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Plating bacteria
and
growing colonies
Fig. 5-2
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)
Chapter 5: Genetics of bacteria and their viruses
Fig. 5-1
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
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
Conjugating
E. coli
pili
Fig. 5-6
Conjugation in E. coli is based on the F (fertility) plasmid
Replication-coupled transfer of F
Fig. 5-7
F can integrate into the bacterial chromosome
Hfr: high frequency recombination derivative
Fig. 5-8
Transfer of integrated F includes donor chromosome
Unidirectional
transfer……
Recombination…..
Partial transfer…..
Crossing over of exo/endogenote results in recombinant genome
(replacement of a segment of recipient genome with the homologous segment of the donor genome)
Fig. 5-10
DNA transfer during conjugation is time-dependent
Transfer 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
Hfr azir tonr lac+ gal+ strs X F- azis tons lac- gal- strr
Fig. 5-11
Hfr azir tonr lac+ gal+ strs X F- azis tons lac- gal- strr
Fig. 5-11
Genetic map generated by interrupted mating experiment
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
Fig. 5-13
Mapping of small regions by recombination
Fig. 5-16
F integration by
recombination
of IS element
Excision using
another IS element
results in F bearing
chromosome fragment (F’)
Transfer create partial diploid
Fig. 5-17
…at least
10 species
ancestors.
Fig. 5-18
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
Fig. 5-19
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)
Fig. 5-22
Bacteriophage lytic cycle
Fig. 5-23
Plaques (infection bursts) of bacteriophage  on a lawn of E. coli
Fig. 5-24
Generalized transduction
Fig. 5-27
Random DNA fragments are transferred
Linkage mapping of a segment of the E. coli chromosome
by co-transduction experiments with phage P1
Fig. 5-28
Lysogenic infection: integration of a viral genome into
one of many sites within the host cell chromosome where
it quiescently resides
Fig. 5-30
Upon specific cues, the process may be reversed,
resulting in lytic infection
Specialized transduction
(genes nearest the insertion
site are most efficiently
transferred)
Fig. 5-31
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