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Sheela SrivastavaDepartment of GeneticsUniversity of Delhi, South CampusNew Delhi, DelhiIndia
ISBN 978-81-322-1089-4 ISBN 978-81-322-1090-0 (eBook)DOI 10.1007/978-81-322-1090-0Springer New Delhi Heidelberg New York Dordrecht London
Library of Congress Control Number: 2013930625
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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
xi
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
xii Contents
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’’.
xv