Plating bacteria and growing colonies Fig 5 2

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 2. 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

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 P 1 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

Fig. 5 -