Bacterial Genetics

Objectives
To understand the structure and organisation of
genetic materials.
To understand the fidelity DNA replication
Termination of DNA replication
 Bacterial Genetics
Genetic information enables the bacteria to
 Maintain viability
 Adapt to the environment
 Multiply
 Be virulent/pathogenic
 Resist antimicrobial agents
To appreciate these aspects we need to understand:
 Structure and organization of genetic material
 Replication and expression of genetic materials
 The mechanisms by which genetic information is
changed and exchanged among bacteria.
 NUCLEIC ACID STRUCTURE AND
ORGANISATION

Hereditary information is encoded in the

nucleic acids:
Deoxyribonucleic Acid (DNA)
Ribonucleic Acid (RNA)
DNA/RNA STRUCTURE
Consists deoxyribose/ribose sugars connected by phosphodiester
bonds.
The bases that are convalently linked to each deoxyribose/ribose
sugar are the key to the genetic code within the DNA molecule.
These bases are:

 Adenine Purines
 Guanine

 Thymine in DNA
 Uracil in RNA Pyrimidines
 Cytosine
Phosphodiester bonds link successive nucleotides in nucleic
acids end 5’ end
5’
O O

O P O O P O

O O
A,T,G,C U,G,C,A
5’ CH2 O 5’ CH2 O
H H H H
3’ H 3’ H
H H
O H O OH
Phosphodiester
O P O O P O
bond
O O
A,T,G,C U,G,C,A
5’ CH2 O 5’ CH2 O
H H H H
3’ H 3’ H
H H
O H O OH

H H
3’ end
3’ end

DNA RNA
7
 DNA/RNA STRUCTURE cont’d
Nucleotide
A single unit consists of sugar, base and the phosphate
Therefore DNA and RNA are nucleotide polymers.
Base sequence
Order of the bases in the DNA/RNA strand
The intact DNA molecule usually is composed of two
nucleotides polymers
DNA/RNA STRUCTURE cont’d
These polymers run antiparallel with the 5’ terminus
of one strand opposite the 3’ terminal of the other
They form a unique structural conformation often
referred to as a “twisted ladder”
The two strands are also complementary (A – T or U
by two H-bonds, and G – C by three H-bonds)
DNA/RNA STRUCTURE cont’d
RNA rarely exists as a double stranded molecule.

Similarly, DNA rarely exits as a single stranded molecule.

The three major types of RNA are:

 Messenger RNA (mRNA) - Intermediates that carry genetic

information from one or few genes to a ribosome, where the

corresponding proteins can be synthesized
 Transfer RNA (tRNA) - adaptor molecules that translate the

information in mRNA into a specific sequence of amino acids

DNA/RNA STRUCTURE cont’d
Ribosomes (rRNA) - structural components of

ribosomes, the complexes that carry out the

synthesis of proteins
 These play a key role in gene expression.
Genes and the genetic code
A gene is a segment of DNA that encodes for a specific
product (RNA or protein) which play essential metabolic
roles in the cells.
 The genome is the complete set of sequences in the
genetic material of an organism. It includes the sequence
of each chromosome plus any DNA in organelles.
The size of a gene or genome is expressed in the number
of base pairs. i.e. kilobases (103 bases) or Megabases (106
bases)
Genes and the genetic code
Certain genes are widely distributed while others are
limited to particular species and the base pair
sequences may be highly conserved or widely
variable.
Basis for the molecular tests used to identify, detect
and characterise microorganisms.
A chromosome is a discrete unit of the genome
carrying many genes. Each chromosome consists of a
very long molecule of duplex DNA.
Biopolymers: Nucleic acids and Proteins

Transformation: the genetic alteration of a cell

resulting from the uptake and expression of foreign
genetic material (DNA).
DNA Replication
Bacteria multiply by cell division which results in two
daughter cells
Therefore the genome has to replicate for the
daughter cells to receive the same complement of
functional DNA.
Replication is a complex process that involves several
enzymes and cofactors.
Enzymes -
 Polymerases - Various enzymes that catalyze the
formation of polynucleotides of DNA or RNA using an
existing strand of DNA or RNA as a template.
Include:
DNA polymerase,
RNA polymerase, or
Reverse transcriptase.
Primase - Together with other proteins (forming a
Primeosome) makes short pieces of RNA (RNA
primers) that are recognised by DNA polymerase III to
initiate replication.
Topoisomerases: -Introduce negative supercoils and

relieve strains in the double helix at either end of the

bubble

Helicase: - Unwinds and unzips the DNA helix by

breaking the Hydrogen bonds between the base pairs,

thus allowing the two strands to separate.
DNA ligases: - Repairs the gaps between DNA

Okazaki fragments.

The SSB proteins (Single Strands Binding): - Stabilize

the single strands thus preventing them from zipping

back together and to form hairpin loops.
DNA Replication cont’d
In addition to being complementary, the two strands of DNA
are antiparallel: they are orientated in opposite directions.
DNA polymerase can only synthesize DNA in one direction by
adding nucleotides to the 3' end of a DNA strand, implying
that synthesis of the new strand is in the 5’ to 3’ direction.
Therefore, the two chains replicate in different direction.
Steps of DNA Replication
Origin of Replication
Are particular points within the DNA, which are targeted by proteins
that separate the two strands and initiate DNA synthesis.
Origins contain DNA sequences recognized by replication initiator
proteins.
Initiator proteins attach to a specific initiation site, recognized as a
particular sequence of DNA nucleotides.
DNA Helicase attaches at initiation site. Other proteins called
SSB(Single Strand Binding proteins), prevent reannealing of the
DNA. DNA Helicase is an enzyme that unravels the DNA double
helix and breaks the hydrogen bonds.
These initiator proteins recruit other proteins to separate the two
strands and initiate replication forks.
Origins tend to be "AT-rich" (2 hydrogen bonds).
 Replication
fork
Once strands are separated:
RNA primers are created on the template strands.
(DNA primase is an enzyme that generates an RNA
sequence (primer) that serves as a starting point for
synthesis of the new DNA chain)
DNA Polymerase extends these to create newly
synthesized DNA.

As DNA synthesis continues, the original DNA

strands continue to unwind on each side of the
bubble, forming Replication forks.
The Replication fork refers to the region on
the DNA that is the leading edge of DNA
replication as the DNA unzips in a
particular region.

At this stage the replication fork has a

complex of proteins including the enzyme
helicase and another enzyme, DNA
primase.
Replication fork
The two single strands of DNA are replicated in different
ways, therefore there is:

 A leading strand, the one synthesized in the 5'→3' direction in a

continuous manner.

 A lagging strand, synthesized also in the 5'→3' direction but in a

discontinuous manner. The lagging strand is synthesized in short

segments known as Okazaki fragments.
 An Okazaki fragment is a stretch of non parental DNA produced

along the lagging strand of parental DNA by the DNA polymerase

beginning at primer.
 DNA ligases repair the gaps between DNA Okazaki fragments.
Steps of DNA Replication
The combination of DNA polymerases with some of
the accessory proteins yields an activity identified as
DNA polymerase holoenzyme.

The entire activity located in the replication fork is

called a Replisome
Control of Replication
Eukaryotes
Within eukaryotes, DNA replication is
controlled within the context of the cell cycle.
Bacteria
Most bacteria do not go through a well-defined
cell cycle and instead continuously copy their
DNA; during rapid growth this can result in
multiple rounds of replication occurring
concurrently.
Termination of Replication
Eukaryotes

Eukaryotes initiate DNA replication at multiple points in

the chromosome, so replication forks meet and
terminate at many points in the chromosome; these
are not known to be regulated in any particular
manner.
Bacteria
Because bacteria have circular chromosomes,
termination of replication occurs when the two
replication forks meet each other on the
opposite end of the parental chromosome.
E coli regulate this process through the use of
termination sequences which, enable only one
direction of replication fork to pass through. As
a result, the replication forks are constrained to
always meet within the termination region of the
chromosome.
Notes edited on 28/09/2017