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					MICROBIAL GENETICS

Mutations

Change in the base sequence of the DNA that usually results in insertion of a different amino acid in to a protein and the appearance of altered phenotype Due to three types of molecular changes

Base substitution
Frameshift mutation Transposons

Base substitution

Occurs when one base is inserted in place of another Take place at the time of DNA replication Missense mutation : Base substitution results in a codon that simply causes a different amino acid to be inserted Nonsense mutation: base pair substitution generates a termination codon that stops protein synthesis prematurely

Frameshift mutation
Occurs when one or more base pairs are added or deleted, which shift the reading frame on the ribosome and results in incorporation of wrong amino acids Production of inactive protein

Transposons Insertion sequences are integrated in to the DNA Can cause profound change in the genes

Can also be caused by chemicals, radiation or viruses

Action of chemicals

Alter the existing base so that it forms a hydrogen bond preferentially with the wrong base
Nitrous acid and alkylating agents : AC pairing

Base pair analogues : 5 – bromouracil can be inserted in place of thymine
Benzpyrene present in tobacco smoke intercalate between the adjacent bases there by distorting and offsetting the DNA sequence

X- rays By breaking the covalent bonds that hold the ribose phosphate chain together By producing free radicals that can attack the bases By altering the electrons in the bases and thus changing their hydrogen bonding Ultraviolet radiation Causes the cross – linking of the adjacent pyrimidine bases to form dimers results in inability of the DNA to replicate properly Bacterial viruses can cause frame shift mutations or deletions

Conditional lethal mutations – influenza vaccines

How do you say there are any mutant colonies in the culture?

Positive selection Growing the culture on a medium that will allow the growth of mutant colonies e.g. Penicillin resistant mutants will grow well in media containing penicillin

Negative selection Used to identify mutants that have lost the ability to perform certain functions that their parents had e.g. microbes that lost their function to synthesis histidine can grow well in histidine rich medium

TRANSFER OF DNA WITHIN BACTERIAL CELLS

Transposons – jumping genes
Drug resistance gene Transposase gene Repressor gene IR

IR

Programmed rearrangements
Transfer of DNA within the bacteria Accounts for any of the antigenic variation

Seen in N. gonorrhoeae, Borrelia recurrentis and Trypanosomes
Expression locus 1 mRNA 2 3 4 N

Protein 1
Expression locus 2 mRNA 2

Programmed rearrangement moves gene 2 in to the expression locus 3 4 N

Protein 2

Transfer of DNA between the bacterial cells

Conjugation
Transduction Transformation

Conjugation

DNA is transferred from one bacteria cell to another, via "sex pilli". The ability to transfer DNA by conjugation is dependent on the presence of a cytoplasmic entity termed the fertility factor, or F. Cells carrying F are termed F+; cells without F are F-. It is an example of a class of elements termed plasmids, which are self-replicating extrachromosomal DNA molecules. F contains approximately 100 genes; these give F several important properties

Observed by Lederberg and Tatum in 1946 Definition: Gene transfer from a donor to a recipient by direct physical contact between cells

Donor contains F factor (Fertility factor)
Recipient -Lacks an F factor

• Some F+ cells have their F plasmid integrated in to the bacterial DNA – Hfr cells

• During the process single strand of F factor enters first followed by bacterial chromosome and then by remainder of F.
• Some times only a portion of donor chromosome enters the recipient – breaking of attachment • Newly acquired DNA recombine in to recipient’s DNA and becomes the genetic material

In the mating of Hfr and F -, the F - cell almost never becomes F + or Hfr

Part of the F factor usually get transferred to other cells

To become F + or Hfr the recipient cell must receive the entire F factor

The frequency of F - cells to become Hfr is very rare – 1/10,000

F ’ cells

Produced when an F factor does excise from the bacterial chromosome Contain a small amount of bacterial chromosome Mating of F ’ and F - cells give rise to 2 F ’ cells

Resistance transfer factor

Mediated by plasmids called as R factor

Contains two components
Resistance transfer factor (RTF) – responsible for conjugational transfer

Resistance determinant – for several drugs
The RTF sometimes may dissociate from the r determinant In that case, though the cell remains drug resistant, the resistance is not transferable Seen in various pathogenic bacteria

Resistance Plasmids

Significance • Rapid spread of drug resistance • Resistance to multiple drugs • Inter species and inter genus spread of resistance • Transfer of virulence genes

Transduction
• Definition: Gene transfer from a donor to a recipient by way of a bacteriophage • Produced during the growth of phage inside the bacterial cell • Integrate in to new bacteria during infection – lysogenic conversion • Lytic or virulent phage: Phage that can only multiply within bacteria and kill the cell by lysis. • Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell.

Some times a small pieces of bacterial DNA, will be packaged into the bacteriophage genome- generalized transduction At the same time, some phage genes are left behind in the bacterial chromosome – specialized transduction Transformation Transfer of DNA it self from one cell to another

Dying cells release their DNA, taken up by others
DNA extracted from encapsulated smooth pneumococci could transform nonencapsulated rough pneumococcci in to encapsulated smooth organisms

Recombination • Refers to the exchange between two DNA molecules, results in new combinations of genes on the chromosome.

• Most familiar recombination event is known as crossing over.

• A chromosome segment entering the cell and aligning with its homologous segment on the bacterial chromosome.

• The two break at corresponding point, switch fragments and rejoin - two recombinant chromosomes

Genotypic and phenotypic variation
Phenotypic expression – depends on the environmental factors No synthesis of flagella by Salmonella when grown in phenol agar

Operon concept

Mutational drug resistance Involves resistance to one drug at a time

Transferable drug resistance Simultaneous resistance to multiple drugs

Degree of resistance usually low Degree of resistance is high May be overcome by high drug dosage High drug dosage ineffective

Can be prevented by treatment with combination drugs
Resistance does not spread

Combination of drugs can not prevent
Resistance spreads

virulence may be low

virulence not decreased


				
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posted:9/29/2008
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