Biology 212 General Genetics Spring 2007Lecture 16: Bacterial Genetics II: ConjugationReading: Chap. 7 pp. 253-259Lecture Outline:1. Bacterial mating: F+ x F-2. Rolling circle replication3. Hfr strains4. Conjugation mappingLecture1. Bacterial mating- ability of bacteria to mate depends on presence of F factor (fertility factor- F+ cell contains F plasmid=donor- F- cell lacks F plasmid=recipientF factor- Low copy number plasmid- Has its own replication origin- 100 kb circular plasmid- encodes gene for pilus protein--forms conjugation bridge- other genes also encodedProcess of conjugation:1) contact between F+ cell and F- cell2) pilus from F+ cell connect the two cells3) copy of F plasmid is produced in F+ cell4) copy is transferred across pilus into F- cell5) F- cell is converted to F+ cell by transfer of the plasmid2. Rolling circle replication- mechanism of replication of the F plasmid and some bacterial virusessee Fig. 7.121. DNA backbone of one strand is broken2. DNA strand separates from the complementary strand3. New DNA is added to the 3' end of the broken strand4. Newly synthesized strand is transferred into recipient cell5. Second strand is copied from the first; F- cell becomes F+3. Hfr strainsHfr = High frequency of recombinationDepends on integration of F plasmid into chromosome1F factor usually integrates via IS elements Fig. 7.11- IS elements in F factor and chromosome pair- Homologous recombination occurs- A large circle is formed = cointegrate- Process is reversible: Hfr can revert to an F+ strainIntegration produces an Hfr strain, able to transfer E. coli genes with high frequencyReview structures:F+ cellF- cellHfr cellFeatures of Hfr mating with F- cell: (Fig. 7.12)1. contact between Hfr and F- cell2. pilus produced by Hfr3. replication (rolling circle model) initiated within the F factor4. transfer of a region of the single stand of Hfr chromosome into recipient cell5. usually mating interrupted before the entire chromosome is transferred6. recombination between the introduced DNA containing bacterial genes with chromosome of F- strain7. donor is still Hfr; recipient is usually still F-, but now contains bacterial genesUse selective plating methods to determine which genes are transferred4. Conjugation mapping2- Genes in bacterial chromosome can be mapped using conjugation- Gene order is determined by transfer order- Distances are time in minutes of transfer- Usually mating is interrupted at fixed time points "interrupted mating"Example of mapping experiment Fig. 7.13Cross Hfr a+b+c+d+e+ str-s x F-a-b-c-d-e- str-rSample the mating at different times.Results: See Fig. 7.13A b a e c dMap: Fig. 7.13 B6 min 6 min 12 min 8 minWith results from additional Hfr strains can assemble a full map: Fig. 7.13 FOverall genetic map of E. coli Fig. 7.14- 100 min. map- genes for enzymes that work in same pathways often clustered- each strand encodes some genes- Hfr stains: many different strains because IS elements (site of F factor integration)can be located at random in the bacterial