1. Genetic Variability in Bacteria Mutation spontaneous vs inducible point mutations silent, missense, nonsense, frameshift forward vs reverse (reversion & suppression) macrolesions Genetic Exchange Transformation Conjugation Transduction Transposable elements

2. Genetic Exchange Recombination: –Reciprocal: 2 x dsDNA with common sequence regions swap (cross-over) –Nonreciprocal: ssDNA forms heteroduplex with dsDNA –Site-specific –Transposition Transformation Conjugation Transduction Transposons See Holliday Model (Fig. 13.2 )

3. Griffith’s Transformation (1928) Avery, MacLoed & McCarty (1944) concluded DNA!

4. Transformation DNA fragments from environment get recombined into the chromosome. Natural mechanisms of DNA uptake vary among bacteria. Artificially facilitated by chemically treating cells (competent cells), or by electrical pulse.

5. Conjugation (“Bacterial Sex”) F+ cell is donor. F- cell is recipient. F+ x F- mating. Bound by F+ cell pilus Transfer by rolling circle mechanism.

6. F-Plasmid Replication for Transfer “Rolling Circle Mechanism” –Relaxosome nicks DNA –Extend at 3’-OH. –Displace parent strand. –Synthesize complement for displaced strand. Viral Replication –Same mechanism –Multiple copies made from a single initiation.

7. F-factor Integration F-factor recombines (single crossover) with F+ cell chromosome at an insertion sequence (IS). High frequency of recombination Hfr cell created. Precise de-integration reverses Hfr back to F+. Imprecise de-integration converts Hfr to F’. IS

8. IS Facilitated Plasmid Integration

9. What is an F’? HfrF’

10. Chromosomal Gene Exchange Hfr or F’ cells may result in transfer and recombination of chromosomal genes to F- cell. F’ factor has chromosomal DNA; transfers like a normal F factor to the recipient, making a new F’. Hfr can initiate transfer via the rolling circle mechanism; typically transfer of the chromosome is incomplete; only a fragment gets incorporated.

11. Transduction Viruses of bacteria are called a phage. Phage protein coat surrounding DNA is called a capsid. During phage replication and assembly, capsids may package chromosomal or plasmid DNA by mistake. When transferred to a new host it may recombine. Transducing particle

12. Transposable Elements: “Jumping Genes” Transposable elements (insertion sequences and transposons) can tranfer copies of themselves within or to other DNA molecules (chromosome, pDNA, or vDNA). Antibiotic resistance genes rapidly spread within and between bacterial populations by composite transposons carried on F factors called R plasmids.

13. Self-replicative recombination Transposon or IS self-replicates copy to splice into DNA at a specific target sequences. Endonuclease activity cuts target sequence, leaving single strand overhanging ends. Transposon is ligated to ends. Gaps are filled by DNA polymerase to yield a target sequence at each side of the transposon (called direct repeats). Specific details of the process are more involved (Fig 13.10).

14. Effects of Transposon Activity Insert to mutate genes; change phenotype. Influence transcription or translation (on/off) –Promoters activate –Stop codons and terminator sequences inactivate –Useful tool in gene purification and function studies Facilitate plasmid fusion. Conjugative transposons with transfer genes. Transfer antibiotic resistance. –Plasmid to plasmid –Plasmid to chromosome

15. Consequences of Genetic Exchanges Facilitate macrolesions; mutations of large sequence regions of DNA: –deleted abcdefg → abfg –inserted abcdefg → abcxyzefg –Inverted abcdefg → abedcfg –duplicated. abcdefg → abcdecdefg Spread beneficial trait: –Intraspecific –Interspecific Antimicrobial resistance