30. Genetics and recombination in bacteria Lecture Outline 11/16/05. The Bacterial Genome and Its Replication The bacterial chromosome

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1 30. Genetics and recombination in bacteria Lecture Outline 11/16/05 Replication in bacteria Types of recombination in bacteria Transduction by phage Conjugation ( mating ) F+ plasmids Hfr s Transformation of raw DNA Evidence for recombination in nature Resistance plasmids 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 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+) 1

2 Replication of the circular chromosome Bacterial cells usually divide asexually by binary fission Origin of replication Replication always starts at a certain place Replication fork Normal replication fork for DNA synthesis but they can occasionally exchange genes: EXPERIMENT Mix two mutant s: Arg+ Trp- and Arg- Trp+. Grown them on complete media. After a short while, test them on culture medium without Trp or Arg. Mixture Termination of replication arg + trp arg trp + Figure Figure Now test them on minimal culture medium RESULTS Mixture Why do they need the control plates? Four ways bacteria can exchange genes arg + trp arg trp + No colonies (control) Colonies grew No colonies (control) CONCLUSION 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. 2

3 1. Phage virus infects A+B+ cell 1. Transduction 2. Reproduction and lysis Once in a while host DNA is mistakenly packaged in a capsid Phage can transfer bacterial genes between cells 3. Transfer of a+ DNA from phage to new cell 2. Conjugation direct transfer of genetic material between bacterial cells that are temporarily joined Conjugation and transfer of an F plasmid from an F+ donor to an F recipient F Plasmid Donor cell contains F+ plasmid Bacterial chromosomes Recipient cell is F(has no plasmid) F+ cell F+ cell Mating bridge F+ cell F cell F+ cell can form a mating bridge with an F cell and transfer its F plasmid. One way transfer Figure Sex pilus 1 µm Single strand of the F plasmid breaks at a specific point and moves into the recipient cell. Both cells are now F+. Figure 18.18a 3

4 Structure of F plasmid Donor F+ cell Synthesis of complementary strand in recipient These genes play a role in the transfer of DNA They are thus designated tra and trb followed by a capital letter Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3. High Frequency Recombination (Hfr cells) Conjugation of Hfr cell with F- cell) F factor can sometimes become integrated in to a bacterial chromosome. F+ Cell is F+ because it has all of the F factor genes MUCH more likely to transfer chromosomal genes to F- cell during conjugation See this in action Usually carries some chromosomal DNA along with it when it is transferred to an F cell 4

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

6 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 Spread of Atrizine decomposing bacteria A few bacterial species are capable of metabolizing the synthetic herbicide Atrizine All have nearly identical genes. 6

7 Atrizine catabolism plasmid Genes DEF in an operon Dispersed atrizine catabolism genes (ABC) acquired separately? Resistance mechanisms Antibiotic Method of resistance Chloramphenicol reduced uptake into cell Tetracycline active efflux from the cell B-lactams, Erythromycin, eliminates or reduces binding of antibiotic to target B-lactams, Erythromycin hydrolysis Aminoglycosides, Chloramphenicol, inactivation of antibiotic by enzymatic modification B-lactams, Fusidic Acid sequestering of the antibiotic by protein binding Sulfonamides, Trimethoprim metabolic bypass of inhibited reaction Sulfonamides, Trimethoprim overproduction of antibiotic target (titration) Bleomycin binding of specific immunity protein to antibiotic Martinez et al. J Bact. Oct 2001 Transposons flank these genes 7