major phenotypic groups of prokaryotes2

Group 1 SpirochetesGroup 2 Aerobic/microaerophilic, motile, helical/vibrioid, Gram negative bacteriaGroup 3 Nonmotile or rarely motile, curved Gram-negative bacteriaGroup 4 Gram-negative aerobic/microaerophilic rods and cocciGroup 5 Facultatively anaerobic Gram-negative rodsGroup 6 Anaerobic, straight, curved, and helical Gram-negative rodsGroup 7 Dissimilatory sulfate- or sulfite-reducing bacteriaGroup 8 Anaerobic Gram-negative cocciGroup 9 Symbiotic and parasitic bacteria of vertebrate and invertebrate speciesGroup 10 Anoxygenic phototrophic bacteriaGroup 11 Oxygenic phototrophic bacteria

1 Two phyla (A1 and A2) occur within the Archaea and B1-B23 within the Bacteria. These two prokaryotic domains were subdivided into these phyla on the basis of DNA sequence data,principally 16S and 23S rDNA. Earlier treatment of prokaryote taxonomy subdivided some 590 genera into major phenotypic groups (represented above as Groups 1-37). Assignment tothese phenotypic groups was based on readily recognizable phenotypic or metabolic characters that could be used for the presumptive identification of species [see Holt, J.G. et al. (eds.)(1994). Bergey’s Manual of Determinative Bacteriology, 9th Edition, Baltimore: Williams & Wilkins].

2 The group number refers to the phenotypic group used in the Bergey’s Manual of Determinative Bacteriology, 9th Edition.

17



radiation. Others, surprisingly, carry genes coding for functions that have“. . . [I]t is becoming clear that thebiodiversity is much greater thanexpected. When numerous strainsare analyzed and grouped by vari-ous methods, as in Xanthomonas, itappears that this genus constitutes acontinuum of geno- and phenotypeswith cloudy condensed nodes repre-senting ecologically more successfultypes. Thus, any attempt to divide bio-logical populations into discrete taxa,as is done in the current classifica-tion systems, will always be more orless artificial because of its inconsis-tency with the real continuous natureof biodiversity. Obviously, this situa-tion will be more pronounced in onegenus than another.”

Source: Vauterin, L., and Swings, J. (1997).Are classification and phytopathologicaldiversity compatible in Xanthomonas? Jour-nal of Industrial Microbiology & Biotechnology,19, 77–82.

BOX 1.4

been thought to be a distinguishing characteristic of the host species. Forexample, the most characteristic trait of the fluorescent Pseudomonas (seebelow) is thought to be its ability to degrade a wide range of organiccompounds; however, many of the genes that make these degradations pos-sible are located on plasmids. The same is true of the genes for nitrogen fix-ation in the species that carries out much of the biological nitrogen fixationon Earth – Rhizobium – and of the genes for disease-causing factors (toxins,proteases, or hemolysins; i.e., the proteins that lyse red blood cells and otheranimal cells) in many pathogenic bacteria. Because plasmids sometimesconfer highly noticeable phenotypic traits on their hosts, they may influ-ence the classification of the host organism. For example, certain strains ofStreptococcus lactis, classified as S. lactis subsp. diacetylactis, carry a plasmidthat allows them to utilize citrate. These are the strains responsible for thecharacteristic aroma of cultured butter, which results from the diacetyl theyproduce when fermenting citrate in milk.

Some plasmids have the ability to transfer themselves from one bacte-rial host cell into another. Sometimes the host is of a different species orgenus. On the other hand, the plasmid genes can become integrated intothe host’s chromosome and become a part of the permanently inheritedgenetic makeup of the cell. This “lateral” transfer of genetic informationinto different groups of bacteria, if it were to occur frequently, would makeevery bacterium into an extremely complex hodgepodge of genes comingfrom many different sources. Experimental studies, however, have shownthat lateral exchange certainly has not occurred to the extent of obliteratingthe phylogenetic lines of descent of various organisms.

The ability of plasmids to replicate themselves has been utilized in theconstruction of cloning vectors, many of which contain a replication func-tion derived from plasmids and can therefore be maintained indefinitely

A Bradyrhizobium elkanii cell with aBradyrhizobium 16S rRNA gene lineage

Lateral transfer (probably plasmid-mediated)of a 16S rRNA gene from a cell with aMesorhizobium sp. 16S rRNA gene lineage

Incorporation through recombinationof a short segment of the Mesorhizobiumgene into the B. elkanii gene

FIGURE 1.8

Diagrammatic representation of lateralgene transfer and recombination eventsleading to the incorporation of a short seg-ment of the 16S rRNA gene of Mesorhizo-bium mediterraneum (Upm-Ca 36) into the16S rRNA gene of Bradyrhizobium elkanii toproduce the present day B. elkanii (USDA 76)16S rRNA gene. [Based on data from vanBerkum, P., et al. (2003). Discordant phylo-genies within the rrn loci of Rhizobia. Journalof Bacteriology, 185, 2988–2998.]



Heterologous hybrid

Homologous hybrid

Rel

ativ

e A

bso

rban

ce a

t 2

60

nm 1.3

1.2

1.1

1.060 70 80 90 100

Temperature (°C)

Tm = 84°C

FIGU RE 1.9

Temperature dependence of the absorbanceof a solution of a perfectly complementaryDNA hybrid duplex at 260 nm (A260). Theseparation of the two strands (also termedthe “melting” of the DNA) is accompanied byan increase in the absorbance at 260 nm.The temperature at which the change inA260 is 50% complete is designated as themelting temperature (Tm). The Tm is sensi-tive to the pH and ionic strength of thebuffer. In the representation of a heterolo-gous hybrid (upper diagram), the arrows pointto noncomplementary positions in the twoDNA sequences. Such a hybrid would havea much lower Tm than the perfectly comple-mentary hybrid duplex whose melting curveis shown here.

in the cytoplasm of the host bacteria. However, in many cases, the replica-tion of plasmids requires the participation of host functions too. This is oneof the reasons why plasmids can survive in only a limited range of hosts.One way to construct a vector that can replicate in a wide range of hostsis to use the replication genes from a plasmid that has a broad host range.Here again, a knowledge of phylogenetic relationships will help us in pre-dicting the range of host bacteria that would support the replication of suchvectors. For example, many broad–host range plasmids isolated from theGram-negative bacteria of the “purple bacteria” group (see below) are likelyto replicate in most of the members of this group, or at least in the membersof the same subgroup.

ANALYSIS OF MICROBIAL POPULATIONSIN NATURAL ENVIRONMENTS

We have seen above how the sequence of 16S rRNA is utilized to clas-sify prokaryotes and to assign phylogenetic relationships. The universalacceptance of this molecular marker has resulted in the determinationof a very large number of 16S rDNA sequences. As of March 1, 2007, theRibosomal Database Project provides over 335,800 small ribosomal subunitrRNA sequences from a wide variety of prokaryotic taxa. By amplifying andsequencing the 16S rDNA of an unknown organism, it is now possible todetermine its phylogenetic relationship to the 16S rDNA sequences charac-teristic of the known genera of prokaryotes.

As of March 20, 2007, the curated Swiss-Prot database contains over1,500,000 distinct protein sequences from all kingdoms of organisms. Thecomplete sequences of over 480 prokaryote genomes have been determinedas of January, 2007, and contribute substantially to the total content ofthis database. Comparative sequence information on protein coding genesallows the use of molecular markers other than 16S rDNA to explore incomplementary ways the taxonomy, phylogeny, and functional diversity ofprokaryotes and has opened the way to powerful in situ analyses of microbialpopulations in natural environments.

NUCLEIC ACID SEQUENCE–BASED METHODSIN ENVIRONMENTAL MICROBIOLOGY

We examine three powerful methods selected from among many sequence-based approaches to the study of natural microbial populations. Labelednucleic acid probes allow sensitive detection and enumeration of cells in amixed population that contain a particular nucleic acid sequence. Where thesequences of flanking regions of a DNA sequence of interest are known, thepolymerase chain reaction (PCR) allows completely selective amplificationof that sequence from a complex mixture of nucleic acids. Finally, whole-genome shotgun sequencing of the DNA of microbial populations providesunique insights into both the complexity of natural microbial populations



Frac

tion

of

pro

be

bo

un

d

1.00

0.75

0.50

0.25

0Tm(3) Tm(2) Tm(1)

Temperature

1 Perfect match between probe and target

Probe 3' -5'Target 5' -3' ---CGGGCCUCUUGCCAUCGGAUGUGCCCAGAU---

2 One mismatch between probe and target near 5' end of probe

Probe 3' -5'Target 5' -3' ---CGGGCCUCUUGCCAUCGGAUCUGCCCAGAU---

3 Internal mismatch between probe and target

Probe 3' -5'Target 5' -3' ---CGGGCCUCUUGCGAUCGGAUGUGCCCAGAU---

GAGAACGGUAGCCUACAC•



FIGURE 1.10

Hybridization of oligonucleotide probes toa target DNA sequence. The sequence ofprobe 1 is perfectly complementary to thatof the target DNA, whereas there is one mis-match (position indicated in boldface in thesequence of the target DNA) in each of theoligonucleotide probes 2 and 3. The blackdot at the 5′ end of each probe indicatesa covalently attached fluorescent label. Thelower panel illustrates the dissociation pro-file of each of the hybrids. Note that thehigher the Tm the higher the stringency ofhybridization.

and the functions of some of the organisms present. Together, these methodsprovide information on the microbial diversity, gene content, and relativeabundance of organisms in environmental samples.

NUCLEIC ACID PROBES

The interactions between three related oligonucleotide probes and a targetDNA sequence are illustrated in Figure 1.10. There is a perfect complemen-tarity between the probe and the target in case 1 and a single mismatch, indifferent positions, between the probe and the target in cases 2 and 3. Asillustrated, under appropriate conditions, each of these probes can base-pair (hybridize) with the denatured target DNA. However, the hybrids withmismatches will be less stable than the one with the perfectly complemen-tary probe and as illustrated in the lower panel of Figure 1.10, will dissociateat a lower temperature (Tm) than the perfectly matched probe. The stabilityof the hybrid is affected by both the location and the character of the mis-match. Appropriate selection of the composition of the hybridization bufferand the temperature is critical to optimize the stringency of the hybridization,that is, the selection of the conditions under which the perfectly matchedprobe will remain bound to immobilized target nucleic acid while hybridswith mismatches either will not form or will dissociate upon washing with



probe-free hybridization buffer. The optimum temperature for hybridiza-tion is thus slightly below the Tm. Note that the probes illustrated in Figure1.10 bear fluorescent labels that allow optical detection with very high sen-sitivity.

The huge database of 16S rDNA sequences guides the design of PCRoligonucleotide primers that are complementary to different sets of targetsequences. Universal primers can be selected that are complementary to aregion of 16S rDNA sequence that is perfectly conserved in all 16S rDNAgenes. Using the same approach, primers unique to archaeal and eukaryalsequences or those complementary only to bacterial sequences can be chosenand enable the selective amplification of these sets of sequences. Any oneof such PCR primers can be labeled with a fluorophore (or other label) andutilized as a probe for the target sequence. At the highest level of selectivity,if a 16S rDNA sequence is fully known, an oligonucleotide label can be syn-thesized that will bind with high stringency exclusively to this unique targetsequence.

CATALOGS OF 16S rDNA SEQUENCES: MEASURES OF MICROBIALDIVERSITY IN NATURAL ENVIRONMENTS

Total DNA can be readily obtained from diverse natural environments. PCRprimers, chosen as described above, are then utilized for the amplifica-tion of the 16S rDNA sequences present in the DNA sample. The amplifiedsequences are then cloned and sequenced, and the resulting catalog of 16SrDNA sequences is a set of molecular signatures for the organisms presentin the environment under study and a measure of the diversity of the popu-lation. The individual 16S rDNA sequences also provide information on thetaxonomic affinities and phylogenetic relationships between these organ-isms and well-studied microorganisms. A minute fraction of microorganismspresent in any natural environment i

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