Genetics of Bacteria

Sheela Srivastava

Genetics of Bacteria

123

Sheela SrivastavaDepartment of GeneticsUniversity of Delhi, South CampusNew Delhi, DelhiIndia

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Preface

From the time microorganisms could be seen, described, and studied,

they have provided a useful system to gain insight into the basic prin-

ciples of life, though we are still far from understanding them fully. The

relative simplicity, which may often be deceptive, made microbes ideally

suited for answering some very fundamental questions in science.

Microorganisms have been employed in almost all fields of biological

studies, including the science of Genetics.

The whole edifice of classical genetics centers around three processes

viz, the generation, expression, and transmission of biological variation.

Thus, the most crucial requirement of genetic analysis is to select or

introduce variations in a specific gene (mutation\s). Even with the rapid

growth of modern molecular biology, the relevance of genetic analyses

that depends on finding the mutants and using them to elucidate the

normal structure and operation of a biological system has not been lost.

Mutation may lead to an altered trait in an organism and if the change

takes place in the observable characteristics (phenotype) of that

organism, this could be used to follow the transmission of the said gene.

The genetic composition (genotype) of the organism could be inferred

from the observable characteristics. With the improved biochemical

techniques and instrumentation, and the revelation of the chemical

nature of the gene (DNA, RNA in some viruses), it became possible to

dissect the gene expression/function at the molecular level. In all these

studies, gene mutations occupied the center stage.

Gene cloning and other techniques of gene manipulation provided a

new direction, as the genes could be isolated and studied without a prior

requirement of obtaining mutants. Moreover, these genes could be

altered specifically and in a desired manner in vitro (site-directed

mutagenesis). However, deriving the gene function by its alteration

never lost its relevance. Also, it should become clear that most genes do

not function in isolation and its real understanding can come through

in vivo analysis only.

When microorganisms, first fungi and then bacteria, were employed

as model systems in genetic analysis, they offered immediate advantages

in studying all the three aspects of heredity: being haploid and struc-

turally simpler it became easy to isolate mutations of various kinds

and relate them to a specific function. Though very few morphological

v

mutants could be obtained, a whole range of biochemical mutants

became available in a very short time. The availability of these mutants

and their amenability to detail genetic and biochemical analyses led to

the generation of a whole lot of information about gene expression and

its regulation. So much so, that they provided the first clues, and the

platform for studying the complex eukaryotic systems.

It was when transmission of biological variation was to be studied

that a different strategy had to be employed. While in higher organisms,

such a line of study would require phenotypic markers in a controlled

hybridization, in microorganisms, especially bacteria, a more genetic

approach needed to be employed.

Both bacteria and their viruses and fungi have been extensively

exploited for genetic analyses. The information so generated became so

vast that creation of a branch of Microbial Genetics became thoroughly

justified. Microorganisms have not attempted to alter any established

genetic concept but the technique applied to them and the way the

results are to be interpreted are so different from higher organisms that

their clubbing together may cause some confusion. In the same vein,

fungi and bacteria represent two entirely different types of biological

systems, i.e., eukaryotic and prokaryotic, respectively. Thus, it would

not be inappropriate to treat them separately.

Bacteria, the simplest of the living organisms, have provided enough

material on all aspects of genetics. In any compendium, however, the

treatment of these aspects may be very different. In most basic genetics

books, bacterial genetics may occupy the place of a chapter with

information about mutation and expression combined with other

eukaryotes. Some books dealing with microbes or more correctly with

bacteria alone are also available and have served the purpose of a useful

resource book on Microbial Genetics to teachers as well as students.

While teaching a course on Microbial Genetics for the last 25 years to

post-graduate students at Delhi University, I have realized that a book

on Bacterial Genetics may be very handy to students, researchers, and

teachers alike. However, a new format has been planned for this book

where emphasis has been on the transmission aspects, along with giving

due share to the generation of biological variation, because without the

latter, the former is not possible. The omission of expression part has

indeed been intentional. And the reason: a large volume of information

available on this aspect in books dealing with genetics, biochemistry,

cells biology, molecular biology, and biotechnology. Thus, the inclusion

of such information would only have amounted to repetition.

Bacterial genetics is moving through an important phase in its history.

While on the one hand, this field of study continues to remain instru-

mental in the development of new tools and methodologies for better

understanding of molecular biology, on the other, it provides scientists

with a strong handle whose ultimate impact is hard to foresee. In

addition to providing an insight into basic biological questions, genetic

knowledge can also be used to manipulate biological systems for sci-

entific or economic reasons. Traditionally, genetic manipulation

vi Preface

requires mutagenesis, gene transfer, and genetic recombination followed

by selection for desired characteristics. However, with such techniques,

geneticists are forced to work with random events with selection often

quite complex to detect rare mutations with the desired genotypes.

Moreover, the nature of the gene and its function in most cases often

remain unclear.

The application of microbial genetics led to the accumulation of a

huge body of knowledge and a continually greater understanding of the

nature of genes. The basic research in microbial genetics has not ceased

but continues to reveal phenomena important to the understanding of

life and its processes as a whole. So, while bacterial genetics paved the

way for studying genetic systems other than bacteria, it also ventured to

provide solutions for specific industrial, environmental, ecological,

pharmaceutical, and other problems. In the early 1970s, microbial

genetics itself underwent a revolution with the development of the

recombinant DNA (r-DNA) technology. Through these remarkable but

straightforward biochemical techniques usually called genetic engi-

neering or gene cloning, the genotype of an organism can be modified in

a directed and pre-determined way. The r-DNA technology, in fact,

ushered in the era of manipulation of DNA outside the cell, recombi-

nation in vitro, and reintroduction of recombinant DNA into a new cell.

In this way, novel organisms with characteristics drawn from distant

species and genera can be created. For example, human genes can be

transferred to a bacterium, and a bacterial gene placed into plants or

animal cells. In fact, the glitter of this technology has led to the con-

version of hundreds of research laboratories into gene cloning factories,

and to the development of a new industry known as bioengineering or

biotechnology. Biotechnologists, however, draw heavily from the clas-

sical as well as molecular genetics, when it comes to get the required

information, and realizing the applications of this technology. Once

again this aspect has also not been touched upon in the present treatise,

as innumerable information is available elsewhere. In this era of

genomics, bacteria figure extensively in genome sequencing projects,

adding on to loads of new information. A large number of bacterial

genomes have been sequenced, but several gene functions even in the

best-studied organisms, such as Escherichia coli and Bacillus subtilis,remain unknown. Many of these genes do not resemble the other genes

characterized in the database, throwing the whole field open for

discovering new pathways.

The contents of this book are spread over seven chapters. In Chap. 1,

the readers are familiarized with the genetic terminology and some of

the basic genetic tools applied to bacteria. Chapter 2 deals with the basic

mechanism of mutation, not unique to bacteria, but to which bacteria

have made seminal contributions. The next three chapters describe three

different pathways through which the inter-bacterial gene transfer is

materialized. All these have been essential to generate the genetic vari-

ability that has profoundly impacted the bacterial evolution. Chapter 3

describes Conjugation, Chap. 4, Transformation, and Chap. 5 deals

Preface vii

with Transduction. Chapter 6 is devoted to the discussion on different

aspects of an extra-chromosomal genetic unit, the Plasmid and its

Biology. The last chapter describes the Transposable Elements and their

contribution to bacterial evolution. A set of important references has

been provided and an Index has been appended at the end.

This book intends to initiate the readers into the field of bacterial

genetics. Familiarizing them with the tools and techniques of both

classical and molecular genetics and exposing them to the strength of

bacterial systems in analyzing basic concepts of Genetics, on the one

hand, and prepare them to confront newer and newer challenges that

bacteria continue to throw at the scientific community.

viii Preface

Acknowledgments

To document comprehensive information on any aspect of bacteria,

the invisible, tiny, omnipresent organisms, is a herculean task. These

organisms have turned out to be more of a boon for the scientific com-

munity, as they can be exploited to gain knowledge on various facets of

biology. I have selected the aspects dealing with ‘‘Genetics’’ for this

treatise, because this is one area in which they have made their presence

strongly felt. Inclusion of all the information that is becoming available

through bacteria, in a few hundred pages, however, turned out to be not

so easy. I hope that this book will promote better understanding of the

subject and ignite young minds to venture into scientific research taking

bacteria as model system. While completing this book, information has

been drawn from various sources. Therefore

I acknowledge:

• All the authors and researchers who have contributed extensively in

this area of science. Their work has not only served as an important

resource but also helped format the framework of this book.

• The scientists and investigators, whose work continues to document

the usefulness of the bacterial system to answer fundamental ques-

tions on life. The immense body of knowledge generated with the help

of these tiny organisms has been instrumental in the sharp growth of

biological sciences as a whole.

• The students over the years, who posed probing and sometimes

uncomfortable questions. Their inquisitiveness has helped keep their

perspective in mind.

• My colleagues and friends for their valuable discussions and inputs on

the subject. Prof. P. S. Srivastava for critically going through the

manuscript.

• The help rendered by Ms. Vandana at various stages in the prepa-

ration of this manuscript.

Sheela Srivastava

ix

Contents

1 Bacteria and Science of Genetics . . . . . . . . . . . . . . . . . . . . 11.1 Bacterial Nucleoid . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Genetic Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . 51.3 Methods of Genetic Analysis. . . . . . . . . . . . . . . . . . . . 61.4 What is a Bacterial Cross? . . . . . . . . . . . . . . . . . . . . . 101.5 Genetic Exchange in Bacteria . . . . . . . . . . . . . . . . . . . 12Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2 Gene Mutation: The Basic Mechanism for GeneratingGenetic Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.1 What is Mutation? . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2 Why Mutation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.3 Detection of Mutation. . . . . . . . . . . . . . . . . . . . . . . . . 192.4 Characterization of Mutation . . . . . . . . . . . . . . . . . . . . 202.5 Biochemical Nature of Mutation . . . . . . . . . . . . . . . . . 212.6 Spontaneous Mutations . . . . . . . . . . . . . . . . . . . . . . . . 232.7 Induced Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.8 DNA Damage and Repair Pathway . . . . . . . . . . . . . . . 322.9 General Repair Mechanisms . . . . . . . . . . . . . . . . . . . . 362.10 Site-Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . 422.11 Why are Mutations Important? . . . . . . . . . . . . . . . . . . 512.12 Reversion and Suppression . . . . . . . . . . . . . . . . . . . . . 542.13 Directed Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3 Conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.1 The Historical Cross. . . . . . . . . . . . . . . . . . . . . . . . . . 593.2 Compatibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.3 Formation of Recombinants. . . . . . . . . . . . . . . . . . . . . 633.4 High Frequency Recombination Donors . . . . . . . . . . . . 643.5 Kinetics of Gene Transfer and Mapping . . . . . . . . . . . . 653.6 Generation of Different Hfr Strains . . . . . . . . . . . . . . . 723.7 F-Prime Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . 733.8 Structure of F Plasmid . . . . . . . . . . . . . . . . . . . . . . . . 743.9 Structure of the DNA Transfer Apparatus . . . . . . . . . . . 793.10 Chromosome Transfer and Recombination . . . . . . . . . . 813.11 Conjugation in Other Gram-Negative Bacteria. . . . . . . . 83

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3.12 Conjugation in Gram-Positive Bacteria . . . . . . . . . . . . . 853.13 Conjugation in Genetic Analysis . . . . . . . . . . . . . . . . . 88Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

4 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914.1 Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914.2 The Nature of Transforming Principle . . . . . . . . . . . . . 924.3 Transformation as a Method of Gene Transfer. . . . . . . . 934.4 Natural Transformation. . . . . . . . . . . . . . . . . . . . . . . . 944.5 Artificial Transformation. . . . . . . . . . . . . . . . . . . . . . . 1034.6 Transformation in Genetic Analysis . . . . . . . . . . . . . . . 105Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

5 Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1095.1 Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1095.2 Vegetative Growth of Phage . . . . . . . . . . . . . . . . . . . . 1095.3 Generalized Transduction . . . . . . . . . . . . . . . . . . . . . . 1105.4 Specialized or Restricted Transduction . . . . . . . . . . . . . 1185.5 Transduction in Genetic Analysis. . . . . . . . . . . . . . . . . 122Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

6 Plasmids: Their Biology and Functions . . . . . . . . . . . . . . . . 1256.1 Detection and Nomenclature . . . . . . . . . . . . . . . . . . . . 1256.2 Plasmid Organization . . . . . . . . . . . . . . . . . . . . . . . . . 1276.3 Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286.4 Copy Number Control . . . . . . . . . . . . . . . . . . . . . . . . 1296.5 Plasmid Stability and Maintenance. . . . . . . . . . . . . . . . 1356.6 Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446.7 Plasmid Incompatibility . . . . . . . . . . . . . . . . . . . . . . . 1446.8 Plasmid Amplification . . . . . . . . . . . . . . . . . . . . . . . . 1456.9 Plasmid Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456.10 F Plasmid: A Prototype Model System . . . . . . . . . . . . . 1466.11 Other Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

7 Transposable Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . 1537.2 Insertion Sequences (IS) . . . . . . . . . . . . . . . . . . . . . . . 1547.3 Transposons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1577.4 Bacteriophage Mu . . . . . . . . . . . . . . . . . . . . . . . . . . . 1587.5 Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607.6 Target Site Duplications (TSD)/Repeats . . . . . . . . . . . . 1617.7 Influence on Gene Expression . . . . . . . . . . . . . . . . . . . 1617.8 IS1 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627.9 Transposon Tn3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627.10 Transposon Tn5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

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7.11 Transposon Tn10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647.12 Transposon Tn7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647.13 Bacteriophage Mu . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667.14 Delivery of Transposable Elements . . . . . . . . . . . . . . . 1667.15 Mechanisms of Transposition . . . . . . . . . . . . . . . . . . . 1677.16 Regulation of Transposition. . . . . . . . . . . . . . . . . . . . . 1717.17 Transposition Immunity . . . . . . . . . . . . . . . . . . . . . . . 1737.18 Target Site Selection . . . . . . . . . . . . . . . . . . . . . . . . . 1747.19 Conjugative Transposons . . . . . . . . . . . . . . . . . . . . . . 1757.20 Integrons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1777.21 Transposable Elements as Genetic Tools. . . . . . . . . . . . 1807.22 Transposable Elements and their Impact on the Host . . . 182Further Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Additional Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Contents xiii

About the Author

Sheela Srivastava is currently Professor of Genetics at University of Delhi,South Campus, India, where she has been on the faculty since 1984. Withher initial training in Botany with specialization in Genetics at Master’slevel, she achieved her Ph.D. in Biochemical Genetics of the fungus,Aspergillus nidulans. Her post-doctoral stint in bacterial molecular geneticsled to her scientific interest focusing on bacteria. Her major area of researchis in genetics of metal–microbe interaction, plant growth promotingcharacteristics of rhizospheric bacteria, peptide antibiotic production bylactic acid bacteria, and metagenomics. Being associated with the Depart-ment of Genetics since its inception, she has served as Head of theDepartment, Dean, Faculty of Interdisciplinary and Applied Sciences,Chairman, Board of Research Studies, and Chairman, Committee ofCourses. She has co-authored two books: Understanding Bacteria (KluwerAcademic Publishers, 2003) and Introduction to Bacteria (Vikas PublishingHouse, 1983) besides co-editing a few volumes. She is currently teachingcourses on introductory prokaryotic genetics and advanced courses onbacterial and bacteriophage genetics to post-graduate, M.Phil, and Ph.D.students. Her tag line is: ‘‘the most challenging job of a teacher in this fieldis how to make young students learn genetics’’.

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