Chapter 3.4-Bacterial Genetics

General Bacteriology:
Bacterial Genetics
Learning objectives
At the end of the session, the students will be able to understand:
▰ The principles of bacterial genetics
▰ Concept of transformation, transduction, lysogenic conversion, conjugation

and transposition
▰ Gene transfer
3
Essentials of Medical Microbiology
PRINCIPLES OF
BACTERIAL GENETICS
4
PRINCIPLES OF BACTERIAL
GENETICS
▰ Study of heredity and gene variations seen in bacteria.
▰ All hereditary characteristics of the bacteria - encoded in their DNA

(deoxyribonucleic acid).
▰ Bacterial DNA is present in chromosome as well in extrachromosomal

genetic material as plasmid.
5
BACTERIAL DNA
6
BACTERIAL DNA
▰ Bacteria possess - single haploid chromosome - comprising of super coiled
circular double stranded DNA.
▰ Bacterial DNA lacks basic proteins.
▰ Bacteria do not have a true nucleus; but the genetic material is located in an
irregularly-shaped region called the nucleoid.
▰ No nuclear membrane or nucleolus.

7
Structure of DNA (Watson and Crick
Model)
▰ Composed of two strands - complementary
nucleotides that are coiled together in the
form of a double helix.
▰ Described first by Watson and Crick.
8
Model) (Cont..)
▰ Each strand is composed of three elements
 Backbone of deoxyribose sugar and phosphate groups
 Nitrogenous bases are attached to the sugar group
 Nucleotide and nucleoside - components of the DNA strand.
 Nucleoside = Sugar + nitrogenous base
 Nucleotide = Sugar + nitrogenous base + phosphate
9
Model) (Cont..)
▰ There are four nitrogenous bases,
 Two purines: adenine (A) and guanine (G)
 Two pyrimidines: thymine (T) and cytosine (C)
10
Model) (Cont..)
▰ Pairing- Two DNA strands - held together by hydrogen bonds. Pairing
follows specific rule-
 Adenine of one strand binds with thymine (A-T) of other strand by

double hydrogen bonds.
 Guanine of one strand binds with cytosine (G-C) of other strand by

triple hydrogen bonds.
11
Model) (Cont..)
▰ In a molecule of DNA, the number of A=T; G=C.
▰ The ratio of A + T to G + C is constant for each species but varies widely

from one bacterial species to another.
12
DNA Replication
▰ In eukaryotes, during DNA replication, the two strands of the double helix
unwind from one another and separate.
▰ Each strand acts as template for a new DNA strand which is synthesized
through complementary base pairing- A with T, and G with C.
13
DNA Replication (Cont..)
Bidirectional replication:
▰ DNA helix is unwound at a region called replication fork, which is the site
at which the DNA synthesis occurs and individual strands are replicated.
▰ Seen in E. coli.
14
Rolling-circle mechanism:
▰ Replication occurs during bacterial conjugation and during the

reproduction of viruses.
15
Replication Enzymes Functions
Helicases DNA unwinding
Topoisomerase (e.g. DNA gyrase in Relieves the tension generated by rapid unwinding by
E.coli) removing the super twists.
DNA polymerase  Forms complementary strand synthesis by adding
nucleotides to the growing end of the strand (5′ to 3′
direction).
 DNA polymerase III plays the major role in
replication, although it is probably assisted by
polymerase I.
 Polymerases I and II participate - repair of damaged
DNA.
DNA ligase- Helps in joining of the fragments
16
BACTERIAL RNA
17
BACTERIAL RNA
▰ RNA (ribonucleic acid) is structurally similar to DNA, except for
 In sugar- ribose is present instead of deoxyribose
 In nitrogenous base- uracil replaces thymine.
▰ Three different types of RNA in a cell:
 Messenger RNA (mRNA)
 Ribosomal RNA (rRNA)
 Transfer RNA (tRNA).
18
POLYPEPTIDE
SYNTHESIS
19
POLYPEPTIDE SYNTHESIS
▰ Gene - segment of DNA that stores information for a particular polypeptide
synthesis.
▰ Genetic information - stored in DNA is transcribed into RNA and then

translated to form the particular polypeptide.
20
Genetic Code
Terminology Explanation
Codon (Nirenberg and Khorana – Sequence of three nucleotide bases present on mRNA that
1968) stores the information of an amino acid synthesis.
Sense codons  There are 64 codons, out of which 61 are sense codons.
 Each codon directs the production of a single amino acid.
 There are only 20 amino acids, so more than one codon
exist for the same amino acid.
Non-sense codons  Three codons (UGA, UAG, and UAA) do not code for any
amino acids.
 Involved in the termination of translation.
21
Genetic Code (Cont..)
Terminology Explanation
Start codon  First codon of an mRNA from which the translation begins.
 Most common start codon - AUG (codes for methionine in
eukaryotes and modified methionine [N-Formyl methionine
(fMet)] in prokaryotes).
Anticodon  Set of three nucleotide bases present on tRNA that is
complementary to the nucleotide bases of codon on mRNA.
22
Transcription
▰ Process in which a particular segment of DNA is copied into RNA by the

enzyme RNA polymerase.
▰ DNA acts as a template for synthesis of m-RNA, therefore, the bases in m-

RNA are complementary to one strand of DNA.
23
Translation
▰ mRNA transcribed from DNA is decoded by a ribosome to produce a
specific amino acid chain (cytoplasm).
Translation phases Function
Initiation First tRNA is attached at the start codon of mRNA.
Elongation tRNA transfers an amino acid to the adjacent tRNA corresponding to the next
codon.
Translocation Ribosome then moves (translocates) to the next mRNA codon to continue the
process, creating an amino acid chain
Termination: When a stop codon is reached, the ribosome releases the polypeptide.
24
PLASMID
25
PLASMID
▰ Extra chromosomal ds circular DNA molecules that exist in free state in the
cytoplasm of bacteria and also found in some yeasts.
▰ May be present singly or in multiple numbers- up to >40 plasmids per cell.
▰ Plasmids are capable of replicating independently.
26
PLASMID (Cont..)
▰ Episome - Plasmid may integrate with chromosomal DNA of bacteria and

such plasmids are called as episomes. They replicate along with bacterial
chromosome.
27
PLASMID (Cont..)
▰ Curing - process of eliminating the plasmids from bacteria.
 Occur spontaneously or may be induced by treatment with acridine,

radiations, thymine starvation, and growth at higher temperatures (does
not destroy host cell).
28
Classification of Plasmids
1. Based on ability to perform conjugation:
 Conjugative plasmids- Possess ability to transfer themselves to other

bacteria by means of conjugation (self transmissible or conjugative
plasmids).
 Non-conjugative plasmids-They cannot transfer themselves (non-

transmissible plasmids).
29
Classification of Plasmids (Cont..)
2. Based on compatibility between the plasmids:
 Compatible plasmids - Different plasmids can exist in a single bacterial

cell only if they are compatible to each other.
 Incompatible plasmids - If two plasmids are not compatible, one or the

other will be rapidly lost from the cell.
30
Classification of Plasmids (Cont..)
3. Based on function:
▰ Fertility or F-plasmids
▰ Resistance (R) plasmids
▰ Col plasmids
▰ Virulence plasmids
▰ Metabolic plasmids
31
Plasmid as Vector
▰ Ability to transfer DNA from one cell to another.
▰ Important vectors in genetic engineering.
▰ Plasmids contain certain sites where genes can be inserted artificially by

recombinant DNA technology.
▰ Uses - protein production, gene therapy.
32
Gene Transfer in
Bacteria
33
Gene Transfer in Bacteria
▰ Bacteria undergo genetic variation and acquire new gene through a

mechanism called mutation.
▰ Following which - newly acquired genes are transferred either vertically to

their off-springs during cell devision or horizontally to other bacteria in the
surrounding.
34
MUTATION
▰ Mutation - Random, undirected heritable variation caused by change in

nucleotide sequence of the genome of the cell.
▰ Frequency of mutation -10- 2 to 10-10 per bacterium per division.
35
MUTATION (Cont..)
Mutations occur two ways:
▰ Spontaneous mutations - Occur naturally in any dividing cells - without
adding any mutagen.
▰ Induced mutations - Result of exposure of the organism to a mutagen.
Examples of mutagens include-
 Physical agent- e.g. ultraviolet radiations- cytosine and thymine are
more vulnerable to UV rays.
 Chemical agents-alkylating agents, 5-bromouracil.
36
MUTATION (Cont..)
▰ Most mutants go unrecognised as the mutation may be lethal or may

involve some minor functions that may not be expressed.
▰ Mutation is best appreciated when it involves a function, which can be

readily observed by experimental methods.
37
MUTATION (Cont..)
▰ Mutation can affect any gene and hence may modify any characteristic of
the bacterium.
 Sensitivity to bacteriophages
 Loss of ability to produce capsule or flagella
 Loss of virulence
 Alteration in colony morphology
 Alteration in pigment production
38
Classification of Mutation Types
▰ Small-scale mutations
▰ Large-scale mutations
39
Types of mutations
Forward mutations - Substitutions at single nucleotide base pair
At DNA Level
Transition Point mutation that changes a purine nucleotide to another purine (A ↔ G) or a pyrimidine
nucleotide to another pyrimidine (C ↔ T).
Transversion Substitution of a purine for a pyrimidine or vice versa -DNA, (C/T ↔ A/G).
At codon level
Silent mutation New codon codes for the same amino acid, e.g. aGG « CGG, both code for arginine
neutral mutation New codon forms different but functionally equivalent amino acid: aaa (lysine) aGa
(arginine)
Missense New codon codes for a different amino acid
mutation
nonsense New codon is a stop codon which causes termination, e.g. CaG (Glutamine) « UaG (stop)
mutation
40
Types of mutations (Cont..)
Addition or Deletion at single or many nucleotide base pairs:
Frameshift Any addition or deletion of base pairs that is not a multiple of three results in a shift
mutation in the normal reading frame of the coded message forming new set of triplet codon
Reverse mutations A second mutation which nullifies the effect of the first mutation that results in
gaining back the function of the wild phenotype is called as reverse mutation.
True reversion A true reverse mutation converts the mutant nucleotide sequence back to the wild-
type sequence.
AAA (Lysine) forward mutation GAA(Glutamine) reverse mutation AAA(Lysine)
(wild type) (mutant) (wild type)
41
Types of mutations (Cont..)
Addition or Deletion at single or many nucleotide base pairs:
Equivalent Second mutation produces a different codon which codes for the same aminoacid of
reversion wild type sequence.
UCC (Serine) forward mutation GAA (Cystine) reverse mutation AAA(Serine)
(wild type) (mutant) (wild type)
Suppressor It is a second mutation in a different gene, that reverts the phenotypic effects of an
mutation already existing mutation.
42
Detection and Isolation of
Mutants
Recognized - by genetic and phenotypic methods.
▰ Gene sequencing - method of choice currently used.
▰ Phenotypic methods - less commonly used now.

 Fluctuation test - demonstrates spontaneous mutations in bacteria
 Replica plating method - used to demonstrate auxotrophic mutants
 Ames test - used to test the carcinogenicity of a mutagen.
43
HORIZONTAL
GENE TRANSFER
44
HORIZONTAL GENE
TRANSFER
Occurs in bacteria by several methods:
▰ Transformation (uptake of naked DNA)
▰ Transduction (through bacteriophage)
▰ Lysogenic conversion
▰ Conjugation (plasmid mediated via conjugation tube).
45
TRANSFORMATION
▰ Definition - Process of random uptake of free or naked DNA fragment

from the surrounding and incorporated into its bacterial chromosome in a
heritable form.
▰ Natural transformation studied in -Streptococcus, Bacillus, Haemophilus,

Neisseria, Acinetobacter and Pseudomonas.
46
Mechanism of Transformation
▰ When bacteria lyse, they release large amounts of dsDNA into the
surrounding environment. Their uptake is usually mediated by the
competent bacteria present in the surroundings
47
Competency for transformation
▰ Bacteria expressing competence factors (S. pneumoniae) can uptake any

DNA fragment irrespective of source.
▰ If competence factors not expressed (Haemophilus influenza) - uptake of

DNA occurs only from the closely related species.
48
STEPS IN TRANSFORMATION
49
Griffith Experiment
▰ Griffith experiment (1928) on mice using pneumococci strains provided the
direct evidence of existence of transformation.
50
Griffith Experiment (Cont..)
• Stated that the live non-capsulated strains were transformed into the
capsulated strains due to transfer of the capsular genes released from the lysis
of the killed capsulated strains which was confirmed later by Avery, Macleod
and McCarty in 1944.
51
TRANSDUCTION
52
Definition
▰ Transmission of a portion of DNA from one bacterium to another by a

bacteriophage
53
Mechanism of Transduction
▰ During the transmission of bacteriophages from one bacterium to other - a

part of the host DNA may accidentally get incorporated into the
bacteriophage and then gets transferred to the recipient bacterium.
▰ This leads to - acquisition of new characters by the recipient bacterium

coded by the donor DNA.
54
Bacteriophages -Life cycle
1. Lytic or virulent cycle:
▰ Bacteriophage multiplies in host cytoplasm - produces large number of

progeny phages, which subsequently, are released causing death and lysis
of the host cell.
55
Bacteriophages -Life cycle (Cont..)
2. Lysogenic or temperate cycle:
▰ Host bacterium is unharmed. The
▰ Phage DNA remains integrated with the bacterial chromosome as the

prophage, which multiplies synchronously with bacterial DNA.
56
Bacteriophages -Life cycle (Cont..)
2. Lysogenic or temperate cycle (Cont..)
▰ When the phage DNA tries to come out, it is disintegrated from host
chromosome, comes out into the cytoplasm, and behaves as a lytic phage.
▰ It replicates to produce daughter phages, which are subsequently released

by host cell lysis.
57
Types of Transduction
1. Generalized Transduction
2. Restricted or Specialized Transduction
58
Generalized Transduction
▰ Involves transfer of any part of the donor bacterial genome into the
recipient bacteria.
▰ Occurs as a result of defective assembly during the lytic cycle of virulent

and some temperate phages.
59
Generalized Transduction (Cont..)
60
Restricted or Specialized Transduction
61
Role of Transduction
▰ Method of transfer of episomes and plasmids.
▰ Drug resistance
▰ Treatment- Transduction has also been proposed as a method of genetic

engineering in the treatment of some inborn metabolic defects.
62
LYSOGENIC
CONVERSION
63
LYSOGENIC CONVERSION
▰ During the temperate or lysogenic life cycle - phage DNA remains
integrated with the bacterial chromosome as prophage - multiplies
synchronously with the bacterial DNA.
▰ Prophage acts as an additional chromosomal element - encodes for new

characters and is transferred to the daughter cells - known as lysogeny or
lysogenic conversion.
64
Phage Coded Toxins
▰ Diphtheria toxin
▰ Cholera toxin
▰ Verocytotoxin of E. coli
▰ Streptococcus pyrogenic exotoxin (SPE)— A and C
▰ Botulinum toxin C and D
65
CONJUGATION
66
CONJUGATION
▰ Refers to - transfer of genetic material from one bacterium (donor or male)
to another bacterium (recipient or female) by mating or contact with each
other and forming the conjugation tube.
▰ Discovered first by Lederberg and Tatum (1946).
67
F + X F - mating
68
HFR Conjugation
69
F’ X F - mating
70
Fate of the Donor DNA
▰ Following horizontal gene transfer, the donor DNA either gets degraded by
host nucleases or may integrate with recipient chromosome by a method
called recombination.
▰ The donor DNA integrates with the recipient chromosome either as a

replacement piece (usually occurs in transformation) or as an extra piece
71
Role of conjugation in bacterial drug
resistance
▰ R factor (or the resistance factor) is a plasmid which has two components.
(R factor = RTF+ r determinants).
 Resistance transfer factor (RTF)- is the plasmid responsible for
conjugational transfer (similar to F factor)
 Resistance determinant (r) - An R factor can have several r
determinants and each r determinant coding for resistance to one drug.
72
TRANSPOSITION
73
TRANSPOSITION
▰ Transposons or transposable elements are the bacterial genes that are
capable of intracellular transfer between:
 Chromosome to chromosome
 Plasmid to plasmid
 Chromosome to plasmid or vice versa
▰ Process of such intracellular transfer - Transposition.
74
Types of Transposons
▰ Insertion Sequence Transposon
▰ Composite Transposon
75
Types of Transposons (Cont..)
A. Insertion sequence transposon; B. Hairpin loop structure of transposon; C. Composite

transposon
76
GENE TRANSFER BY
ARTIFICIAL
METHODS
77
GENETIC ENGINEERING
▰ Refers to deliberate modification of an organism’s genetic information by

directly altering its nucleic acid genome.
▰ Accomplished by a precise mechanism known as recombinant DNA

technology.
78
GENETIC ENGINEERING (Cont..)
▰ Gene coding for any desired protein is isolated from an organism, and then
inserted into suitable vector, which is then cloned in such a way that it can
be expressed in the formation of specific (desired) protein.
79
Recombinant DNA Technology
▰ Procedure of recombinant DNA technology involves the following steps:
 Treatment with restriction enzyme
 Southern blot
 Recombination with a vector
 Introduction of the vector into bacteria
 Cloning
80
Applications of Genetic
Engineering
▰ Production of vaccines
▰ Production of antigens used in diagnostic kits
▰ Production of proteins used in therapy
▰ Transgenic animals
▰ Gene therapy
81
Vector
▰ Small piece of DNA, into which a foreign DNA fragment can be inserted
and that can be stably maintained in an organism and used for cloning
purposes.
▰ Four major types of vectors, such as: 1. Plasmids, 2. bacteriophages,
3. cosmids, 4. artificial chromosomes
82
BLOTTING
TECHNIQUES
83
BLOTTING TECHNIQUES
▰ Blot, in molecular biology - method of transferring DNA , RNA, or

proteins, from gel onto a carrier (e.g. nitrocellulose membrane), followed
by their detection by using specific nucleic acid probes (for DNA or RNA
detection) or enzyme immunoassay (for protein detection).
84
BLOTTING TECHNIQUES
(Cont..)
▰ Southern blot - to detect DNA
▰ Northern blot - to detect RNA
▰ Western blot - to detect proteins
▰ Eastern blot - Modification of Western blot - analyze proteins for post-

translational modifications using probes that may detect lipids,
carbohydrate, phosphorylation.
85
Questions:
▰ Q1. Mechanism of direct transfer of free DNA:
a. Transformation
b. Conjugation
c. Transduction
d. Transposition
86
Questions:
▰ Q2. Plasmids have following functions except:
a. Transposition
b. Conjugation
c. Resistance
d. Virulence
87
Questions:
▰ Q3. Griffith experiment on mice using pneumococci strains provides the
direct evidence of:
a. Transformation
b. Transduction
c. Conjugation
d. Transposition
88
Questions:
▰ Q4. Horizontal transmission of ‘R’ factor is by:
a. Transformation
b. Transduction
c. Conjugation
d. Fusion
89

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Chapter 3.4-Bacterial Genetics

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General Bacteriology:

At the end of the session, the students will be able to understand:

▰ The principles of bacterial genetics

▰ Concept of transformation, transduction, lysogenic conversion, conjugation

▰ Study of heredity and gene variations seen in bacteria.

▰ All hereditary characteristics of the bacteria - encoded in their DNA

▰ Bacterial DNA is present in chromosome as well in extrachromosomal

▰ Bacterial DNA lacks basic proteins.

▰ No nuclear membrane or nucleolus.

▰ Described first by Watson and Crick.

▰ There are four nitrogenous bases,

 Two purines: adenine (A) and guanine (G)

 Two pyrimidines: thymine (T) and cytosine (C)

 Adenine of one strand binds with thymine (A-T) of other strand by

 Guanine of one strand binds with cytosine (G-C) of other strand by

▰ In a molecule of DNA, the number of A=T; G=C.

▰ The ratio of A + T to G + C is constant for each species but varies widely

▰ Replication occurs during bacterial conjugation and during the

▰ Genetic information - stored in DNA is transcribed into RNA and then

▰ Process in which a particular segment of DNA is copied into RNA by the

▰ DNA acts as a template for synthesis of m-RNA, therefore, the bases in m-

▰ May be present singly or in multiple numbers- up to >40 plasmids per cell.

▰ Plasmids are capable of replicating independently.

▰ Episome - Plasmid may integrate with chromosomal DNA of bacteria and

▰ Curing - process of eliminating the plasmids from bacteria.

 Occur spontaneously or may be induced by treatment with acridine,

1. Based on ability to perform conjugation:

 Conjugative plasmids- Possess ability to transfer themselves to other

 Non-conjugative plasmids-They cannot transfer themselves (non-

2. Based on compatibility between the plasmids:

 Compatible plasmids - Different plasmids can exist in a single bacterial

 Incompatible plasmids - If two plasmids are not compatible, one or the

▰ Ability to transfer DNA from one cell to another.

▰ Important vectors in genetic engineering.

▰ Plasmids contain certain sites where genes can be inserted artificially by

▰ Uses - protein production, gene therapy.

▰ Bacteria undergo genetic variation and acquire new gene through a

▰ Following which - newly acquired genes are transferred either vertically to

▰ Mutation - Random, undirected heritable variation caused by change in

▰ Frequency of mutation -10- 2 to 10-10 per bacterium per division.

▰ Most mutants go unrecognised as the mutation may be lethal or may

▰ Mutation is best appreciated when it involves a function, which can be

▰ Gene sequencing - method of choice currently used.

▰ Phenotypic methods - less commonly used now.

▰ Transformation (uptake of naked DNA)

▰ Transduction (through bacteriophage)

▰ Conjugation (plasmid mediated via conjugation tube).

▰ Definition - Process of random uptake of free or naked DNA fragment

▰ Natural transformation studied in -Streptococcus, Bacillus, Haemophilus,

▰ Bacteria expressing competence factors (S. pneumoniae) can uptake any

▰ If competence factors not expressed (Haemophilus influenza) - uptake of

▰ Transmission of a portion of DNA from one bacterium to another by a

▰ During the transmission of bacteriophages from one bacterium to other - a

▰ This leads to - acquisition of new characters by the recipient bacterium

1. Lytic or virulent cycle:

▰ Bacteriophage multiplies in host cytoplasm - produces large number of

2. Lysogenic or temperate cycle:

▰ Host bacterium is unharmed. The

▰ Phage DNA remains integrated with the bacterial chromosome as the