The Ministry of Health Protection and Social Development
The Stavropol State Medical Academy
Microbiology, Virology, Immunology Department
I.A.Bazikov, I.V.Klimanovich
The Manual
for the Students of General Medicine
of the English-speaking Medium
Stavropol 2010
МИНИСТЕРСТВО ЗДРАВООХРАНЕНИЯ И
СОЦИАЛЬНОГО РАЗВИТИЯ РФ
Ставропольская государственная медицинская академия
Кафедра микробиологии, вирусологии и иммунологии
The Ministry of Health Protection and Social Development
The Stavropol State Medical Academy
Microbiology, Virology, Immunology Department
Базиков И.А., Климанович И.В.
I.A.Bazikov, I.V. Klimanovich
Генетика бактерий
Учебное пособие для студентов англоязычного отделения
Bacterial Genetics
The Manual for the Students
of the English-speaking Medium
Ставрополь 2010
Stavropol 2010
2
УДК 535.317.68 (07.07)
ББК 52.64 я 73
G 36
Генетика бактерий. Учебное пособие для студентов англоязычного
отделения (на английском языке). – Ставрополь: Изд-во СтГМА. – 2010. –
32с.
Авторы:
Базиков Игорь Александрович, доктор медицинских наук, профессор,
зав. кафедрой микробиологии, вирусологии и иммунологии СтГМА.
Климанович Инна Викторовна, ассистент кафедры микробиологии
вирусологии и иммунологии СтГМА.
Учебное пособие включает в себя основные темы курса «Генетика
бактерий» для студентов англоязычного отделения. Оно состоит из
следующих
разделов:
«Организация
генетического
материала
микроорганизмов», «Фенотипические вариации», «Генотипические
вариации», «Генетические рекомбинации» и «Молекулярная генетика».
Рецензенты:
Ходжаян Анна Борисовна, доктор медицинских наук, профессор, зав.
кафедрой биологии с экологией СтГМА;
Знаменская Стояна Васильевна, кандидат педагогических наук,
доцент, зав. кафедрой иностранных языков с курсом латинского языка, декан
факультета иностранных студентов СтГМА;
Тимченко Людмила Дмитриевна, доктор ветеринарных наук,
профессор кафедры общей биологии СГУ.
УДК 535.317.68 (07.07)
ББК 52.64 я 73
G 36
Рекомендовано к изданию Цикловой методической комиссией
Ставропольской государственной медицинской академии по англоязычному
обучению иностранных студентов.
Разрешено к печати редакционно-издательским Советом СтГМА
© Ставропольская государственная
медицинская академия, 2010
3
УДК 535.317.68 (07.07)
ББК 52.64 я 73
G 36
Bacterial Genetics. Manual for the students of the English-speaking Medium
(in English). – Stavropol. – Publisher: Stavropol State Medical Academy. – 2010.
– 32 p.
Authors:
I.A. Bazikov, Professor, D.M.S., the Head of Microbiology, Virology,
Immunology Department of the Stavropol State Medical Academy,
I.V. Klimanovich, assistant of the Microbiology, Virology, Immunology
Department of the Stavropol State Medical Academy
Presented manual includes the basic themes of course “Bacterial Genetics”
for the students of the English-speaking Medium. It consists of following themes:
“Genome Organization of Microorganisms”, “Phenotypic Variation”,
“Genotypic Variation”, “Gene Transfer” and “Molecular Genetics”.
Reviewers:
Hodzhayan Anna Borisovna, Professor, D.M.S., the Head of Biology with
Ecology Department of the Stavropol State Medical Academy,
Znamenskaya Stoyana Vassilievna, C.P.S., Associate Professor, the Head
of Latin and Foreign Languages Department, Dean of the foreign students’ faculty
of the Stavropol State Medical Academy;
Timchenko Lyudmila Dmitrievna, Professor, D.V.S., of the General
Biology Department of the Stavropol State University.
УДК 535.317.68 (07.07)
ББК 52.64 я 73
G 36
The Textbook is recommended to publishing Cyclic Methodical Commission
Stavropol State Medical Academy on English education foreign student.
The Textbook is allowed to seal editorial-publishing Advice StSMA
© Stavropol State Medical
Academy, 2010
4
ВВЕДЕНИЕ
Учебное пособие по микробиологии на английском языке предназначено
для студентов англоязычного отделения. Оно включает основные темы из
курса «Бактериальная генетика».
Цель учебного пособия – знакомство студентов второго курса с общими
понятиями молекулярной биологии, генотипическими и фенотипическими
вариациями
микроорганизмов,
особенностями
передачи
генетической
информации и возникновения антибиотикорезистентности бактерий. В
пособии подробно изложены современные аспекты бактериальной генетики,
биотехнологии и новые методы диагностики инфекционных заболеваний.
Знание основных аспектов данной темы облегчит восприятие материала
предмета в дальнейшем и поможет сформировать клиническое мышление
студента для осуществления правильных и своевременных лечебнодиагностических и профилактических мероприятий.
«Бактериальная
генетика»
вызывает
затруднения
у
студентов
второкурсников. Поэтому в методическом пособии кратко и в доступной
форме изложены теоретические аспекты данной темы. Кроме того,
представлен обширный наглядный материал в виде таблиц и схем, которые
облегчают восприятие данной темы.
5
INTRODUCTION
The manual in microbiology in English is for students of the English-speaking
medium. It includes the basic themes of a course “Bacterial Genetics”.
The purpose of the manual is an acquaintance of the second course students
with basic principles of molecular biology, genotypic and phenotypic variation of
microorganisms, transmission of genetic material and genetic mechanisms of drug
resistance in bacteria. The study of bases of the bacterial genetics will make easier
the comprehension of the subject for the students and will help to the doctor in
diagnostics, prevention and treatment of infection diseases.
“Bacterial Genetics” causes some difficulties in mastering this theme by the
second-year students. Therefore, theoretical aspects of the given theme are stated
briefly and accessibly in the manual. Besides, there is the additional evident
material as tables and schemes in the manual.
6
1. Genome Organization
The character of a cell is basically determined by the specific polypeptides that
make up its enzymes and other proteins. The genetic information in bacteria, as in all
cells, is contained in the specific sequence of nuсleotides in the cell's deoxyribonucleic
acid (DNA). The DNA acts as template for the replication of the DNA so that two
copies are available at cell division and it also acts as template for the transcription of
ribonucleic acid (RNA) for protein production within the cell. The sequence of
nueleotides in the DNA determines the corresponding sequence of nueleotides in the
RNA, and this is translated into the appropriate sequence of amino acids by
ribosomes. The sequence of amino acids in the resultant polypeptide chain in turn
determines the specific configuration into which this chain folds itself in forming the
completed molecule of protein, and thus the specific enzymic or structural properties of
the protein. Figure 1 illustrates this 'central dogma' of molecular biology; the model is
generally true for prokaryotic cells, though there are some important variations, e.g.
with certain viruses in eukaryotic cells.
Figure 1 The central dogma of molecular biology
7
1.1. Bacterial Chromosome
A segment of DNA that specifies the production of a particular polypeptide
chain is called a gene and the total complement of genes in a cell is known as the cell's
genome.
Most bacteria contain enough DNA to code for the production of 1000 to
3000 different types of polypeptide chains i.e. 1000 to 3000 genes. In the bacteria that
have been most intensively studied, notably Escherichia coli, it has been found that the
DNA is present as a single circular double-stranded molecule about 1000 to 1300 mm
long. The DNA is not associated with protein or histones as in eukaryotic cell
chromosomes. Since the DNA is about 1000 times longer than the cell, it is obvious
that it is not arranged as a simple circle; electron microscopy of thin sections of
bacteria shows the nuclear body as an irregular coiled bundle of DNA lying free in the
cytoplasm, like a skein of thread. The terms nucleus and chromosome were
originally restricted to the structures that could be seen in eukaryotic cells. There
are many differences in detail between the organization and control of the DNA in
prokaryotic and in eukaryotic cells, but it is accepted that the basic functions are the
same, that the nuclear body is the functional equivalent of a nucleus and that its single
circular molecule of naked DNA is the functional equivalent of a chromosome.
The prokaryotic cell has a single chromosome and, after replication, only a
single molecule of DNA has to be directed into each daughter cell. The mechanism of
this partition is not clearly understood but it may involve attachment of the DNA to a
site on the cytoplasmic membrane or on the mesosome. Eukaryotic cells have multiple
chromosomes held together in a nuclear membrane and they have a complex mitotic
apparatus for separating the two sets of chromosomes after replication. The advantage
of the eukaryotic type of organization is that it can handle much morе genetic
information; this has allowed the evolution of complex multicellular organisms with
diploid chromosomes (i.e. two sets of chromosomes) and sexual methods of
reproduction. Prokaryotic cells, which are normally haploid (i.e. with one copy of each
gene), have the advantages of simplicity, small size and rapid replication.
1.2. Bacterial Plasmids
A. Definition - Plasmids are extrachromosomal genetic elements capable of
autonomous replication. An episome is a plasmid that can integrate into the bacterial
chromosome.
B. Classification of Plasmids
1. Transfer properties
a. Conjugative plasmids - Conjugative plasmids are those that mediated conjugation.
These plasmids are usually large and have all the genes necessary for autonomous
replication and for transfer of DNA to a recipient (e.g. genes for sex pilus).
b. Nonconjugative plasmids - Nonconjugative plasmids are those that cannot mediate
conjugation. They are usually smaller than conjugative plasmids and they lack one or
more of the genes needed for transfer of DNA. A nonconjugative plasmid can be
transferred by conjugation if the cell also harbors a conjugative plasmid.
2. Phenotypic effects
a. Fertility plasmid (F factor)
8
b. Bacteriocinogenic plasmids - These plasmids have genes which code for
substances that kill other bacteria. These substances are called bacteriocins or
colicins.
c. Resistance plasmids R factors) - These plasmids carry antibiotic resistance genes.
Origin - The origin of the R factors is not known. It is likely that they evolved for
other purposes and the advent of the antibiotic age provided a selective advantage for
their wide-spread dissemination.
3.Structure - R plasmids are conjugative plasmids in which the genes for replication
and transfer are located on one part of the R factor and the resistance genes are
located on another part as illustrated in Figure 2.
Figure 2. Structure of an R plasmid showing the RTF which mediates transfer and
the R determinant which carry the antibiotic resistance genes
a) RTF (Resistance Transfer Factor) - carries the transfer genes.
b) R determinant - carries the resistance genes. The resistance genes are often parts
of transposons.
1.3. Transposable Genetic Elements
A. Transposable Genetic Elements - Transposable genetic elements are segments of
DNA that have the capacity to move from one location to another (i.e. jumping
genes).
B. Properties of Transposable Genetic Elements
1. Random movement - Transposable genetic elements can move from any DNA
molecule to any DNA other molecule or even to another location on the same
molecule. The movement is not totally random; there are preferred sites in a DNA
molecule at which the transposable genetic element will insert.
2. Not capable of self replication - The transposable genetic elements do not exist
autonomously (exception - some transposable phages) and thus, to be replicated they
must be a part of some other replicon.
9
3. Transposition mediated by site-specific recombination - Transposition requires
little or no homology between the current location and the new site. The transposition
event is mediated by a transposase coded for by the transposable genetic element.
Recombination that does not require homology between the recombining molecules
is called site-specific or illegitimate or nonhomologous recombination.
4. Transposition can be accompanied by duplication - In many instances
transposition of the transposable genetic element results in removal of the element
from the original site and insertion at a new site. However, in some cases the
transposition event is accompanied by the duplication of the transposable genetic
element. One copy remains at the original site and the other is transposed to the new
site.
C. Types of Transposable Genetic Elements
1. Insertion sequences (IS)- Insertion sequences are transposable genetic elements
that carry no known genes except those that are required for transposition.
a. Nomenclature - Insertion sequences are given the designation IS followed by a
number. e.g. IS1
b. Structure (Figure 3)
Figure 3. Structure of an IS with inverted repeats at the ends.
Insertion sequences are small stretches of DNA that have at their ends repeated
sequences, which are involved in transposition. In between the terminal repeated
sequences there are genes involved in transposition and sequences that can control
the expression of the genes but no other nonessential genes are present.
c. Importance
1) Mutation - The introduction of an insertion sequence into a bacterial gene will
result in the inactivation of the gene.
2) Plasmid insertion into chromosomes - The sites at which plasmids insert into the
bacterial chromosome are at or near insertion sequence in the chromosome.
2. Transposons (Tn) - Transposons are transposable genetic elements that carry one
or more other genes in addition to those which are essential for transposition.
a. Nomenclature - Transposons are given the designation Tn followed by a number.
b. Structure - The structure of a transposon is similar to that of an insertion sequence.
The extra genes are located between the terminal repeated sequences. In some
instances (composite transposons) the terminal repeated sequences are actually
insertion sequences. (See Figure 4).
Figure 4. Structure of a composite Tn with direct or inverted IS at the ends.
10
c. Importance - Many antibiotic resistance genes are located on transposons. Since
transposons can jump from one DNA molecule to another, these antibiotic resistance
transposons are a major factor in the development of plasmids which can confer
multiple drug resistance on a bacterium harboring such a plasmid. These multiple
drug resistance plasmids have become a major medical problem because the
indiscriminate use of antibiotics have provided a selective advantage for bacteria
harboring these plasmids.
2. Phenotypic Variation
The sum total of the gens that make up genetic apparatus of a cell (genome)
establishes its genotype, which is the hereditary constitution of the cell that is
transmitted to its progeny. The genotype includes the complete genetic potential of
the cell, all of which may or may not be expressed in a given environment situation.
The phenotype is the physical expression of genotype in the given
environment. It follows, therefore, that a cell may exhibit different phenotypic
appearances in different situation. Phenotypic variations are limited in range by the
genotype, temporary and not heritable.
2.1. Regulation of Gene Expression
Bacteria do not make all the proteins that they are capable of making all of
the time. Rather, they can adapt to their environment and make only those gene
products that are essential for them to survive in a particular environment. e.g.
Bacteria do not synthesize the enzymes needed to make tryptophan when there is an
abundant supply of tryptophan in the environment. However, when tryptophan is
absent from the environment the enzymes are made. Similarly, just because a
bacterium has a gene for resistance to an antibiotic does not mean that that gene will
be expressed. The resistance gene may only be expressed when the antibiotic is
present in the environment. Bacteria usually control gene expression by regulating
the level of transcription. In bacteria, genes with related function are generally
located adjacent to each other and they are regulated coordinately (i.e. when one is
expressed they all are expressed). Coordinate regulation of clustered genes is
accomplished by regulating the production of a polycistronic mRNA (i.e. a large
mRNA containing the information for many genes). Thus, bacteria are able to
"sense" their environment and expresses the appropriate set of genes needed for that
environment by regulating transcription of those genes.
A. Inducible Genes - The Operon Model
1. Definition - Inducible genes are those in which the presence of a substance (an
inducer) in the environment turns on the expression of one or more genes (structural
genes) involved in the metabolism of that substance. e.g. Lactose induces the
expression of the lac genes. An antibiotic induces the expression of a resistance gene.
Induction is common in metabolic pathways that result in the catabolism of a
substance and the inducer is normally the substrate for the pathway.
11
2. Lactose Operon - The lactose operon is illustrated in Figure 5.
Figure 5. The Lactose Operon.
a. Structural genes - The lactose operon contains three structural genes that code for
enzymes involved in lactose metabolism. The lac z gene codes for ß-galactosidase,
an enzyme that breaks down lactose into glucose and galactose, the lac y gene that
codes for a permease, which is involved in uptake of lactose, and the lac a gene,
which codes for a galactose transacetylase. These genes are transcribed from a
common promoter into a polycistronic mRNA, which is translated to yield the three
enzymes.
b. Regulatory gene - The expression of the structural genes is not only influenced by
the presence or absence of the inducer, it is also controlled by a specific regulatory
gene. The regulatory gene may be next to or far from the genes that are being
regulated. The regulatory gene codes for a specific protein product called a
REPRESSOR.
c. Operator - The repressor acts by binding to a specific region of the DNA called
the operator which is adjacent to the structural genes being regulated. The structural
genes together with the operator region and the promoter is called an OPERON.
However, the binding of the repressor to the operator is prevented by the inducer and
the inducer can also remove repressor that has already bound to the operator. Thus,
in the presence of the inducer the repressor is inactive and does not bind to the
operator, resulting in transcription of the structural genes. In contrast, in the absence
of inducer the repressor is active and binds to the operator, resulting in inhibition of
transcription of the structural genes. This kind of control is referred to a NEGATIVE
CONTROL since the function of the regulatory gene product (repressor) is to turn
off transcription of the structural genes.
d. Inducer - Transcription of the lac genes is influenced by the presence or absence
of an inducer (lactose or other ß-galactosides) (Figure 6).
12
Figure 6 (+) inducer – expression (-) inducer - no expression
3. Catabolite repression (Glucose Effect)
Many inducible operons are not only controlled by their respective inducers and
regulatory genes, but they are also controlled by the level of glucose in the
environment. The ability of glucose to control the expression of a number of different
inducible operons is called CATABOLITE REPRESSION. Catabolite repression is
generally seen in those operons which are involved in the degradation of compounds
used as a source of energy. Since glucose is the preferred energy source in bacteria,
the ability of glucose to regulate the expression of other operons ensures that bacteria
will utilize glucose before any other carbon source as a source of energy.
a. Mechanism - There is an inverse relationship between glucose levels and
cAMP levels in bacteria. When glucose levels are high cAMP levels are low and
when glucose levels are low cAMP levels are high. This relationship exists because
the transport of glucose into the cell inhibits the enzyme adenyl cyclase which
produces cAMP. In the bacterial cell, cAMP binds to a cAMP binding protein called
CAP or CRP. The cAMP-CAP complex, but not free CAP protein, binds to a site in
the promoters of catabolite repression sensitive operons. The binding of the complex
results in a more efficient promoter and thus more initiations of transcriptions from
that promoter as illustrated in Figures 7 and 8.
Glucose transport inhibits Adenylate Cyclase, lowers cAMP levels, and fails to
activate CAP. Low Glucose allows cAMP levels to increase, bind to CAP, and
stimulate expression of the lac operon.
Since the role of the CAP-cAMP complex is to turn on transcription this type of
control is said to be POSITIVE CONTROL. The consequences of this type of control
is that toachieve maximal expression of a catabolite repression sensitive operon
glucose must be absent from the environment and the inducer of the operon must be
present. If both are present the operon will not be maximally expressed until glucose
13
is metabolized. Obviously, no expression of the operon will occur unless the inducer
is present.
Figures 7 and 8. Activation of an inducer.
B. Repressible Genes - The Operon Model
1. Definition - Repressible genes are those in which the presence of a substance (a
co-repressor) in the environment turns off the expression of one or more genes
(structural genes) involved in the metabolism of that substance. e.g., Tryptophan
represses the expression of the trp genes.
Repression is common in metabolic pathways that result in the biosynthesis of a
substance and the co-repressor is normally the end product of the pathway being
regulated.
2. Tryptophan operon -The tryptophan operon is illustrated in Figure 9.
Figure 9. the structure of the trp operon. E,D,C,B and A are structural genes
14
a. Structural genes - The tryptophan operon contains five structural genes that code
for enzymes involved in the synthesis of tryptophan. These genes are transcribed
from a common promoter into a polycistronic mRNA, which is translated to yield the
five enzymes.
b. Regulatory gene - The expression of the structural genes is not only influenced by
the presence or absence of the co-repressor, it is also controlled by a specific
regulatory gene.
The regulatory gene may be next to or far from the genes that are being regulated.
The regulatory gene codes for a specific protein product called a REPRESSOR
(sometimes called an apo-repressor). When the repressor is synthesized it is
inactive. However, it can be activated by complexing with the co-repressor (i.e.
tryptophan).
c. Operator - The active repressor/co-repressor complex acts by binding to a specific
region of the DNA called the operator which is adjacent to the structural genes being
regulated. The structural genes together with the operator region and the promoter
iscalled an OPERON. Thus, in the presence of the co-repressor the repressor is active
and binds to the operator, resulting in repression of transcription of the structural
genes. In contrast, in the absence of co-repressor the repressor is inactive and does
not bind to the operator, resulting in transcription of the structural genes. This kind of
control is referred to a NEGATIVE CONTROL since the function of the regulatory
gene product (repressor) is to turn off transcription of the structural genes.
d. Co-repressor - Transcription of the tryptophan genes is influenced by the
presence or absence of a co-repressor (tryptophan) (illustrated in Figure 10). e.g. (+)
co-repressor - no expression (-) co-repressor – expression.
Figure 10. Trp co-repression of the trp operator. The trp repressor is synthesized in
an inactive form (apo-repressor). Tryptophan binds to the inactive repressor,
activating it. The active repressor can then bind to and block transcription of the trp
operon.
15
3. Attenuation
In many repressible operons transcriptions that initiate at the promoter can terminate
prematurely in a leader region that precedes the first structural gene. (i.e. the
polymerase terminates transcription before it gets to the first gene in the operon. This
phenomenon is called ATTENUATION; the premature termination of transcription.
Although attenuation is seen in a number of operons, the mechanism is best
understood in those repressible operons involved in amino acid biosynthesis.
2.2. Phase variation
Phase Variation - The flagellar antigens are one of the main antigens to which
the immune response is directed in our attempt to fight off a bacterial infection. In
Salmonella there are two genes which code for two antigenically different flagellar
antigens. The expression of these genes is regulated by an insertion sequences. In one
orientation one of the genes is active while in the other orientation the other flagellar
gene is active. Thus, Salmonella can change their flagella in response to the immune
systems' attack. Phase variation is not unique to Salmonella flagellar antigens. It is
also seen with other bacterial surface antigens. Also the mechanism of phase
variation may differ in different species of bacteria (e.g. Neisseria; transformation).
3. Genotypic Variation
Genotypic variations are stable, heritable and not influenced by the
environment. They may occur by mutation or by one of the mechanisms genetic
transfer or exchange, such as transformation, transduction and conjugation. As
bacteria reproduce by asexual binary fission, the genome is normally identical in all
the progeny. The DNA is a double helix with complementary nucleotide sequences in
the two strands. At replication the strands separate and new complementary strands are
formed on each of the originals so that two identical double helices are produced, each
with the same nucleotide sequence and hence the same genetic information as the
original. This process is very accurate, but occasional inaccuracies produce a slightly
altered nucleotide sequence in one of the daughter cells. One of the fundamental
requirements for evolution is that although gene replication must normally be
completely accurate to ensure stability there must also be occasional variation to
produce new or altered characters that could prove to be of selective value to the
organism. The mechanism of DNA replication has evolved not only to ensure accuracy
of replication, but also to provide occasional 'mistakes'. Mutation is the commonest
source of genetic variation in bacteria, but in addition the genome may occasionally be
changed by acquisition of DNA from outside the cell.
3.1 Mutation
Biochemically, a mutation is an alteration in the nucleotide sequence at some
point in the organism's DNA. This may lead to the production of a protein that has an
altered amino acid sequence. Such an alteration usually does not produce a readily
observable change in the function of the protein, A small proportion of mutations lead
16
to the production of a protein that is altered in a minor way so that its function is only
slightly changed, e.g. an enzyme with altered specificity for substrates. This is the kind
of mutation that is most likely to be of evolutionary value to an organism; many
examples of drug resistance acquired in the laboratory have been shown to be of this
type. Occasional mutations alter a gene so that a non-functional protein is formed: if
this protein is essential, the mutation is lethal.
Since mutation may occur in any of the cell's several thousand genes and
different mutations in the same gene may produce different effects in the cell, the
number of possible mutations is very large. Bacteria are normally haploid and therefore
the effect of a mutated gene can be expressed immediately and is not masked by the
presence of another copy of the gene as often happens in higher diploid organisms.
Particular mutations occur spontaneously at fairly constant rates, usually in the range
of once per 104 to once per 1010 cell divisions. A large bacterial colony contains about
104 cells all derived from a single organism by repeated cell division. For many
purposes it is valuable to consider such a colony as a clone of genetically identical cells,
but it should be realized that after 109 cell divisions, many thousands of different
mutations will have occurred, affecting very many of the genes in the cell. Thus any
bacterial colony contains a small proportion of a variety of mutants, some of which are
viable and might be selected by particular environmental conditions during subculture.
For the same reason, in every infected patient, a variety of mutants arise spontaneously
in the population of, say, 10(8) to 1014 progeny that soon grow from the single or few
bacteria originally entering the body.
3.2. Selection of mutants
Whether in culture or in a patient's body, a mutant will become sufficiently
numerous to be observable and to produce significant effects only if its new character
makes it better fitted to grow under the prevailing conditions in the culture medium or
host's tissues than the parental bacteria and so enable it to outgrow and outnumber
the latter. Thus mutation is significant only when conditions are selectively favourable
to the mutant and bring about its natural or artificial selection. An antibiotic-resistant
mutant, for example, will outgrowths sensitive parental bacteria in a culture medium
containing antibiotic or in the body of a patient receiving antibiotic therapy. A mutant
with enhanced ability to grow in the body of a particular host species, and thus with
greater virulence for it, will be selectively increased in the course of a natural or
experimental infection. A mutant with altered surface antigens will escape the
restraining effect of immunity previously developed in an individual or a community
against the parental form and so be able to cause a recurrence of the infection.
3.3. Mechanisms of mutation
The rate of mutation can be greatly increased artificially by exposing bacteria to
irradiation by X-rays or ultra-violet light or by growing bacteria in the presence of
certain mutagenic chemicals that interfere with DNA replication. Mutations are
normally permanent and stably inherited by the progeny, but further mutations may
occur and these may occasionally restore the original nucleotide sequence. The rate at
17
which such back-mutation can occur depends on the extent of the original mutation.
The chance of a single nucleotide substitution being reversed is much higher than the
chance of replacing a deleted sequence of a few amino acids, whereas mutations caused
by deletion of a substantial portion of a gene are effectively irreversible by further
mutation. Some organisms have particularly effective mechanisms for the repair of
damaged portions of DNA.
The mutations first studied by bacteriologists were those producing effects that
were detectable by the experimental methods then in use, e.g. mutations producing
alteration in colonial morphology or pigmentation; variation in cell surface antigens or
in sensitivity to bacteriophages or bacteriocines; loss of the ability to produce
capsules, spores, flagella; or changes in virulence towards particular hosts. It was not
originally possible to characterize these mutations in molecular terms. In some cases
the mechanism is now understood: for example, in pneumococci a mutation leads to
failure to produce a capsule; loss of the capsule in this organism is directly related to
failure to synthesize the type-specific (capsular) antigen, alteration of the normal
smooth colony to a rough form (S-R variation) and loss of resistance to phagocytosis
with consequent loss of virulence when the organisms are injected into mice. Thus a
single mutation produces a variety of biological phenomena. The mechanism of
biosynthesis of pneumococcal capsular polysaixharide is now known in some detail and
it is possible to ascribe this mutation to the loss of a specific enzyme involved in
polysaccharide synthesis. Many mutations, however, still cannot be characterized
biochemically. The structure of normal bacterial cell walls is seldom well enough
understood to permit a molecular description of altered somatic antigens or
bacteriophage receptors. Some organisms have been subcultured in the laboratory for
many generations until they have lost their virulence for man, e.g. in the production of
live attenuated vaccines. Such strains have lost, by a series of mutant characters that
confer a selective advantage on the organism in vivo, but are of no advantage in vitro,
e.g. aggressins, toxins or capsules that enable virulent strains of certain species to resist
the actions of the body's phagocytes or other defence mechanisms. Mutants that no
longer produce such factors can multiply a little faster in vitro since they do not waste
nutrients and metabolites on the production of these unnecessary substances, and they
may gradually outgrow the wild-type cells during repeated subculture. However, since
the normal mechanisms of pathogenicity can seldom be described in molecular terms, it
is usually impossible to identify the biochemical changes underlying the alterations in
biological behaviour of the attenuated strains.
Mutations may occur in any gene but, since many genes code for production of
substances essential for cell survival and multiplication, many individual mutations are
lethal. Other mutations affect gene products that are essential only under particular
cultural conditions.
3.4. Mutation that affect biochemical pathways
Amongst the mutations that have been most studied are those in which the
organism loses the ability to synthesize an essential metabolite (or regains it by backmutation). Many organisms, e.g. Esch. coli, can grow in simple defined culture media
18
because they have genes for the production of all the enzymes they require for the
synthesis from simple nutrients of all the large variety of organic compounds essential
for the construction and functioning of their cells. These are called prototrophic cells.
Mutants that can no longer synthesize a particular essential metabolite, such as an
ammo acid, and are unable to grow in a medium that does not contain the essential
ammo acid are called auxotrophic mutants. Studies of mutants blocked at different
stages in a synthetic pathway (i.e. with mutations affecting different enzymes in the
pathway) are very valuable in the elucidation of biosynthetic pathways.
Auxоtrophic cells that revert to the prototrophic state by back-mutation can be
selected from the rest of the auxotrophic cells by culture in the simple defined medium;
prototrophic revertants are the only cells able to grow in a medium that does not contain
the essential metabolite.
3.5. Drag-resistance Mutation
For many antibiotics, a particular mutation produces only a slight increase in
resistance but a series of consecutive mutations can produce a stepwise increase in
resistance. For some drugs, e.g. penicillin, such stepwise mutations are easily selected
in the laboratory but the final level of resistance is still below the levels of antibiotic that
are attainable in the tissues or body fluids during treatment of a patient. Many drugresistant mutants produced in the laboratory grow less efficiently than the original drugsensitive cells and may be less virulent. However, clinically important drug resistance in
siaphylococci has arisen largely by the selection of spontaneous mutants with increased
drug resistance. Resistance to many antibiotics can be produced only by the sequential
selection of multiple 'small-step' mutations but resistance to streptomycin arises quite
readily by a single 'large-step' mutation in a variety of species and it is probably
because of this that acquisition of resistance to streptomycin by mutation is a serious
clinical problem, e.g. in the treatment of tuberculosis.
Drug resistance of clinical significance is often found to be due not to
mutation but to the production of inactivating enzymes. Penicillin resistance in
staphylococci is due to the production of penicillinase (/S-lactamase) and resistance to
ampicillin, chloramphenicol, streptomycin and other aminoglycoside antibiotics in
Gram-negative bacilli is often due to the production of enzymes that break down or
modify the antibiotic. The origin of such resistance genes is unclear but they cannot be
produced by simple mutation in a drug-sensitive cell.
4. Gene Transfer Mechanisms in Bacteria
4.1. Transformation - Transformation is gene transfer resulting from the uptake by a
recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus,
Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and
the DNA that is taken up can be incorporated into the recipient's chromosome.
1. Factors affecting transformation
5
a. DNA size state - Double stranded DNA of at least 5 X 10 daltons works best.
Thus, transformation is sensitive to nucleases in the environment.
19
b. Competence of the recipient - Some bacteria are able to take up DNA naturally.
However, these bacteria only take up DNA a particular time in their growth cycle
when they produce a specific protein called a competence factor. At this stage the
bacteria are said to be competent. Other bacteria are not able to take up DNA
naturally. However, in these bacteria competence can be induced in vitro by
treatment with chemicals (e.g. CaCl2).
2. Steps in transformation
a. Uptake of DNA - Uptake of DNA by Gram+ and Gram- bacteria differs. In Gram
+ bacteria the DNA is taken up as a single stranded molecule and the complementary
strand is made in the recipient. In contrast, Gram- bacteria take up double stranded
DNA.
b. Legitimate/Homologous/General Recombination - After the donor DNA is taken
up, a reciprocal recombination event occurs between the chromosome and the donor
DNA. This recombination requires homology between the donor DNA and the
chromosome and results in the substitution of DNA between the recipient and the
donor as illustrated in Figure 11.
Recombination requires the bacterial recombination genes (recA, B and C) and
homology between the DNA's involved. This type of recombination is called
legitimate or homologous or general recombination. Because of the requirement for
homology between the donor and host DNA, only DNA from closely related bacteria
would be expected to successfully transform, although in rare instances gene transfer
between distantly related bacteria has been shown to occur.
Figure 11.Homolgous recombination during transformation.
3. Significance - Transformation occurs in nature and it can lead to increased
virulence. In addition transformation is widely used in recombinant DNA
technology.
4.2. Transduction
Transduction is the transfer of genetic information from a donor to a recipient by way
of a bacteriophage. The phage coat protects the DNA in the environment so that
transduction, unlike transformation, is not affected by nucleases in the environment.
20
Not all phages can mediate transduction. In most cases gene transfer is between
members of the same bacterial species. However, if a particular phage has a wide
host range then transfer between species can occur. The ability of a phage to
mediated transduction is related to the life cycle.
Phage multiplication cycle
1. Lytic or Virulent Phages - Lytic or virulent phages are phages which can only
multiply on bacteria and kill the cell by lysis at the end of the life cycle.
2. Lysogenic or Temperate Phage - Lysogenic or temperate phages are those that can
either multiply via the lytic cycle or enter a quiescent state in the cell. In this
quiescent state most of the phage genes are not transcribed; the phage genome exists
in a repressed state. The phage DNA in this repressed state is called a prophage
because it is not a phage but it has the potential to produce phage. In most cases the
phage DNA actually integrates into the host chromosome and is replicated along with
the host chromosome and passed on to the daughter cells. The cell harboring a
prophage is not adversely affected by the presence of the prophage and the lysogenic
state may persist indefinitely. The cell harboring a prophage is termed a lysogen.
a) Events Leading to Lysogeny - The Prototype Phage: Lambda
1) Circularization of the phage chromosome - Lambda DNA is a double stranded
linear molecule with small single stranded regions at the 5' ends. These single
stranded ends are complementary (cohesive ends) so that they can base pair and
produce a circular molecule. In the cell the free ends of the circle can be ligated to
form a covalently closed circle as illustrated in Figure 12.
Figure 12. Circularization of the lambda chromosome during the establishment of
lysogeny.
2) Site-specific recombination - A recombination event, catalyzed by a phage coded
enzyme, occurs between a particular site on the circularized phage DNA and a
particular site on the host chromosome. The result is the integration of the phage
DNA into the host chromosome as illustrated in Figure 13.
Repression of the phage genome - A phage coded protein, called a repressor, is made
which binds to a particular site on the phage DNA, called the operator, and shuts off
transcription of most phage genes EXCEPT the repressor gene. The result is a stable
repressed phage genome which is integrated into the host chromosome. Each
temperate phage will only repress its own DNA and not that from other phage, so
that repression is very specific (immunity to superinfection with the same phage).
21
Figure 13. Site-specific recombination during establishment of lysogeny in
bacteriophage lambda.
Repression of the phage genome - A phage coded protein, called a repressor, is made
which binds to a particular site on the phage DNA, called the operator, and shuts off
transcription of most phage genes EXCEPT the repressor gene. The result is a stable
repressed phage genome which is integrated into the host chromosome. Each
temperate phage will only repress its own DNA and not that from other phage, so
that repression is very specific (immunity to superinfection with the same phage).
b. Events Leading to Termination of Lysogeny
Anytime a lysogenic bacterium is exposed to adverse conditions, the lysogenic state
can be terminated. This process is called induction (Figure 6). Conditions which
favor the termination of the lysogenic state include: desiccation, exposureto UV or
ionizing radiation, exposure to mutagenic chemicals, etc. Adverse conditions lead to
the production of proteases (rec A protein) which destroy the repressor protein. This
in turn leads to the expression of the phage genes, reversal of the integration process
and lytic multiplication.
Figure 14. Inactivation of the repressor and excision of the prophage
22
4.2.1. Generalized Transduction
Generalized transduction is transduction in which potentially any bacterial gene
from the donor can be transferred to the recipient. The mechanism of generalized
transduction is illustrated in Figure 15.
Figure 15. Mechanism of generalized transduction of bacterial genes by phage.
Phages that mediate generalized transduction generally breakdown host DNA into
smaller pieces and package their DNA into the phage particle by a "head-full"
mechanism. Occasionally one of the pieces of host DNA is randomly packaged
into a phage coat. Thus, any donor gene can be potentially transferred but only
enough DNA as can fit into a phage head can be transferred. If a recipient cell is
infected by a phage thatcontains donor DNA, donor DNA enters the recipient. In
the recipient a generalized recombination event can occur which substitutes the
donor DNA and recipient DNA (See Figure 11).
4.2.2. Specialized transduction
Specialized transduction is transduction in which only certain donor genes can be
transferred to the recipient. Different phages may transfer different genes but an
individual phage can only transfer certain genes. Specialized transduction is
mediated by lysogenic or temperate phage and the genes that get transferred will
depend on where the prophage has inserted in the chromosome. The mechanism of
specialized transduction is illustrated in Figure 16.
Figure 16. Generation of a
specialized transducing phage; left
normal excision of the prophage,
right abnormal excision resulting
in a specialized transducing phage
23
During excision of the prophage, occasionally an error occurs where some of the
host DNA is excised with the phage DNA. Only host DNA on either side of where
the prophage has inserted can be transferred (i.e. specialized transduction). After
replication and release of phage and infection of a recipient, lysogenization of
recipient can occur resulting in the stable transfer of donor genes. The recipient
will now have two copies of the gene(s) that were transferred. Legitimate
recombination between the donor and recipient genes is also possible.
3. Significance - Lysogenic (phage) conversion occurs in nature and is the source
of virulent strains of bacteria.
4.3. Conjugation
Conjugation - transfer of DNA from a donor to a recipient by direct physical
contact between the cells. In bacteria there are two mating types a donor (male)
and a recipient (female) and the direction of transfer of genetic material is one
way; DNA is transferred from a donor to a recipient.
1. Mating types in bacteria
a. Donor - The ability of a bacterium to be a donor is a consequence of the
presence in the cell of an extra piece of DNA called the F factor or fertility factor
or sex factor. The F factor is a circular piece of DNA that can replicate
autonomously in the cell; it is an independent replicon. Extrachromosomal pieces
of DNA that can replicate autonomously are given the general name of plasmids.
The F factor has genes on it that are needed for its replication and for its ability to
transfer DNA to a recipient. One of the things the F factor codes for is the ability to
produce a sex pilus (F pilus) on the surface of the bacterium. This pilus is
important in the conjugation process. The F factor is not the only plasmid that can
mediated conjugation but it is generally used as the model.
b. Recipient - The ability to act as a recipient is a consequence of the lack of the F
factor.
2. Physiological states of the F factor
a. Autonomous (F+) - In this state the F factor carries only those genes necessary
for its replication and for DNA transfer. There are no chromosomal genes
+
associated with the F factor in F strains.
+
-
-
+
+
+
In crosses of the type F X F the F becomes F while F remains F . Thus, the F
factor is infectious. In addition, there is only low level transfer of chromosomal
genes.
b. Integrated (Hfr) - In this state the F factor has integrated into the bacterial
chromosome via a recombination event as illustrated in the Figure 16. In crosses of
-
-
the type Hfr X F the F rarely becomes Hfr and Hfr remains Hfr. In addition, there
is a high frequency of transfer of donor chromosomal genes.
c. Autonomous with chromosomal genes (F') - In this state the F factor is
autonomous but it now carries some chromosomal genes. F' factors are produced
by excision of the F factor from an Hfr, as illustrated in Figure 16. Occasionally,
when the F factor is excising from the Hfr chromosome, donor genes on either side
24
of the F factor can be excised with the F factor generating an F'. F' factors are
named depending on the chromosomal genes that they carry.
In crosses of the type F' X F the F becomes F' while F' remains F'. In addition
there is high frequency of transfer of those chromosomal genes on the F' and low
frequency transfer of other donor chromosomal genes.
+
Figure 16. Physiological states of the F factor; F -autonomous, Hfr - integrated
into the chromosome, and F’ - autonomous and carrying some chromosomal genes.
3. Mechanism of conjugation
a. F+ X F- crosses (Figure 17)
1) Pair formation - The tip of the sex pilus comes in contact with the recipient and
a conjugation bridge is formed between the two cells. It is through this bridge that
the DNA will pass from the donor to the recipient. Thus, the DNA is protected
from environmental nucleases. The mating pairs can be separated by shear forces
and conjugation can be interrupted. Consequently, the mating pairs remain
associated for only a short time.
+
-
Figure 17. Mechanism of F X F conjugation
2) DNA transfer - The plasmid DNA is nicked at a specific site called the origin of
transfer and is replicated by a rolling circle mechanism. A single strand of DNA
25
passes through the conjugation bridge and enters the recipient where the second
strand is replicated. This process explains the characteristics of F+ X F- crosses.
The recipient becomes F+, the donor remains F+ and there is low frequency of
transfer of donor chromosomal genes. Indeed, as depicted in Figure 10 there is no
transfer of donor chromosomal genes. In practice however, there is a low level of
transfer of donorchromosomal genes in such crosses.
b. Hfr X F- crosses (Figure 18)
1)Pair Formation
-
Figure 18. Mechanism of Hfr X F conjugation
2) DNA transfer - The DNA is nicked at the origin of transfer and is replicated by a
rolling circle mechanism. But the DNA that is transferred first is the chromosome.
Depending upon where in the chromosome the F factor has integrated and in what
orientation, different chromosomal genes will be transferred at different times.
However, the relative order and distances of the genes will always remain the
same. Only when the entire chromosome is transferred will the F factor be
transferred. Since shearing forces separate the mating pairs it is rare that the entire
chromosome will be transferred. Thus, the recipient does not receive the F factor in
a Hfr X F- cross.
3) Legitimate recombination - Recombination between the transferred DNA and
the chromosome results in the exchange of genetic material between the donor and
recipient.
4) This mechanism explains the characteristics of Hfr X F crosses. The recipient
remains F-, the donor remains Hfr and there is a high frequency of transfer of
donor chromosomal genes.
c. F' X F- crosses (Figure 19)
1) Pair Formation.
+
2) DNA transfer - This process is similar to F X F crosses. However, since the F'
has some chromosomal genes on it these will also be transferred.
3) Homologous recombination is not necessary although it may occur.
26
-
This mechanism explains the characteristics of F' X F crosses. The F- becomes F',
the F' remains F' and is the high frequency transfer of donor genes on the F' but
low frequency transfer of other donor chromosomal genes.
’
-
Figure 19. Mechanism of F X F conjugation
4. Significance - Among the Gram negative bacteria this is the major way that
bacterial genes are transferred. Transfer can occur between different species of
bacteria. Transfer of multiple antibiotic resistance by conjugation has become a
major problem in the treatment of certain bacterial diseases. Since the recipient cell
becomes a donor after transfer of a plasmid it is easy to see why an antibiotic
resistance gene carried on a plasmid can quickly convert a sensitive population of
cells to a resistant one.
Gram positive bacteria also have plasmids that carry multiple antibiotic resistance
genes, in some cases these plasmids are transferred by conjugation while in others
they are transferred by transduction. The mechanism of conjugation in Gram +
bacteria is different than that for Gram -. In Gram + bacteria the donor makes an
adhesive material which causes aggregation with the recipient and the DNA is
transferred.
Mutational drug resistance
Transferable resistance
One drug resistance at a time
Multiple drug resistance
Low degree resistance
High degree resistance
Can overcome by high drug dose
High dose ineffective
Drug
combinations
can
prevent Combinations cannot prevent
resistance
Does not spread
Spreads to same or different species
Mutants may be defective
Not defective
Virulence may be low
Virulence not decrease
Table 1. Comparison of mutational and transferable drug resistance
27
5. Molecular Genetics
Discoveries in microbial genetics have provided the basis for the discipline
of molecular genetics, which is concerned with the analysis and manipulation of
DNA using biochemical and microbiological techniques. It has been stated that
these techniques have revolutionised the study of biology and medicine, probably
more than any technique since the development of the light microscopes. Some
techniques and applications of molecular genetics are discussed below.
5.1.Genetic engineering
The most important application of molecular genetics in biotechnology is
genetic engineering or recombinant DNA technology. This consists of isolation of
the genes coding for any desired protein from microorganisms or from cells of
higher forms of life including human beings, and their introduction into suitable
microorganisms, in which the genes would be functional, directing the production of
the specific protein. Such cloning of genes in microorganisms enables the
preparation of the desired protein in pure form, in large quantities and at a
reasonable cost.
Different strategies have been employed for obtaining the desired genes. For very
small proteins, such as the pituitary hormone somatostatin whose complete aminoacid
sequences are known, the genes can be synthesised in the laboratory. With larger
proteins, this is not possible. The DNA can be cleaved by specific enzymes called
restriction endonucleases and the fragments containing the desired genes isolated.
This does not work with DNA of higher organisms as they contain introns. In such
cases, the messenger RNA concerned can be isolated from cells producing the desired
protein. A DNA copy is made from the mRNA using the enzyme reverse transcriptase.
The double-stranded DNA gene is then prepared using DNA polymerase. This is
incorporated into suitable vectors or carriers, such as plasmids or temperate
bacteriophages, for insertion into microorganisms. The microorganism commonly
employed is E. coli K12, though many other bacteria and yeasts have also been used.
Genetic engineering has become an established branch of biotechnology with
great scope for commercial exploitation. Cloned human insulin, interferons,
somatostatin, growth hormones and many other biologicals have already been
marketed. Safer vaccines can be produced by cloning the protective antigens of
pathogens, as has already been done, as in the case of foot and mouth disease, and
hepatitis В and rabies viruses. This versatile technique has many extramedical
applications also. Restriction endonucleases: (restriction enzymes) are
microbial enzymes which cleave double-stranded DNA at specific oligo nucleotide
sequences. The natural function of restriction enzymes in bacteria may be the
destruction of foreign DNA that may enter the bacterial cell.
Restriction enzymes split DNA strands into fragments of varying lengths.
These can be separated by gel electrophoresis and stained with ethydium bromide
and photographed. DNA probes: The specificity of the interaction in base pairing
during DNA or RNA synthesis enables the production of specific DNA probes.
28
These are radioactive, biotinylated or otherwise labelled copies of cloned singlestranded DNA fragments, usually 20-25 nucleotides long and containing unique
nucleotide sequences which can be used for the detection of homologous DNA by
hybridisation. DNA probes are being used increasingly in the diagnosis of
infectious diseases. Probes containing sequences unique to the microbe (strain, species
or group) to be detected can be added to microbial cultures, body fluids, tissues or other
materials suspected to contain the microbe or its DNA. The DNA probe hybridises
with the complementary specific sequences on the microbe's DNA. The advantages of
DNA probes for diagnosis are their high degree of specificity, ability to detect minute
quantities of complementary DNA even in the presence of other microbes, and the
capacity to recognise microbes that are either difficult or impossible to culture. DNA
probes for the detection of many pathogens are now commercially available.
5.2. Polymerase chain reaction (PCR)
This is a rapid automated method for the amplificationof specific DNA
sequences for genes), invented by Kary В Mullis in 1983, for which he won the Nobel
Prize in Chemistry in 1993. PCR consists of several cycles of sequential DNA
replication where the products of the first cycle become the template for the next cycle.
It makes available abundant quantities of specific DNA sequences starting from sources
containing minimal quantities of the same.
The technique is as follows: two oligonucleotide primers complementary to the
flanking region of the DNA sequence to be amplified are incubated with die target
DNA, nudeorides and DNA polymerase. The reaction consists of three essential steps:
1. heat denaturation of the sample DNA to single strand;
2. annealing of sequence-specific oligonucleotide primers to the boundaries of the
DNA segment;
and
3. extension of the primers by DNA polymerase to form new double-stranded DNA
across the segment by sequential addition of deoxynucleotides.
These three steps constitute one cycle of the reaction. These cycles are repeated
several times, usually for 20-50 cycles in the thermocycler, at the end of which
hundreds of thousands of copies of the original target sequences are available. As the
reation steps take place at high temperature (50-95оC), a heat-stable polymerase, such
as Taq I has to be employed.
With its enormous capacity to amplify DNA, PCR is a versatile tool useful in
diverse areas such if diagnosis of infectious, genetic or neoplastic diseases, in
forensic investigations, in archeobiological studies of ancient specimens and in
the examination of phylogenetic relationships in evolution.
Molecular epidemiology: One offshoot of molecular genetics is molecular
epidemiology. Here molecular methods such as plasmid profile analysis, genomic
fingerprinting and PCR are used for the identification and matching of microbial
isolates for epidemiological purposes. Genetic mapping: As a result of the
remarkable advances in molecular genetics, it has been possible to delineate the
complete genomic sequences of bacteriophages and other viruses, bacteria and their
29
plasmids, and even of some eukaryotes including mammals. Quite apart from the
useful information it has provided in microbiology, its success emboldened the
international scientific community to venture on the 'human genome project', the most
expensive and ambitious scientific project so far undertaken in biology. The results of
this mammoth study became available by the dawn of the twenty-first century and
have opened vistas in human biology and medicine, as well as controversies and
dilemmas that transcend medicine.
30
List of literature.
1. Microbiology. V.M. Korshunov. Moscow, 2002.
2. Textbook of Microbiology. R. Ananthanarayan. Orient Longman, 2007.
3. Medical Microbiology. Mackie and McCartney Longman Group (FE) Ltd 13
th edition
4. Hardy K. 1986. Bacterial Plasmids. 2nd edn. Reinhold
5. Mullis KB. 1993. ‘The polymerase chain reaction.’ Nobel Lecture,
Stockholm
31
Contents:
Introduction………………………………………………………….....6
1 Genome Organization………………………………………………...7
1.1. Bacterial Chromosome…………………………………………...8
1.2. Bacterial Plasmids………………………………………………..8
1.3. Transposable Genetic Elements………………………………….9
2. Phenotypic Variation……………………………………………….11
2.1. Regulation of Gene Expression…………………………………11
2.2. Phase variation………………………………………………….16
3. Genotypic Variation………………………………………………..16
3.1 Mutation………………………………………………………….16
3.2. Selection of mutants …………………………………………….17
3.3. Mechanisms of mutation………………………………………...17
3.4. Mutation that affect biochemical pathways……………………..18
3.5. Drag-resistance Mutation………………………………………..19
4. Gene Transfer………………………………………………………19
4.1. Transformation …………………………………………………19
4.2. Transduction…………………………………………………….20
4.2.1. Generalized Transduction…………………………………...23
4.2.2. Specialized Transduction……………………………………23
4.3. Conjugation……………………………………………………..24
5. Molecular Genetics…………………………………………………28
5.1. Genetic Engineering……………………………………………28
5.2. Polymerase Chain Reaction (PCR)……………………………29
List of literature ……………………………………………………...31
32
Bacterial Genetics
The Manual for the Students
of the English-speaking Medium
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