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30. Genetics and recombination in bacteria
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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
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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
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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+)
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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
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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 :
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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?
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Four ways bacteria can exchange genes
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1. Transduction Phage can transfer bacterial genes between cells
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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
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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
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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
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Donor F+ cell Synthesis of complementary strand in recipient
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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
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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
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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)
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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
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Integration of F+ plasmid into a chromosome
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Transformation –uptake of naked, foreign DNA from the surrounding environment Remember Griffith’s experiment with heat killed bacteria and mice?
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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
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Spread of Atrizine decomposing bacteria A few bacterial species are capable of metabolizing the synthetic herbicide Atrizine All have nearly identical genes.
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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
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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/
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