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Objective
By the end of this presentation we expect you to
know:
About bacterial chromosome and plasmids
About translation and transcription
Replication
Genetic variation in bacteria
Uses of bacteria in genetic engineering
Introduction
Genetics is the study of genes including the
structure of genetic materials, what information is
stored in the genes, how the genes are
expressed and how the genetic information is
transferred.
Genetics is the study of heredity and variation.
Two essential function of genetic material is
replication and expression.
The genome of an organism is defined as the
totality of its genetic material.
Genotype vs. phenotype
Introduction
Bacteria are one of the three domains in
taxonomy, they are also prokaryotes;
Bacteria store DNA in two places the
chromosome and the small circular plasmid.
Bacteria’s DNA is about thousand times the size
of the cell.
Has both DNA and RNA
Structure of bacterial genetic materialChromosome
Most bacteria's has a single haploid circular chromosome. But some may have more than one:
For example, Vibrio cholerae and Brucella melitensis,leptospira, have two or more dissimilar chromosomes. There are also exceptions to this rule of circularity because some prokaryotes (eg, Borreliaburgdorferi and Streptomyces coelicolor) have been shown to have a linear chromosome.
Plasmids
are autonomously replicating DNA molecules of varying size located in the cytoplasm. Could contain transposons,
Virulence plasmid
plasmid
Resistance plasmid
Significance of plasmids:
1. Codes for resistance to several antibiotics. Gram-negative
bacteria carry plasmids that give resistance to antibiotics such
as neomycin, kanamycin, streptomycin, chloramphenicol,
tetracycline, penicillins and sulfonamides.
2. Codes for the production of bacteriocines.
3. Codes for the production of toxins
4. Plasmids carry virulence determinant genes. Eg, the plasmid
Col V of Escherichia coli contains genes for iron sequestering
compounds.
5. Codes resistance to uv light (DNA repair enzymes are coded
in the plasmid).
6. Codes for colonization factors that is necessary for their
attachment.
7. Contains genes coding for enzymes that allow bacteria unique
or unusual materials for carbon or energy sources. Some
strains are used for clearing oil spillage.
Transcription
Transcription enables the DNA to direct the
synthesis of RNA,
Types of RNA:
mRNA rRNA
tRNA snRNA
siRNA miRNA
The process will be explained in the next slide
DNA damageCauses
Endogenous –e.g. Reactive oxygen (metabolic byproducts)
Exogenous - external agents (radiations)
Types of DNA damage:• Deamination: (e.g. C->U )• Depurination : ( purine base A or G lost)• T-T and T-C dimers: (bases become cross-linked)
• Alkaylation : ( an alkyle group e.g. CH3 added to base
• Oxidative damage: (guanine oxidizes to 8 –oxo-guanine• cause SS and DS breaks
• Replication error: wrong nuceotide or modified neuclotideinserted
• Double strand breaks (DSB): induced by ionizing radiations, transposons,, endonucleases, mechanical stress, etc
Repair against damage
Damage caused by UV
Mismatch
Proofreading
Nucleotide Excision repair
SOS response ( there are over set of 17 genes
that are involved tin this)
Base excision repair
mutation
Mutation is random it can happen to any gene
and to any bacteria.
But in bacteria it may seem like the environment
has a role in changing the genotype but it
doesn’t.
Lederberg’s concept
There are different types of mutation
Mutation on a coding stand
Point mutation Nonsense
Missense
Frame shift
Lethal mutation
Conditional
Resistance mutation
Auxotrophic mutation
Reversion
suppression
Genetic exchange
Bacteria can not adopt simply because of
mutation, because mutation is to rare, they need
other system.
Sharing of genetic material
Donor Vs. recipient, exogenoate Vs.
endogenoate
This along with mutation is the cause of genetic
variation amongst bacteria's.
A type of horizontal transfer
Transformation
Transformation involves the uptake of free or
naked DNA released by donor by a recipient. By
using surface proteins that recognize and snatch
naked DNA.
The competence is determined by genes that
become active under certain conditions. But can
also be artificial.
N.B. not all bacteria’s are capable of carrying out
only competent bacteria’s like ( Bacillus,
Haemophilus, Neisseria, Pneumococcus) can.
The classic example for transformation is the
griffith experiment.
steps The steps involved in transformation are:
1. A donor bacterium dies and is degraded.
2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to DNA binding proteins
on the cell wall of a competent, living recipient bacterium.
3. Nuclease enzymes then cut the bound DNA into fragments.
4. One strand is destroyed and the other penetrates the recipient bacterium.
3. The Rec A protein promotes genetic exchange (recombination) between a fragment of the donor's DNA and the
recipient's DNA.
Conjugation Bacterial conjugation is the transfer of DNA from
a living donor bacterium to a recipient bacterium.
It requires intimate cell contact
The male donates its plasmid to the female
Conjugate(f) plasmid directs it’s own process, of conjugation
Because it has tra genes.
Transfer replication
Plasmid mobilization
There are various types
Of conjugation
Conjugation Bacterial conjugation is the transfer of DNA from
a living donor bacterium to a recipient bacterium.
It requires intimate cell contact
The male donates its plasmid to the female
Conjugate(f) plasmid directs it’s own process, of conjugation
Because it has tra genes.
Transfer replication
Plasmid mobilization
There are various types
Of conjugation
F+ conjugation
In which plasmid called F plasmid induces f pilli,
formation, and enzymes involved in the process
and the donor bateria gives F plasmid to the
recipient.
the bacterial plasmid to be transferred will be
separated in to two single strands, one to be
exchanged the other will stay with in the cell and
both of them will replicate to be double stranded
DNA,
Exchange occurs across the conjugation tube
Then both bacteria’s will be F+
Hfr conjugation Plasmids may integrate into the bacterial
chromosome by a recombination event depending upon the extent of DNA homology between the two. The plasmid is called episome.
And forms HFR cell because they are able to pass both chromosomal and plasmid genes.
Process is their will be a nick around the area of origin of transfer, and the chromosome will be replicated, and pass through the conjugation tube to the F- cell but most of the time the conjugation bridge collapse.
The integration of the chromosome with the plasmid isn’t stable so the chromosome and the plasmid separate, but through this process the plasmid might take some genes of the chromosome along with it.
Transduction
Transduction is virus-mediated transfer of genetic
information from donor to recipient cell.
This is mediated by bacteriophages.
Bacteriophage’s can be of two types
Lytic
Lysogenic
Depending on the type of bacteriophage used
transduction can be classified as general and
specialized transduction.
General transduction
Lytic bacteriophage infects
the bacteria, the process is
descibed in the image to the
right.
Pseudovirus:- virus that
contains bacterial DNA
Happens 1 in 1000 virus
formed
Specialized transduction
Caused by lysogenic bacteria
The viral DNA attaches to a particular site of the
chromosome, and it may drag along with it the
genes next to it but this is of low probability but as
this integration replicates it becomes of high
probability and then when it infects other bacteria
it would have that gene.
Is valuable for sequencing genes, knowing about
the function and regulation of that gene, cloning
and antibiotic resistance.
transposable DNA elements
Transposable genetic elements are segments of
DNA that have the capacity to move from one
location to another
They synthesize transposase protein that aids
transposition,
The three major kinds of transposable elements
are insertion sequence elements, transposons
and transposable bacteriophage;
Insertion sequences
Transposon are like insertion sequences but they
also cary additional genes between the
transposition gene/ IS segment.
They are important because they might carry
genes for antibiotic resistance
While IS can deactivate a gene by inserting with
in it.
Direct transposition vs. replicative transposition
One such conjugative transposon is Tn916,
found originally in a strain of E. faecalis.
Mechanism of drug resistance Decreased uptake (or increased efflux) of antibiotic: For
example, gram-negative organisms can limit the penetration of certain agents, including β-lactam antibiotics, tetracyclines, and chloramphenicol, as a result of alteration in the number and structure of porins (proteins that form chanels) in the outer membrane.
Alteration of the target site for antibiotic: For example, Staphylococcus pneumoniae resistance to β-lactam antibiotics involves alterations in one or more of the major bacterial penicillin-binding proteins (see p. 75), which results in decreased binding of the antibiotic to its target.
Acquisition of the ability to destroy or modify the antibiotic: Examples of antibiotic inactivating enzymes include: 1) β-lactamases that hydrolytically inactivate the β-lactam ring of penicillins, cephalosporins, and related drugs; 2) acetyltransferases that transfer an acetyl group to the antibiotic, inactivating chloramphenicol or aminoglycosides; 3) esterases that hydrolyze the lactonering of macrolides.
Genetic engineering
Development of vectors or vehicles allowing the
cloning of any DNA sequences
Eucaryotic genes may be expressed in
procaryotic systems
Many genetic diseases are caused by lack of
protein
Production in bacteria of recombinant vaccines
Replacement therapy - bacterial interference
Molecular technologies in diagnosis
Use of nucleic acid (DNA) probes to diagnose
and study diseases
DNA of interest is inserted to bacterium and
amplified to high copy numbers and labeled - in
situ hybridization
PCR - generation of millions copies of specific
pieces of nucleic acid of suspected
microorganism
References
Prescott’s microbiology
Sherri’s microbiology
Lippincott's illustrated review of microbiology