1. 30. Genetics and recombination in bacteria

2. Lecture Outline 11/16/05 Replication in bacteria Types of recombination in bacteria –Transduction by phage –Conjugation (“mating”) F+ plasmids Hfr strains –Transformation of raw DNA Evidence for recombination in nature –Resistance plasmids

3. The Bacterial Genome and Its Replication The bacterial chromosome –Is usually a circular DNA molecule with few associated proteins In addition to the chromosome –Many bacteria have plasmids, smaller circular DNA molecules that can replicate independently of the bacterial chromosome

4. Genetics fo Bacteria Use huge numbers of individuals (billions) To find very rare events Few morphological traits –Antibiotic resistance –“Auxotrophs” cannot synthesize essential nutrients (arg - or trp-) –“Prototrophs” have normal synthesis (arg+, trp+)

5. Replication of the circular chromosome Replication fork Origin of replication Termination of replication Figure 18.14 Replication always starts at a certain place Normal replication fork for DNA synthesis

6. Mutant strain arg  trp + EXPERIMENT Figure 18.15 Mix two mutant strains: Arg+ Trp- and Arg- Trp+. Grown them on complete media. After a short while, test them on culture medium without Trp or Arg. Mutant strain arg + trp – Mixture Bacterial cells usually divide asexually by binary fission but they can occasionally exchange genes :

7. Colonies grew Mutant strain arg + trp – Mutant strain arg – trp + No colonies (control) Mixture To grow on minimal medium, the cell must be able to make both Arginine and Tryptophan (Arg+, Trp+). --> Evidence for genetic transfer of one of those genes to the other strain. CONCLUSION RESULTS Now test them on minimal culture medium Why do they need the control plates?

8. Four ways bacteria can exchange genes

9. 1. Transduction Phage can transfer bacterial genes between cells

10. 1. Phage virus infects A+B+ cell 2. Reproduction and lysis Once in a while host DNA is mistakenly packaged in a capsid 3. Transfer of a+ DNA from phage to new cell

11. 2. Conjugation –direct transfer of genetic material between bacterial cells that are temporarily joined Figure 18.17 Sex pilus 1  m Recipient cell is F- (has no plasmid) Donor cell contains F+ plasmid One way transfer

12. Conjugation and transfer of an F plasmid from an F + donor to an F  recipient Figure 18.18a F + cell can form a mating bridge with an F – cell and transfer its F plasmid. Single strand of the F plasmid breaks at a specific point and moves into the recipient cell. Both cells are now F +. Bacterial chromosomes F + cell Mating bridge F – cell F Plasmid

13. Donor F+ cell Synthesis of complementary strand in recipient

14. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Structure of F plasmid These genes play a role in the transfer of DNA They are thus designated tra and trb followed by a capital letter

15. 3. High Frequency Recombination (Hfr cells) F factor can sometimes become integrated in to a bacterial chromosome. Cell is F+ because it has all of the F factor genes F+ MUCH more likely to transfer chromosomal genes to F- cell during conjugation

16. Usually carries some chromosomal DNA along with it when it is transferred to an F – cell See this in action Conjugation of Hfr cell with F- cell)

17. Conjugation and transfer of part of the bacterial chromosome from an Hfr donor to an F – recipient F + cell Hfr cell F factor The circular F plasmid in an F + cell can be integrated into the circular chromosome by a single crossover event (dotted line). 1 The resulting cell is called an Hfr cell (for High frequency of recombination). 2 Since an Hfr cell has all the F-factor genes, it can form a mating bridge with an F – cell and transfer DNA. 3 A single strand of the F factor breaks and begins to move through the bridge. DNA replication occurs in both donor and recipient cells, resulting in double-stranded DNA 4 The location and orientation of the F factor in the donor chromosome determine the sequence of gene transfer during conjugation. In this example, the transfer sequence for four genes is A-B-C-D. 5 The mating bridge usually breaks well before the entire chromosome and the rest of the F factor are transferred. 6 Two crossovers can result in the exchange of similar (homologous) genes between the transferred chromosome fragment (brown) and the recipient cell’s chromosome (green). 7 The piece of DNA ending up outside the bacterial chromosome will eventually be degraded by the cell’s enzymes. The recipient cell now contains a new combination of genes but no F factor; it is a recombinant F – cell. 8 Temporary partial diploid Recombinant F – bacterium A+A+ B+B+ C+C+ D+D+ F – cell A–A– B–B– C–C– D–D– A–A– B–B– C–C– D–D– D–D– A–A– C–C– B–B– A+A+ B+B+ C+C+ D+D+ A+A+ B+B+ D+D+ C+C+ A+A+ A+A+ B+B+ A–A– B–B– C–C– D–D– A–A– B+B+ C–C– D–D– A+A+ B+B+ B–B– A+A+ Hfr cell D–D– A–A– C–C– B–B– A+A+ B+B+ C+C+ D+D+ A+A+ B+B+ Figure 18.18b

18. Integration of F+ plasmid into a chromosome

19. Transformation –uptake of naked, foreign DNA from the surrounding environment Remember Griffith’s experiment with heat killed bacteria and mice?

21. Does this happen in nature? In E. coli and Salmonella, roughly 17% of their genes have been acquired from other species ( over 100 million years... ) Such “horizontal transfer” is an important issue for the spread of antibiotic resistance

24. Spread of Atrizine decomposing bacteria A few bacterial species are capable of metabolizing the synthetic herbicide Atrizine All have nearly identical genes.

25. Atrizine catabolism plasmid Transposons flank these genes Dispersed atrizine catabolism genes (ABC) acquired separately? Genes DEF in an operon Martinez et al. J Bact. Oct 2001

26. Resistance mechanisms AntibioticMethod of resistance ------------------------------------------------------------------------ Chloramphenicolreduced uptake into cell Tetracyclineactive efflux from the cell B-lactams, Erythromycin, eliminates or reduces binding of antibiotic to target B-lactams, Erythromycinhydrolysis Aminoglycosides, Chloramphenicol, inactivation of antibiotic by enzymatic modification B-lactams, Fusidic Acidsequestering of the antibiotic by protein binding Sulfonamides, Trimethoprimmetabolic bypass of inhibited reaction Sulfonamides, Trimethoprimoverproduction of antibiotic target (titration) Bleomycinbinding of specific immunity protein to antibiotic http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs/