Bacterial%20Genetics

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Bacterial Genetics
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• The rapid development of molecular genetics, in
  conjunction with the ability of scientists to
  manipulate and transfer genes, has elicited
  considerable interest among physicians,
  scientists, and the public in general.
• The ability to manipulate the hereditary material
  of microorganisms and even our own species has
  far reaching consequences.

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Gene Mutation

• Any change in the nucleotide sequence of a gene.
• It may result from base substitutions,
  deletions, insertions and rearrangement.
• Transition Vs transversion of nucleotides
• Mismatch repair by proof reading enzymes (SOS
  system)
• Mutations in bacterial populations can pose a
  problem, bacteria have mechanisms by which genes
  can be transferred to other bacteria. Thus,
• a mutation arising in one cell can be passed
  on to other cells.

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Types of Mutation

• 1) Silent mutation
• 2) Missense mutation
• 3) Nonsense mutation
• 4) Frame-shift mutation
• 5) Null mutation
• 6) Suppressor mutation (intragenic or
• extragenic)

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Spontaneous Mutation

• 10-4 (hot spots) to 10-11. Generally 10-7 in
  progeny of a single cell.
• Mechanism electrochemical rearrangement, or
  moving around of electrons in the nucleotide
  molecule.
• Shift from a keto state to the enol form
• ( tautomerization) promotes abnormal hydrogen
  bonding between nucleotides.
• Thymine (keto) ? adenine
• Thymine (enol) ? guanine

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Induced Mutation

• Mutagens (Chemical and Physical agents)
• Chemical Agents
• 5-Bromouracil analogue of thymine binds to G.
• 2-Aminopurine analogue of adenine binds to C.
• Nitrous acid (HNO2) Nucleotide deamination
• Alkylating agents (eg ethyl-methyl sulfonate)
• Acridine dyes (Proflavin) insertion or deletion
  leading to frame-shift mutation.

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• Physical Agents
• Heat deamination of nucleotides.
• UV light Pyrimidine dimmers (thymine), hydrated
  pyrimidines.
• Gamma and x-rays breaks in the sugar phosphate
  backbone.

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Gene Transfer

• Transfer of DNA from a donor cell to a recipient
  cell with the resultant recombination.
• Gene transfer in bacteria is unidirectional
• Donor ? recipient
• It requires DNA homology
• A small part of DNA is transferred
• It is achieved by three mechanisms
  Transformation, Transduction and Conjugation.

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Transformation

• Uptake of naked fragmented single stranded DNA
• Most primitive mechanism of gene transfer
• It occurs in only a few genera
• Bacillus subtilis
• Streptococcus pneumoniae
• Hemophilus influenzae
• Neisseria gonorrhea
• E. coli (in the presence of a high
  concentration of calcium salts and high
  temperature)

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• The probability that transformation has
  contributed much to genetic variety is very
  small.
• Factors affecting transformation
• - DNA size and state
• - Competence of the recipient
• Steps
• - Uptake of DNA
• - Legitimate/Homologous/General
• recombination

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Transduction

• Transfer of DNA via a bacteriophage
• Lytic Vs lysogenic (temperate) phage life cycle.
• Restricted Vs generalized transduction
• Not affected by nucleases in the environment
• Phages that mediate generalized transduction
  generally breakdown host DNA into smaller pieces
  and package their DNA by a "head-full" mechanism.
• Examples diphtheria toxin, erythrogenic toxin
  and resistance to penicillin in staphylococci.

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Phage Composition and Structure
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Plasmid- Mediated Conjugation

• PIASMIDS
• - Circular ds DNA molecules ranging in size
  
• from 1/1000 to 1/10 of the chromosome.
• - They are usually autonomous (replicons).
• - As few as 1 to as many as 100/cell.
• - May become integrated (episomes).
• - Two classes conjugative and
  nonconjugative

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Conjugation

• Requires the sex (F) factor
• Donor cell (F) and recipient cell (F- )
• F factor is a ds DNA that is 1/40 of the
  chromosome in size, not under the control of the
  bacterial chromosome, ant it can replicate
  autonomously.
• It possesses several genes that code for the
  formation of sex pili.

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Conjugation

• During the conjugation process, a copy of the sex
  factor is made and one of its (single) strands is
  transferred to the recipient cell.
• The complementary strand of the donor single
  strand is synthesized in the recipient cell which
  is now converted to an F cell.

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Mechanism of F x F- Crosses

• Pair formation
• Conjugation bridge

• DNA transfer
• Origin of transfer
• Rolling circle replication

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Physiological States of F Factor

• Integrated (Hfr)
• Characteristics of Hfr x F- crosses
• F- rarely becomes Hfr while Hfr remains Hfr
• High transfer of certain donor chromosomal genes

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• Functions of Plasmids
• R Plasmids Antimicrobial resistance, may be
  multiple but clustered.
• Resistance to heavy metals (Ag, Co, Hg) in
  Staphylococci, coexist with R plasmids.
• Virulence determinants toxins
• Bacteriocinogenic plasmids

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• Mechanisms of Action of Resistance plasmids
• Modification of antibiotic
• Alteration of target site
• Alteration of uptake
• Replacement of a sensitive pathway

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Transposable genetic Elements

• Move from one location to another (jumping genes)
• 1) Random movement (not totallypreferable
  sites)
• 2) Not capable of self replication
• 3) Transposition mediated by site-specific
• recombination (mediated by transposases)
  no
• homology is required
• 4) Transposition can be accompanied by
  duplication

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• Types
• 1) Insertion sequences (Is) Small stretches
  of
• DNA that have at their ends repeated
• sequences
• 2) Transposons (Tn) Carry extra genes

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Genetic Engineering

• Cloning (recombinant DNA)
• - RE sticky ends (passenger DNA)
• - RE sticky ends (vector DNA) of plasmids
• - Annealing (ligase)
• - Insertion to a cell (transformation)