Genetics of Bacteria PDF
Preview Genetics of Bacteria
Sheela Srivastava Genetics of Bacteria Genetics of Bacteria Sheela Srivastava Genetics of Bacteria 123 Sheela Srivastava Department of Genetics Universityof Delhi, SouthCampus New Delhi, Delhi India ISBN 978-81-322-1089-4 ISBN 978-81-322-1090-0 (eBook) DOI 10.1007/978-81-322-1090-0 SpringerNewDelhiHeidelbergNewYorkDordrechtLondon LibraryofCongressControlNumber:2013930625 (cid:2)SpringerIndia2013 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeor part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway, andtransmissionorinformationstorageandretrieval,electronicadaptation,computersoftware, orbysimilarordissimilarmethodologynowknownorhereafterdeveloped.Exemptedfromthis legalreservationarebriefexcerptsinconnectionwithreviewsorscholarlyanalysisormaterial suppliedspecificallyforthepurposeofbeingenteredandexecutedonacomputersystem,for exclusiveusebythepurchaserofthework.Duplicationofthispublicationorpartsthereofis permitted only under the provisions of the Copyright Law of the Publisher’s location, in its currentversion,andpermissionforusemustalwaysbeobtainedfromSpringer.Permissionsfor use may be obtained through RightsLink at the Copyright Clearance Center. 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Printedonacid-freepaper SpringerispartofSpringerScience+BusinessMedia(www.springer.com) Preface From the time microorganisms could be seen, described, and studied, they have provided a useful system to gain insight into the basic prin- ciplesoflife,thoughwearestillfarfromunderstandingthemfully.The relativesimplicity,whichmayoftenbedeceptive,mademicrobesideally 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,thegeneration,expression,andtransmissionofbiologicalvariation. Thus, the most crucial requirement of genetic analysis is to select or introducevariationsinaspecificgene(mutation\s).Evenwiththerapid growth of modern molecular biology, the relevance of genetic analyses that depends on finding the mutants and using them to elucidate the normalstructureandoperationofabiologicalsystemhasnotbeenlost. Mutation may lead to an altered trait in an organism and if the change takes place in the observable characteristics (phenotype) of that organism,thiscouldbeusedtofollowthetransmissionofthesaidgene. 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 newdirection,asthegenescouldbeisolatedandstudiedwithoutaprior 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 neverlostitsrelevance.Also,itshouldbecomeclearthatmostgenesdo not function in isolation and its real understanding can come through in vivo analysis only. When microorganisms, first fungi and then bacteria, were employed asmodelsystemsingeneticanalysis,theyofferedimmediateadvantages 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 vi Preface mutants could be obtained, a whole range of biochemical mutants became availableina veryshorttime. The availability ofthese mutants and their amenability to detail genetic and biochemical analyses led to thegenerationof awhole lot ofinformationaboutgeneexpression 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 thatadifferentstrategyhadtobeemployed.Whileinhigherorganisms, 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 vastthatcreationofabranchofMicrobialGeneticsbecamethoroughly 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 bacteriaalonearealsoavailableandhaveservedthepurposeofauseful 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 whereemphasishasbeenonthetransmissionaspects,alongwithgiving due share to the generation ofbiological variation, because withoutthe latter, the former is not possible. The omission of expression part has indeed beenintentional. Andthe reason: alarge volume of information available on this aspect in books dealing with genetics, biochemistry, cellsbiology,molecularbiology,andbiotechnology.Thus,theinclusion of such information would only have amounted to repetition. Bacterialgeneticsismovingthroughanimportantphaseinitshistory. 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 Preface vii requiresmutagenesis,genetransfer,andgeneticrecombinationfollowed 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 hugebodyofknowledgeandacontinuallygreaterunderstandingofthe nature ofgenes. The basic research inmicrobial geneticshasnotceased but continues to reveal phenomena important to the understanding of life and its processes as a whole. So, while bacterial genetics paved the wayforstudyinggeneticsystemsotherthanbacteria,italsoventuredto 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 recombinantDNA(r-DNA)technology.Throughtheseremarkablebut straightforward biochemical techniques usually called genetic engi- neeringorgenecloning,thegenotypeofanorganismcanbemodifiedin a directed and pre-determined way. The r-DNA technology, in fact, ushered in the era of manipulation of DNA outside the cell, recombi- nationinvitro,andreintroductionofrecombinantDNAintoanewcell. 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- versionofhundredsofresearchlaboratoriesintogenecloningfactories, 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 againthisaspecthasalsonotbeentoucheduponinthepresenttreatise, 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. Thecontentsofthisbookarespreadoversevenchapters.InChap. 1, the readers are familiarized with the genetic terminology and some of thebasicgenetictoolsappliedtobacteria.Chapter 2dealswiththebasic mechanism of mutation, not unique to bacteria, but to which bacteria havemadeseminalcontributions.Thenextthreechaptersdescribethree 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 viii Preface with Transduction. Chapter 6 is devoted to the discussion on different aspects of an extra-chromosomal genetic unit, the Plasmid and its Biology.ThelastchapterdescribestheTransposableElementsandtheir 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. 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,astheycanbeexploitedtogainknowledgeonvariousfacetsof biology. I have selected the aspects dealing with ‘‘Genetics’’ for this treatise,becausethisisoneareainwhichtheyhavemadetheirpresence stronglyfelt.Inclusionofalltheinformationthatisbecomingavailable throughbacteria,inafewhundredpages,however,turnedouttobenot so easy. I hope that this book will promote better understanding of the subjectandigniteyoungmindstoventureintoscientificresearchtaking 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- tionsonlife.Theimmensebodyofknowledgegeneratedwiththehelp 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. • Mycolleaguesandfriendsfortheirvaluablediscussionsandinputson 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 . . . . . . . . . . . . . . . . . . . . 1 1.1 Bacterial Nucleoid. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Genetic Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3 Methods of Genetic Analysis. . . . . . . . . . . . . . . . . . . . 6 1.4 What is a Bacterial Cross? . . . . . . . . . . . . . . . . . . . . . 10 1.5 Genetic Exchange in Bacteria . . . . . . . . . . . . . . . . . . . 12 Further Readings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2 Gene Mutation: The Basic Mechanism for Generating Genetic Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.1 What is Mutation? . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 Why Mutation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Detection of Mutation. . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4 Characterization of Mutation. . . . . . . . . . . . . . . . . . . . 20 2.5 Biochemical Nature of Mutation . . . . . . . . . . . . . . . . . 21 2.6 Spontaneous Mutations. . . . . . . . . . . . . . . . . . . . . . . . 23 2.7 Induced Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.8 DNA Damage and Repair Pathway . . . . . . . . . . . . . . . 32 2.9 General Repair Mechanisms . . . . . . . . . . . . . . . . . . . . 36 2.10 Site-Directed Mutagenesis. . . . . . . . . . . . . . . . . . . . . . 42 2.11 Why are Mutations Important? . . . . . . . . . . . . . . . . . . 51 2.12 Reversion and Suppression . . . . . . . . . . . . . . . . . . . . . 54 2.13 Directed Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Further Readings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3 Conjugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.1 The Historical Cross. . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.2 Compatibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.3 Formation of Recombinants. . . . . . . . . . . . . . . . . . . . . 63 3.4 High Frequency Recombination Donors . . . . . . . . . . . . 64 3.5 Kinetics of Gene Transfer and Mapping. . . . . . . . . . . . 65 3.6 Generation of Different Hfr Strains . . . . . . . . . . . . . . . 72 3.7 F-Prime Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.8 Structure of F Plasmid . . . . . . . . . . . . . . . . . . . . . . . . 74 3.9 Structure of the DNA Transfer Apparatus. . . . . . . . . . . 79 3.10 Chromosome Transfer and Recombination . . . . . . . . . . 81 3.11 Conjugation in Other Gram-Negative Bacteria. . . . . . . . 83 xi
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