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Fig. 5-2
Plating bacteria and growing colonies
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Commonly used genetic markers
• Prototrophic markers: wild-type bacteria are prototrophs(grow on minimal medium)
• Auxotrophic markers: mutants that require additional nutrient(fail to grow on minimal medium)
• Antibiotic-sensitivity: wild-type bacteria are susceptible(fail to grow on antibiotic-containing medium)
• Antibiotic-resistance: mutants that grow in presence of antibiotic(grow on antibiotic-containing medium)
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Fig. 5-1
Chapter 5: Genetics of bacteria and their viruses
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Gene transfer mechanisms in bacteria (especially E. coli)
Conjugation: orderly, deliberate transfer of DNA from one cell to another; programmed by specialized genes and organelles.
Transformation: uptake of environmental DNA into a cell
Transduction: transfer of DNA from one cell to another mediated by a virus
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Properties of gene transfer in bacteria
• All are unidirectional (donor – recipient)
• Recombination requires two steps:
1. Transfer of DNA into the recipient cell, forming a
merozygote (various gene transfer mechanisms)
2. Crossing over that replaces a portion of the recipient genome (endogenote) with the homologous portion of the donor genome (exogenote)
• Transfer is always partial
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Conjugating E. coli
pili
Fig. 5-6
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Fig. 5-7
Conjugation in E. coli is based on the F (fertility) plasmid
Replication-coupled transfer of F
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Fig. 5-8
F can integrate into the bacterial chromosome
Hfr: high frequency recombination derivative
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Fig. 5-10
Transfer of integrated F includes donor chromosome
Crossing over of exo/endogenote results in recombinant genome
(replacement of a segment of recipient genome with the homologous segment of the donor genome)
Unidirectionaltransfer……
Recombination…..
Partial transfer…..
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DNA transfer during conjugation is time-dependentTransfer of an entire E. coli donor genome requires
about 1 hour (F sequence is last to transfer)
Therefore, can map the chromosome as a time function:
• Mix donor Hfr and recipient F- cells
• Interrupt transfer of DNA at various times (violent mixing in a Waring blendor works!)
• Plate out cells to determine which genes were transferred within each timeframe
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Fig. 5-11
Hfr azir tonr lac+ gal+ strs X F- azis tons lac- gal- strr
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Fig. 5-11
Hfr azir tonr lac+ gal+ strs X F- azis tons lac- gal- strr
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Genetic map generated by interrupted mating experiment
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Conjugation map depends upon:
• site of F factor insertion within Hfr chromosome (original F insertion can occur at any one of many sites within chromosome)
• direction/orientation of the F factor within that Hfr strain (clockwise or counter-clockwise)
Mapping using different Hfr strains can provide a map of the entire bacterial chromosome
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Fig. 5-13
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Fig. 5-16
Mapping of small regions by recombination
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Fig. 5-17
F integration by recombinationof IS element
Excision usinganother IS elementresults in F bearing
chromosome fragment (F’)
Transfer create partial diploid
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…at least 10 species ancestors.
Fig. 5-18
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Transformation: DNA in the environment of a cell is taken into the recipient cell forming a merozygote; then recombination occurs
• occurs naturally in some bacteria (e.g., Pneumococcus)
• occurs rarely in others, but can be promoted by treating cells to destabilize their membranes (e.g., in recombinant DNA work)
• can map genes by co-transformation (frequency with which two genes are simultaneously transferred
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Fig. 5-19
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Transduction: Transfer of DNA from one cell to another mediated by a virus; followed by recombination to integrate the DNA into the recipient cell
• can map genes by the frequency of co-transduction (frequency of simultaneous transfer of two genes)
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Fig. 5-22
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Fig. 5-23
Bacteriophage lytic cycle
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Plaques (infection bursts) of bacteriophage on a lawn of E. coli
Fig. 5-24
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Generalized transduction
Random DNA fragments are transferredFig. 5-27
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Linkage mapping of a segment of the E. coli chromosome by co-transduction experiments with phage P1
Fig. 5-28
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Fig. 5-30
Lysogenic infection: integration of a viral genome into one of many sites within the host cell chromosome where it quiescently resides
Upon specific cues, the process may be reversed, resulting in lytic infection
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Fig. 5-31
Specialized transduction(genes nearest the insertion site are most efficiently transferred)
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Fig. 5-
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Fig. 5-
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Fig. 5-
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Fig. 5-