Download BACTERIAL GENETICS

BACTERIAL GENETICS
Learning objectives:
At the end of the lecture the student should be able to:
• Enlist the methods of DNA transfer in microorganisms.
• State the significance of DNA transfer in drug resistance.
• Enlist & describe the types of mutations in bacteria.
• Describe the process of lysogeny.
• Describe the role of mutations in drug resistance in infectious diseases
Understanding Genetics
 We resemble and differ because of Genetic configurations
 Parents - Son - Daughter, how they resemble each other.
They breed true from Generation to Generation
But vary in small proportions in progeny.
Bacteria too obey the laws of Genetics
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DNA
A Complex Structure Makes Life
Knowledge on DNA lead to advances in Molecular Biology
 Central dogma of Life – Deoxyribonucleic acid
 DNA carries the genetic information
 DNA is transcribed to RNA – Polypeptides
Cell Function depends upon specific polypeptides – Proteins – Enzymes
DNA is a store house of Protein synthesis
DNA acts a Template for synthesis of mRNA
Virus differs from other as they contains either DNA or RNA
The Bacterial Genome
 Chromosomes
 Contains a Double stranded molecules of
DNA arranged in circular form.
 Length 1,000 microns.
 Bacterial DNA contains about 4,000
kilobases
 Bacteria are haploid: they have a single
chromosome and therefore a single copy of
each gene.
 In haploid cells, any gene that has mutated
– and is therefore not expressed – results in
a cell that has lost that trait.
Plasmids
 Plasmids are circular DNA molecules
present in the cytoplasm of the Bacteria
 Capable of Autonomous replication
 Can transfer genes from one cell to other
 Act as vectors in Genetic engineering.
 Can also present in Yeasts
Plasmids
 Plasmid seem to be ubiquitous in bacteria, May encode
genetic information for properties
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Resistance to Antibiotics
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Bacteriocins production
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Enterotoxin productions
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Enhanced pathogencity
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Reduced Sensitivity to mutagens
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Degrade complex organic molecules
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Mutations
A mutation is a change in the base sequence of DNA that usually results in
insertion of a different amino acid into a protein and the appearance of an
altered phenotype. Mutations result from three types of molecular changes:
1) Base substitution
2) Frameshift mutation
3) Transposons or insertion sequences
Mutations can be caused by chemical, radiations or viruses
Mutation is a Random, Undirected, Heritable variation
Transfer of DNA within Bacterial cells
 Transposons
 Programmed rearrangement
 These gene rearrangements account for many of the antigenic changes
seen in Neisseria gonorrhoea and Borrelia recurrentis the cause of
relapsing fever.
 A programmed rearrangement consist of movement of a gene from a silent
storage site where the gene is not expressed to an active site where
transcription and translation occur. The insertion of a new gene into the
active site in a sequential repeated programmed manner is the source of the
consistent antigenic variation.
 These movements have the effect of allowing the organism to evade the
immune response.
TRANSPOSONS
 Mobile genetic elements
 The arrangement of genes in the
genome of bacteria and probably all
organisms is not entirely static. Certain
DNA segments called transposons have
the ability to move place to place on the
chromosome and into and out of
plasmids.
 Mobile genetic elements are probably
responsible for most of the genetic
variability in natural bacterial
populations, and for the spread of
antibiotic resistance genes.
Transformation of Genetic material(Gene Transfer)
 Occur by three methods:
 1) Conjugation
 2) Transduction
 3) Transformation
 From a medical point of view the most important
consequence of a DNA transfer is that antibiotic
resistance genes are spread from one bacterium to
another by these processes
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CONJUGATION
 Conjugation is the process by which the bacteria
transfer genes from one cell to another by cell-to-cell
contact.
 The process requires the presence on the donor cells
F+ of hair like projections called sex pili that make
contact with specific receptor sites on the surface of
recipient cells
High frequency recombinant Conjugation
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When it exists as a free plasmid, the F plasmid can
only transfer itself. This isn’t all that useful for
genetics.
However, sometimes the F plasmid can become
incorporated into the bacterial chromosome, by a
crossover between the F plasmid and the chromosome.
The resulting bacterial cell is called an “Hfr”, which
stands for “High frequency of recombination”.
Hfr bacteria conjugate just like F+ do, but they drag a
copy of the entire chromosome into the F- cell.
TRANSDUCTION
 Transduction is defined as transfer of portion of
DNA from one bacterium to another by
Bacteriophages.
 During the growth of virus within the cell a piece of
bacterial DNA is incorporated into the virus particle
and is carried into the recipient cell at the time of
infection.
 Within the recipient cell the phage DNA can
integrate into the cell DNA and the cell can acquire
a new trait, a process called lysogenic
conversion
 This process can change a non pathogenic organism into a pathogenic one.
Bacteriophages
 Are viruses that parasitize bacteria and consists of nucleic acid core and a
protein coat
 A phage particle may have at its core besides its own
nucleic acid and a segment of the Host DNA
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Specialized transduction
Only certain bacterial genes located in the bacterial chromosome in close
proximity to the prophage insertion site of the transducing phage are
transduced.
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Generalised transduction
A random fragment of bacterial DNA resulting from
phage induced cleavage of bacterial chromosome,
is accidentally in a phage protein coat in place of
the phage DNA. When this rare phage particle
infects a cell it injects the bacterial DNA fragment
into the cell, it becomes integrated into the
recipient chromosome by recombination.
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Transformation
Is the transfer of DNA itself from one cell to
another.
In nature, dying bacteria may release their DNA
which may be taken up by recipient cells.
In the laboratory an investigator may extract DNA
from one type of bacteria and inject it into
genetically different bacteria.
When purified DNA is injected into the nucleus of
a eukaryotic cell, the process is called transfection.
Transfection is frequently used in genetic
engineering procedures
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Griffith Phenomenon
Recombination
Once the DNA is transferred from the donor to the recipient cell it can integrate
into the host cell chromosome by recombination.
1) Homologous recombination: in which two pieces of DNA that has extensive
homologous regions pair up and exchange pieces by the process of breakage
and reunion
2) Non homologous recombination in which little if any homology is necessary
What is Gene Therapy?
 Gene therapy is the insertion of genes into an individual's cells and
tissues to treat a disease, such as a hereditary disease in which a
deleterious mutant allele is replaced with a functional one. Although the
technology is still in its infancy, it has been used with some success.
Vivo to Vitro
What Gene therapy can achieve?
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Replacing a mutated gene that causes disease
with a healthy copy of the gene.
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Inactivating, or “knocking out,” a mutated gene that
is functioning improperly.
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Introducing a new gene into the body to help fight
a disease.
Genetically Engineered Products
 Can prepare desired protein in pure form in
economic way
Somatostatin
 Commercial preparations of
Cloned Human Insulin
 Interferons
Hepatitis B vaccine
Restriction Endonucleases
 A restriction enzyme (or restriction endonuclease) is an enzyme that
cuts double-stranded DNA. The enzyme makes two incisions, one through
each of the sugar-phosphate backbones (i.e., each strand) of the double
helix without damaging the nitrogenous bases. They work with cutting up
foreign DNA
Blotting Techniques
 Drug fragments obtained by restriction enzyme digestion on separation Gel
can be transferred to Nitrocellulose or nylon membranes
 Several methods
1 Southern blotting
2 Northern Blotting
3 Western blotting
Western Blot to confirm
HIV Infections made land mark Diagnostic
tool
 Western Blot testing is confirmatory test for
diagnosis of HIV/AIDS
 Identifies antibodies directed against
different antigens in pathogen
Surface,
Core
RT antigen
PCR -Sequences
 PCR consists of several cycles of sequential
DNA replication where the products of first
cycle becomes the template for the Next
 It makes available abundant quantities of
specific DNA sequences starting
Genetic Mapping
 Genetic sequences for Bacteriophages
and virus
 Genetic mapping is done most of the
Human Genes
References
Lippincott’s Illustrated Reviews, Biochemistry
5th edition
Ch. 33 Biotechnology & Human Disease
Pgs. # 465-487