BACTERIAL
GENETICS
Dr.Rouchelle Tellis
Associate Prof, Microbiology
Structure of DNA
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Double stranded (double helix)
Chains of nucleotides
5’ to 3’ (strands are anti-parallel)
Complimentary base pairing
– A-T
– G-C
DNA Structure
Phosphate-P
Sugar-blue
Bases-ATGC
DNA Replication
• Bacteria have closed, circular DNA
• Genome: genetic material in an organism
• E. coli
– 4 million base pairs
– 1 mm long (over 1000 times larger that actual
bacterial cell)
– DNA takes up around 10% of cell volume
DNA Replication-occurs at the
replication fork
• 5’ to 3 ‘
• DNA helicase-unzips + parental DNA strand that is
used as a template
– Leading stand (5’ to 3’-continuous)
*DNA polymerase-joins growing DNA strand after
nucleotides are aligned (complimentary)
– Lagging strand (5’ to 3’-not continuous)
*RNA polymerase (makes short RNA primer)
*DNA polymerase (extends RNA primer then digests RNA primer and
replaces it with DNA)
*DNA ligase (seals Okazaki fragments-the newly formed DNA
fragments)
Replication Fork
Protein Synthesis
• DNA------- mRNA------ protein
transcription
translation
Central Dogma
of Molecular Genetics
Transcription
• One strand of DNA used as a template to make a
complimentary strand of mRNA
• Promoter/RNA polymerase/termination site/5’ to 3’
• Ways in which RNA & DNA differ:
– RNA is ss
– RNA sugar is ribose
– Base pairing-A-U
Transcription
Translation
• Three parts:
– Initiation-start codon (AUG)
– Elongation-ribosome moves along mRNA
– Termination: stop codon reached/polypeptide released
and new protein forms
• rRNA=subunits that form the 70 S ribosomes (protein
synthesis occurs here)
• tRNA=transfers amino acids to ribosomes for protein
synthesis)
PHENOTYPIC VARIATION
• Change in the colony characters , capsule or
flagella
• Phenotypic change = Physical change
GENOTYPIC VARIATION
• Change in the genes resulting in change in
protein synthesis
Mutations – changes in the DNA
• Point mutation – addition, deletion or substitution
of a few bases
• Mis-sense mutation – causes change in a single
amino acid
• Non-sense mutation – changes a normal codon
into a stop codon
• Silent mutation – alters a base but does not
change the amino acid
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Mutations
• Changes in base sequence of DNA/lethal and
inheritable
• Can be:
– Harmful
– Lethal
– Helpful
– Silent
Normal DNA/Missense Mutation
Nonsense Mutation/Frameshift
Mutation
• Ames test: is based on the ability of auxotrophic bacteria
to mutate by reverting to their original synthetic ability.
• Used for screening chemicals for mutagenic properties,
which indicate potential carcinogens
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• Spontaneous mutations occur in the absence of any known
mutagen, due to errors in base pairing during DNA replication.
• Induced mutations are produced by agents called mutagens
that increase the mutation rate.
– Chemical Mutagens: Alkylating agents, deaminating agents,
arcidine derivatives.
– Radiations:
• Repair of DNA Damage Many bacteria have enzymes that can
repair certain damages to DNA
• (1) Light repair uses an enzyme that is activated by visible light
and that breaks bonds between pyrimidines of a dimer.
• (2) Dark repair use several enzymes that do not require light for
activation; they excise defective DNA and replace it with DNA
complementary to the normal DNA strand.
Bacterial gene transfer
TYPES AND SIGNIFICANCE OF GENE TRANSFER:
• Movement of genetic information between organisms.
• Vertical gene transfer passes genes from parent to offspring.
• Lateral gene transfer passes genes to other cells in the same
generation.
• Mechanisms of bacterial gene transfer:
1) Transformation
2) Transduction
3) Conjugation
4) Transposition
• Gene transfer increases genetic diversity within a population,
increasing the likelihood that some members of population will
survive environmental changes.
Transformation
• Definition: Gene transfer resulting from the
uptake of DNA from a donor.
• Factors affecting transformation
– DNA size and state
• Sensitive to nucleases
– Competence of the recipient (Bacillus, Haemophilus,
Neisseria, Streptococcus)
• Competence factor
• Induced competence
• BACTERIAL TRANSFORMATION: discovered in 1928
by Griffith, who showed that a mixed culture of live
rough and heat-killed smooth pneumococci could
produce live smooth Pneumococci capable of killing
mice.
• Avery showed that capsular polysaccharide was
responsible for virulence and that DNA was the
substance responsible for transformation.
• Mechanism of Transformation: Transformation
involves the release of naked DNA fragments and
their uptake by other cells at a certain stage in their
growth cycle:
• (l) Uptake of DNA requires a protein called
competence factor to make recipient cells ready to
bind DNA
Transformation
• Steps
– Uptake of DNA
• Gram +
• Gram -
– Recombination
• Legitimate, homologous
or general
• recA, recB and recC
genes
• Significance
Phase variation in Neiseseria
– Recombinant DNA technology
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The Significance of Transformation
• It contributes to genetic diversity
• It can be used to introduce DNA into an
organism, observe its effects, and study gene
locations
• It can be used to create recombinant DNA.
Transduction
• Definition: Gene transfer from a donor to a recipient by
way of a Bacteriophages
• Phages can be virulent or temperate.
1. Virulent phages: destroy a host cell's DNA and cause
lysis of the host cell in the lytic cycle
2. Temperate phages: can replicate themselves as a
prophage- part of a bacterial chromosome, or eventually
produce new phage particles and lyse the host cell.
• Persistence of the phage in the cell without the
destruction of the host cell is called Lysogeny.
Bacteriophage composition and
Structure
• Composition
– Nucleic acid
Head/Capsid
• Genome size
• Modified bases
– Protein
• Protection
• Infection
• Structure (T4)
– Size
– Head or capsid
– Tail
Contractile
Sheath
Tail
Tail Fibers
Base Plate
Generalized Transduction
• Infection of Donor
• Phage replication and degradation of host DNA
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Assembly of phages particles
Release of phage
Infection of recipient
Legitimate recombination
Transduction
Transduction can specialized or generalized:
1. In specialized transduction, the phage is
incorporated into the chromosome and can transfer
only genes adjacent to the phage
2. In generalized transduction, the phage exists as a
plasmid and can transfer any DNA fragment attached
to it.
The Significance of Transduction
• Transduction is significant because it transfers
genetic material and demonstrates a close
evolutionary relationship between prophage
and host cell DNA.
• Also, its persistence in a cell suggests a
mechanism for the viral origins of cancer, and it
provide a possible mechanism for studying
gene linkage
CONJUGATION
• Large quantities of DNA are transferred from one
organism to another between donor and recipient
cells during contact
• Conjugation was discovered by Lederberg in 1946
when he observed that mixing strains of E. coli
with different metabolic deficiencies allowed the
cells to overcome deficiencies
• Plasmids are extra-chromosomal DNA molecules.
Mechanisms of Conjugation
• 3 mechanisms of conjugation:
1. In the transfer of F plasmids, a piece of extrachromosomal DNA plasmid is transferred.
2. In high-frequency recombination parts of F
plasmids that have been incorporated into the
chromosome (the initiating segment) are
transferred along with adjacent bacterial genes.
3. An F plasmid incorporated into the chromosome
and subsequently separated becomes an F'
plasmid and transfers chromosomal genes
attached to it.
The Significance of Conjugation
• It increases genetic diversity, & may represent
an evolutionary stage between asexual and
sexual reproduction and provides a means of
mapping genes in bacterial chromosomes.
Transposable Genetic Elements
• Definition: Segments of DNA that are able to
move from one location to another
• Properties
– “Random” movement, Not capable of self replication
– Transposition mediated by site-specific recombination
• Transposase
– Transposition may be accompanied by duplication
• Segment of DNA that is capable of independently
replicating itself and inserting the copy into a new
position within the same or another chromosome or
plasmid. Referred to as transposons or jumping genes.
• In bacteria, the transposable elements can be grouped
into two classes, insertion sequences and transposons.
• Transposons encode resistance to many antibiotics &
toxic metals, chemicals.
• Some transposons have the ability to direct the synthesis
of proteins that metabolize carbohydrates, petroleum,
and pesticides.
Types of Transposable Genetic Elements
• Transposons (Tn)
– Definition: Elements that carry other genes
except those involved in transposition
– Nomenclature - Tn10
– Structure
• Composite Tns
– Importance
• Antibiotic resistance
IS
Resistance Gene(s)
IS
IS
Resistance Gene(s)
IS
Plasmids
• Definition: Plasmids are circular, self-replicating.
Double-stranded extra-chromosomal DNA that
carries information that is usually not essential
for cell growth
• Episome - a plasmid that can integrate into the
chromosome
Classification of Plasmids
• Transfer properties
– Conjugative
– Nonconjugative
• Phenotypic effects
– Fertility
– Bacteriocinogenic plasmid
– Resistance plasmid (R factors)
Structure of R Factors
• RTF
RTF
– Conjugative
plasmid
– Transfer genes
• R determinant
– Resistance genes
– Transposons
R determinant
Genetic basis of drug resistance
• Mutational drug resistance – Chromosomal
• Transferable drug resistance – Plasmid mediated
Genetic engineering
• Genetic engineering is the manipulation of
genetic material to alter the characteristics of an
organism.
• Genetic fusion: allows transposition from one
location on a chromosome to another, sometimes
deleting a portion, thereby causing the joining of
genes from two different operons.
• Protoplast Fusion: combines protoplasts (
organisms without cell walls) and allows mixing
of genetic information.
• Gene Amplification: involves the addition of
plasmids to microorganisms to increase yield of
useful substances .
• Recombinant DNA Technology: is DNA produced
when genes from one kind of organism are
introduced into the genome of a different kind of
organism. The resulting organism is transgenic, or
recombinant organism.
• Recombinant DNA has proven especially useful in
medicine, industry and agriculture.
• Hybridisms: are genetic recombination's involving
cells of higher organisms.
• DNA PROBES: Labeled (Radioactive, Biotin etc.)
copies of single-stranded DNA fragments
containing unique nucleotide sequences, which
are used to detect homologous DNA by
hybridization. Highly specific detects even lninute
amounts of target DNA.
• PCR - POLYMERASE CHAIN REACTION: Rapid automated
method for amplification of specific DNA sequences.
• Steps in PCR:
1. De-naturation of DNA to obtain a single strand
2. Annealing of sequence specific oligonucleotide primers.
3. Extension of primers by DNA Polymerase enzyme to form
a new double stranded DNA.
4. This cycle is repeated for 20 - 50 times in a thermo-cycler
to obtain thousands of copies of DNA that can be
identified by DNA probes.
Applications of PCR : Diagnosis of Infectious diseases, genetic
disorders, cancer, forensic investigations and
biotechnology.
RECOMBINANT DNA TECHNOLOGY:
• Isolation of genes coding for any desired protein from
cells or microorganisms –
• Synthesis- small sequences; Enzymes - restriction
endonucleases.
• mRNA - DNA (SS) synthesis by reverse transcriptase &
• polymerization by DNA polymerase to yield DS DNA.
• Vectors - Plasmids, Bacteriophages; E.coli K12 &
Yeast's.