CHAPTER IV

INTRODUCTION TO BACTERIAL
GENETICS
 Learning objective:

At the end of this chapter the students will be able to:

 Describe Bacterial genomic organization

 Express DNA Replication mechanism

 Describe bacterial gene expression and regulation

mechanism
 Recognize Mutation and recombination

 Discuss gene trafer mechanism in bacteria

 The science of genetics defines and analyzes
heredity, or constancy and change in the vast array
of physiologic functions that form the properties of
organisms.
 The unit of heredity is the gene, a segment of DNA
that carries in its nucleotide sequence information
for a specific biochemical or physiologic property.
 The chemical basis for variation in phenotype is
change in genotype, or alteration in the sequence of
DNA within a gene or in the organization of genes.
 Bacterial genomic organization
The Structure of DNA
 A bacterium’s genetic information is stored in its
chromosome and plasmids.
 Each of these structures is made of a single DNA double
helix twisted to the right, then additionally twisted to the left
about its helical axis (supercoiled).
 Plasmids consisting of linear DNA also occur, although this
is rare.
 Cont’d
 Genetic information is stored as a sequence of
bases in deoxyribonucleic acid (DNA).
 DNA molecules is composed of chain of nucleotides.
 The nucleotide has three components; a nitrogenous
base, deoxyribose, and phosphate residue.
 The four nitrogenous bases i.e pyrimdine (thymine
and cytosine) and purine (guanine and adenine).
 Hydrogen bonds unites two nitrogenous bases of
opposite strands, thus making it double stranded.
 Adenine always binds to thymine
 Guanine binds to cytosine.
 Cont’d
 The orientation of the two DNA strands is described
as antiparallel; one strand is chemically oriented in a
5' to 3' direction, while its complementary strand
runs 3' to 5'.
 Ribonucleic acid (RNA) participates in the
expression of the genetic information stored in the
DNA
 The complementarity of the bases enables one
strand (template strand) to provide the information
for copying or expression of information in the other
strand
 The base pairs are stacked within the center of the
DNA double helix and they determine its genetic
information.
 Cont’d
 The negatively charged phosphodiester backbone of
DNA faces the solvent.
 The length of a DNA molecule is usually expressed in
thousands of base pairs, or kilobase pairs (kbp).


The Structure of RNA


 Structure of RNA is similar to that of DNA with

two differences:

– It has ribose sugar instead of deoxyribose

– It has nitrogenous base uracil in place of

thyamine in DNA

 It is usually found as single strand



Types of RNA


There are three types of RNA:
1.Messenger RNA (mRNA): carries information
from the DNA to the ribosome's, where it is
translated
2.R i b o s o m a l R N A ( r R N A ) : c o m p o n e n t o f
ribosomes where protein synthesis occurs
3.Transfer RNA (tRNA): carry amino acids to the
growing peptide
 DNA Replication
 DNA replication is a process by which the DNA copy
itself to produce two identical copies
 It is termed semiconservative because the double
strand of DNA is opened up during replication, where
upon each strand serves as the matrix for synthesis of
a complementary strand.
 Replication uses both strands of the parent DNA as
templates.
 Thus each of the two new double strands “conserves”
one old strand.
 The doubling of each DNA molecule (replicon) begins
at a given starting point, the so-called origin of
replication.
 This process continues throughout the entire fission
cycle.
 DNA Replication … Cont’d
The replication process require different enzymes:
helicase to unwind the DNA at the origin to
expose the DNA
primase to synthesize primers to start the
process
DNA dependent DNA polymerase that copy the
DNA, but only in the 5’ to 3’ direction.
Several other enzymes are involved in the
process of DNA replication
Figure: Semi conservative DNA replication
 Gene expression
• Genes are sequences of nucleotides that have a
biologic function
• Gene expression or synthesis of gene product in
bacteria follows two steps, Transcription and
Translation
Transcription.
• Copying of the sense strand of the DNA into mRNA.
• The continuous genetic nucleotide sequence is
transcribed into mRNA.
• The transcription process can be broken down into the
three phases promoter recognition, elongation, and
termination.
• The promoter region is the site where the RNA
polymerase begins reading the DNA sequence.
Replication of circular bacterial
chromosome
 Gene expression
 Genes that code for functionally related proteins, for
example proteins that act together to catalyze a certain
metabolic step, are often arranged sequentially at
specific locations on the chromosome or plasmid.
– Such DNA sequences are known as operons. Eg. Lac
operon .
 The mRNA synthesized by the transcription of an operon
is polycistronic, i.e., it contains the information
sequences of several genes.
 The information sequences are separated by inter
cistronic regions.
 Each cistron has its own start and stop codon in the
mRNA.
 Gene expression in bacteria
 Gene expression or
synthesis of gene product
in bacteria follows two
steps, transcription and
translation
 DNA codes for RNA, and
RNA codes for protein.
 The DNARNA step is
transcription, and the
RNA protein step is
translation.
 Gene expression…cont’d
Transcription
Copying of the sense
strand of the DNA into
mRNA, after the promoter
region recognized by RNA
polymerase begins reading
the DNA sequence.
The continuous genetic
nucleotide sequence is
transcribed into mRNA, and
stop when reaches
terminator sequence.
 Gene expression…cont’d
Translation
 Transformation of the nucleotide sequence carried by the
mRNA into the polypeptide amino acid sequence at the
ribosomes.
 Nucleotide sequence of mRNA is translated into amino
acid sequence of protein using “three letter words” =
codons
 Translation of mRNA begins at the start codon: AUG, which
also code for amino acid Methionine.
 Translation ends at a stop codon: UAA, UAG, UGA
 In principle, prokaryote and eukaryotic translation is the
same.
 The enzymes and other factors involved do, however, differ
structurally and can therefore be selectively blocked by
antibiotics.


The translation process in protein synthesis

• Initiation: occurs when
the ribosomes binds to
the mRNA, and the first
amino acid attached to
its tRNA.
• Elongation: the
ribosomes adds one
amino acid at a time to
the growing polypeptide
chain.
• Termination: the
ribosomes releases the
mRNA and the
polypeptide,
 Genetic code
 Genetic information is stored in DNA as a code.
 The unit of code is known as codon.
 It consists of three bases. Therefore, code is triplet.
 Permutation of three bases at a time from among 1st
four type of major bases: A, G, C, & U, constitute 43 or
64 codon.
 In total of these, 61 codons encode 20 different amino
acids as eventually some amino acids, are encoded by
more than one codon.
 Each codon specifies or codes for a single amount
acid, but more than one codon may exist for a single
amino acid. Therefore, code is degenerate.
The codons from mRNA specify a given amino acid.

Fig. The Genetic code

 Representation of the codons and their corresponding
amino acids.
 Fig. Interpreting the DNA code
 Genetic code…Cont’d

 There three stop codons (UAA, UAG and UGA)

and one Start codon (AUG) which also code for
amino acid Methionine.
 According to the “central dogma” of molecular
genetics: DNA codes for RNA, and RNA codes
for protein.
Regulation of Gene Expression

 Bacteria demonstrate a truly impressive capacity

for adapting to their environment.
 A number of regulatory bacterial mechanisms are
known, these include:
 Transcription regulation
 Translational regulation
 Post-translational regulation, and
 Termination regulation.
 Cont’d
 The mechanism that has been investigated most
thoroughly is transcriptional regulation of
catabolic and anabolic operons by a repressor or
activator.
 A single regulator protein can also activate or
repress several genes not integrated in an
operon, i.e., at various locations on the DNA.
– Such functional gene groups are called
regulons
 Gene regulation in prokaryotes


 In bacteria, genes are clustered into operons (a
group of genes) that are transcribed at the same
time.
 encode and control the proteins necessary to
perform coordinated function, such as
biosynthesis of a given amino acid
 RNA that is transcribed from prokaryote operons
is polycistronic, found only in bacteria
 Cont’d
 The activity of RNA polymerase at a given promotor is
regulated by interaction with accessary proteins, which
affected its ability to recognize start sites.
 These regulatory proteins can act both positively (activators)
and negatively (repressors)
 The accessibility of promotor region of DNA is regulated by
the interaction of proteins with sequences termed operators.
 Two major modes of transcriptional regulation function in
bacteria (E. coli) to control the expression of operons.
 The one that exerted on operons that produce gene products
necessary for the utilization of energy; these are catabolite-
regulated operons
 The other mode regulates operons that produce gene products
necessary for the synthesis of small biomolecules such as
amino acids
 Cont’d
 During normal growth on a glucose-based medium, the lac
repressor is bound to the operator region of the lac operon,
preventing transcription
 In the presence of an inducer of the lac operon, repressor
protein binds the inducer and is rendered incapable of
interacting with operator region of the operon.
 The lac operon is repressed, even in the presence of lactose,
if glucose is also present.
 This repression is maintained until the glucose supply is
exhausted. The repression of the lac operon under these
condition is termed catabolite repression and is a result of
the low levels of cAMP that result from an adequate glucose
supply
 Cont’d
• The repression of the lac operon is relieved in
the presence of glucose if excess cAMP is
added
• The ability of cAMP to activate expression from
the lac operon results from an interaction of
cAMP with a protein termed CAP (for cAMP
receptor protein)
• The binding of the cAMP-CRP complex to the lac
operon stimulates RNA polymerase activity 20-to
-50-fold
 Reading assignment!
•Gene expression and control: similarity and
difference
in

Eukaryote Vs. Prokaryote

 Mutation

• Change in genetic material

 Genetic disorders are caused by genetic mutations!!!!!!
• Mutation is an inherited change in the nucleotide base
sequence of that genome.
• Mutation are usually brings about only a very small
amount of genetic change in a cell.
• This can fuel the evolutionary process.
• Changes in bacterial DNA
• It occurs as a result of spontaneous mutations in
individual genes as well as recombination processes
resulting in new genes or genetic combinations
 Cont’d
Spontaneous Mutation
• Mutation that occur under natural conditions
• Usually arise due to error in DNA replication or damage
to DNA
• involve substitution of a single nucleotide, deletions,
inversions, or insertions.
 Cont’d
• Spontaneous
mutation rate  10-6
 1 mutation per
million replicated
genes
• May be neutral (silent
), beneficial, or
harmful.
• Mutagens increase
mutation rate by 10
-1000x
 Recombination
• The term recombination designates processes that lead to
the restructuring of DNA, formation of new genes or
genetic combinations.
– Homologous (generalized) recombination
• A precise exchange of DNA between corresponding
sequences.
– Site-specific recombination
• Integration or excision of a sequence in or from
target DNA. Only a single sequence of a few
nucleotides of the integrated DNA needs to be
homologous with the recombination site on the
target DNA.
– Transposition
• The transposition process does not require the
donor and target DNA to be homologous.
• DNA sequences can either be transposed to a
different locus on the same molecule or to a
 Types of mutations
1. Point mutations (Base pair substitution)

• Missense mutation

• Silent mutation

• Nonsense mutation

2. Frameshift mutations
• Insertion

• Deletion of one or more nucleotide pairs



Point mutation

• A change in one base pair in a DNA sequence
Example: AUG=Met
AAG=Lys
• A point mutation can cause an amino acid to
change, which will change the structure of the
protein being made.


Point mutations

Missense mutation
Changes in the first or
second base of the triplet
more often lead to
significant changes in the
polypeptide.
missense mutation
because the informationa
l "sense“ (sequence of
amino acids) in the
ensuing polypeptide has
changed.
 Cont’d
Nonsense mutation
It changes a codon encoding an amino acid to a
stop codon, ribosomes fall off mRNA.
This result in premature termination of
translation, leading to an incomplete polypeptide
that would almost certainly not be functional
Unless the nonsense mutation occurs very near
the end of the gene, the incomplete product will be
completely inactive.
 Cont’d
Silent mutation
•A change at the DNA level which does not result in
the change of amino acid in the encoded protein.
•Note that silent mutations in coding regions
almost always occur in the third base of the codon.


Point mutations in human cell

• Sickle cell anemia is a blood disease caused by
a point mutation.
• A single nucleotide is changed from “A” to “T”
which causes the amino acid to change from
glutamic acid to valine:
Normal: ACT CCT GAG GAG
Amino Acids: Thr – Pro – Glu – Glu
Sickle cell: ACT CCT GTG GAG
Amino acids:Thr – Pro – Val – Glu


Frameshift mutations


• Frameshift mutation: Adding or deleting
nucleotides to a DNA sequence.
• An insertion/ deletion of a number of
nucleotides that is not a multiple of three will
cause a frame to shift, which will change the
amino acid sequence of polypeptide to change
• frameshift mutation is much worse than a point
mutation, because it causes the entire DNA
sequence to be shifted over.


Additions


• Nucleotide added: entire DNA sequence
changed
 Deletions
• Nucleotide missing: entire DNA sequence
changed
 Mutagens
 Nucleoside (base) analogs have altered
base-pairing properties. They can be:
• randomly incorporated into growing cells
(cancer drugs)
• only used by viral enzymes (e.g. Azidothymidine)
 Frameshift mutagens such as intercalating agents
(e.g.:, aflatoxin, ethidium bromide)
 Ionizing radiation (x-rays and -rays) lead to
deletion mutations
 UV rays lead to thymine dimers (intrastrand
bonding)
 e.g: Crohn’s disease is
caused by a frameshift
mutation.
o It causes
inflammation to the
digestive tract


Plasmids


 Extrachromosomal genetic materials
 Most are circular but some (very few) are linear.
 circular double strand DNA molecules present in the
cytoplasm of bacteria, capable of autonomous replication
 There could be more than one type of plasmid or many
copies of each plasmid per bacterial cell.
 varying size (3 x 103 to 4.5 x 105 bp), but are much smaller
than the bacterial chromosome
 Many of them carry genes that code for certain phenotypic
characteristics of the host cell.
 Episomes: a plasmid that can integrate into the cell’s DNA
 Cont’d
 Like cellular DNA, plasmids
 Replicate when the cell grows and usually distributed to
daughter cells upon cell division.
 They do not encode functions important to bacterial growth
or survival/ viability.
 But they can provide gene products that may benefit the
bacteria in certain circumstances.
 Can be transmitted from one bacteria to another.
 Are naked DNA.
The following plasmid types are medically relevant:
• Virulence plasmids: Carry determinants of bacterial
virulence (virulence genes), e.g., enterotoxin genes or
hemolysin genes.
• Resistance (R) plasmids: Carry genetic information bearing
on resistance to anti infective agents.
 Major types of plasmid
 Fertility plasmids (F plasmids): Involved in transfer
of genes between bacteria by conjugation
(bacterial mating)
 Virulence plasmids: Carry determinants of bacterial
virulence (virulence genes), e.g., enterotoxin genes
or hemolysin genes.
 Resistance plasmids (R plasmids): Carry genetic
information bearing one or several antibiotic
resistance or poisons ---- R plasmids may carry several R
genes at once
 Col-plamids: contain genes coding for colicines,
proteins that can kill other bacteria
 Degradative plasmids: able to digest unusual


Hypothetical Model of Plasmid

• Fig. Note the location of some important elements/

sites: promotors, restriction sites, inserted gene,
replication origin, resistant gene, selectable marker
 Genetic exchange in prokaryotes
 Gene transfer => a source of genetic variation =>
alters the genotype of bacteria.
 involve a unilateral transfer of genetic information from a
donor cell to a receptor cell
 Three mechanisms of genetic exchange are known in
prokaryotes:
o Conjugation
o Transformation
o Transduction


Conjugation (mating)


 A process of gene transfer from a living donor cell
to a living recipient cell by cell-to-cell contact.
 Only cells that have plasmid called the fertility
factor, which encode for sex pili can initiate
conjugation.
 A cell that has this fertility factors is called donor
(F+), while the cell that does not have called the
recipient (F-).
 Types of conjugative processes:
1. F+ conjugation
2. Hfr conjugation
3. Resistance plasmid conjugation
 F+ conjugation
process
 When F+ cell (the
donor) is mated with
an F– cell (the
recipient), a copy of
the F+ plasmid is
transferred to the F–
cell,
 so that after the
process is complete,
both cells will be F+.


Hfr conjugation


• E. coli strain discovered as having Hfr (high
frequency of recombination)
• Hfr strain transfers chromosomal DNA to F-
strains
• The amount of DNA transferred depends on the
time of conjugation
 The lengthy of time a mating occurs, the more
DNA is transferred
 By mating for different times, possible to get
DNA of several sizes, and determine the order of
the genes, and how far apart they are (minutes)
Hfr conjugation




Resistant Plasmid Conjugation

• A transfer of R
plasmid (a plasmid
coding for multiple
antibiotic
resistance and
often a sex pilus)
from a donor
bacterium to a
recipient
bacterium.
• Involves a sex
pilus
 Transformation
• A process by which a bacterium acquire DNA
fragments or genes from surroundings.
• Usually this occurs in microbial culture.
• This is the process by a cell takes in a foreign DNA
fragments found outside the cell and integrates
these fragments into its own DNA.
• Bacterial cells able to take up DNA are termed
competent cells
• The foreign DNA replaces the native DNA in the
bacterial chromosome by genetic recombination.
• This transforms the cells into a genetically
different cell.
 e.g, nonpathogenic S. pneumoniae can be
transformed to pathogenic cells causing
pneumonia
• .
 Transduction

• is a method of gene transfer in which a virus
(phage) acts as a vehicle for carrying DNA from
a donor bacterium to recipient bacterium.
• A bacteriophage is a virus that specifically
attacks bacterial cells
– Composed of genetic material surrounded by
a protein coat
– Bacteriophage have 2 life cycles
• Lytic
• Lysogenic


Lytic and lysogenic cycles of phages


• Transduction happens through either the lytic cycle and
the lysogenic cycle.
• If the lysogenic cycle is adopted, the phage chromosome
is integrated into the bacterial chromosome, where it can
remain dormant for thousands of generations.
• If the lysogen is induced (by UV light for example), the
phage genome is excised from the bacterial chromosome
and initiates the lytic cycle, which culminates in lysis of
the cell and the release of phage particles.
• The lytic cycle leads to the production of new phage
particles which are released by lysis of the host.

 Types of transduction
• Generalized:
– Produce some phage particles with DNA only
from host origin, from any part of chromosome
(P22)
– Transduction from lytic phage
• Specialized:
– Produced particles with both phage and host
DNA, linked in a single DNA molecule, from a
specific region of the chromosome (E. coli phage
)
– Transduction occur through lysogenic phage
Steps in Generalized Transduction

1. A lytic phage adsorbs to a susceptible

bacterium.

2. The phage DNA enters the bacterium. The DNA

directs the bacterium's metabolic machinery to
manufacture phage components and enzymes

3. Occasionally, a phage capsid packs a fragment

of donor bacterium's DNA or a plasmid instead of
a phage DNA by mistake.
 4. The phage are released.

5. The phage carrying the donor

bacterium's DNA adsorbs to a
recipient bacterium

6. The phage inserts the donor

bacterium's DNA it is carrying into
the recipient bacterium .

7. The donor bacterium's DNA is

exchanged for some of the
recipient's DNA.


Steps in Specialised Transduction

1. A temperate phage adsorbs to a
susceptible bacterium and injects
its DNA.

2. The phage inserts its DNA into

the bacterium's DNA to become a
prophage.

3. Occasionally during spontaneous

induction, a small piece of the donor
bacterium's DNA is picked up as part
of the phage's genome in place of
some of the phage DNA which
remains in the bacterium's DNA.
 4. As the phage replicates, the
segment of bacterial DNA
replicates as part of the phage's
genome. Every phage now carries
that segment of bacterial DNA.

5. The phage adsorbs to a recipient

bacterium and injects its genome.

6. The phage genome carrying the

donor bacterial DNA inserts into the
recipient bacterium's DNA.