BacterialGenetics powerpt

8/8/2019 BacterialGenetics powerpt

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Bacterial GeneticsMICROBIOLOGY 101/102 INTERNET TEXT: CHAPTER IX: MICROBIAL EXCHANGE OF GENETIC MATERIAL

Immunology and Microbiology On-line - Chapter 18: Exchange of Genetic Information

Chromosome (nucleoid) and plasmids are the source of genotypes


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Bacterial Genetics

Chromosomes structural components and metabolism

Plasmids - antibiotic resistance, most virulence factors, conjugation

Haploid (almost) any change in the genome will be expressed

spontaneous and induced mutations

viral transduction

transformation

deliberate genetic engineering


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Monitoring changes (mutations) in bacterial genome

Replica plating are there mutants in a bacterial culture?

e.g., has the mutant the lost ability to make a critical metabolite

will not grow unless provided with missing metabolite Grow all cells on medium with that metabolite

Make an impression of all colonies that grow (will include the mutants)

Press this replicate onto separate media with and without the metabolite

Compare colonies on both plates - try to find the absence of a colony on

medium without metabolite that correlates with presence of colony on medium

with metabolite

Represents the mutated colony (can pick it off of medium with metabolite)


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Ames TestUsing a bacterial mutation to detect human mutagens/carcinogens

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AmesTest.html

Bruce James UC Berkeley

Uses a bacterium that is auxotrophic for a particular required amino acid

can not make that amino acid itself

Salmonella typhimurium auxotrophic for histidine (designated as his-)

ability to produce histidine was probably lost due to a mutation

will not grow on medium lacking histidine

Inoculate the entire surface of an agar plate medium lacking histidine

Place paper disk soaked with test compound on center of agar plate

IfS. typhinow does grow surrounding the disk (and not elsewhere)

interpreted as a back mutation (mutated to regain ability to produce its own histidine)

compound tested caused that mutation

interpreted as being a human mutagen

possibly a human carcinogen


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Other ways a bacterial genome can change

Transformation update of exogenous DNAhttp://www.slic2.wsu.edu:82/hurlbert/micro101/pages/Chap9.html#Transformation

Can happen naturally

Can be done in controlled lab environment

DNA is released by cell

Dying cell or deliberately lysed cell

Transforming DNA is mixed with cells to be transformed

DNA is bound to cell surface (competent cell)

Some cells need to be made competent

Cold CaCl2

Nucleases cleave dsDNA

Small pieces of ssDNA are taken into the cell

Can often replace portion of one strand of cells DNA Serves as template for making the based-paired strand

Replaces the other original strand

Homologous recombination


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Conjugation & high frequency recombination (Hfr) already discussed


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Viral transduction

Virus (bacteriophage) initially infects a bacterial cell

During assembly of a few viruses - piece of bacterial DNA replaces some or all viral

DNA

Normal viruses (with only viral DNA) lyse infected cell

The viruses with bacterial DNA are defective

Called a transducing particle

Can inject bacterial DNA into another cell

Can not carry out remaining steps of viral replication cycle

Virus injects bacterial DNA into the cell

Homologous recombination of bacterial DNA

Transduction of only a very few bacteria takes place

Defective viruses are not replicated in the original cell

Very few defective viruses are produced


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Viral transduction

Virus (bacteriophage) initially infects a bacterial cell

During assembly of a few viruses - piece of bacterial DNA replaces some or all viral

DNA

Normal viruses (with only viral DNA) lyse infected cell

The viruses with bacterial DNA are defective

Called a transducing particle

Can inject bacterial DNA into another cell

Can not carry out remaining steps of viral replication cycle

Virus injects bacterial DNA into the cell

Homologous recombination of bacterial DNA

Transduction of only a very few bacteria takes place

Defective viruses are not replicated in the original cell

Very few defective viruses are produced


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Specialized viral transduction lab procedure only

Viral DNA is incorporated into bacterial chromosome Incorporation is most often in a very precise location

Lambda phage viral DNA is incorporated next to lac operon gene complex

Viral DNA detaches from bacterial chromosome

Small piece of bacterial DNA remains attached to viral DNA

e.g., lac operon gene complex + a few nearby nucleotides

Virus DNA (+ piece of bacterial DNA) detaches from bacterial chromosome


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Specialized viral transduction lab procedure only

Virus DNA (+ piece of bacterial DNA) detaches from bacterial chromosome These viruses do replicate many times

Viruses then inject viral+bacterial DNA into another cell

Piece of bacterial DNA incorporated into infected cells genome

(homologous recombination)

Much higher frequency of transduction as many replicates of the lambda phage were produced in the

originally infected cell

Can first place a desired nucleotide sequence next to lac operon before carrying out transduction

Desired sequence is then incorporated into the transduced bacterial cell

Can monitor successful transduction by detecting lac operon (beta-galactosidase production)


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Phage conversion

Genetic information is actually a part of the virus own original DNA

Bacteriophage infects bacterial cell Viral DNA becomes permanently incorporated into bacterial genome

Viral DNA is transcribed and translated

New phenotype is expressed

Corynebacterium diphtheriae exotoxin (already discussed)

Inhibits human cell protein synthesis

Modifies EF-2 (elongation factor 2)

Prevents addition of nucleotides by tRNA

Human cell dies

DPT vaccine uses safe version of diphtheria exotoxin (toxoid)

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