Microbial Genetics

1. Microbial Genetics • WHY? • terms • Genotype refers to genetic makeup • Phenotype refers to expression of that genetic makeup • Heritable traits must be encoded in DNA • Mutations occur randomly

2. Mutants resistant to an antibiotic

3. Kinds of mutants • Auxotroph: loss of biosynthetic enzyme • Cold- or temperature-sensitive: enzyme function is restricted to narrower temperature range • Drug resistance or sensitivity: permeability, degradation, or site of action • Morphology: change in capsule or other feature leads to change in colony • Catabolic: loss of a degradative enzyme leads to, for example, inability to ferment a carbohydrate • Virus-resistant: loss of surface feature that is a virus receptor

4. Results of point mutations

5. Frame-shift mutations

6. Reversions • Same-site reversion: may be true revertant (same sequence) or just restore activity • Second-site reversion • Frame-shift • Production of another enzyme that fulfills the function

7. Mutagens • Analogs for bases • 5-bromo-uracil for thymine (5BU can pair with G as well as with A) • 2-aminopurine for adenine (2AP can pair with C as well as with T) • Intercalating agents (ethidium bromide) • UV (260 nm) causes dimerization of adjacent thymines (photoreactivation) • Ionizing radiation causes chemical changes to the DNA • Generation of free radicals that can react with DNA • SS or DS breaks in the DNA molecule • Transposable genetic elements

8. Recombination

9. Recombination

10. Genetic exchange: transformation

11. Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules • Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) • Some plasmids integrate (F+, Hfr) • Most are double-stranded • About 1- 100kb • Code for: • R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance • Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins • Degradation, tol, nah, • Plasmid copy # • Compatibility (inc)

12. Avery Experiment

13. Transformation • Competence: ability to be transformed • Steps in transformation • DNA binding and uptake (SS or DS, depending on species) • Integration (recA) • Competence may be induced by electroporation, Ca

14. Mechanism of transformation

16. Transduction • Generalized—can be carried out by either lytic or temperate phage • Specialized—requires specific integration

17. Generalized transduction

18. Specialized transduction

19. Specialized transduction

20. Phage Conversion • Lysogeny may cause other changes in the host cell • Often the host acquires immunity to additional infection by that phage type • There may be other changes that may be beneficial to the host • Lysogenized Salmonella anatum acquires cell-surface changes • Lysogenized Corynebacterium diphtherium acquires toxin

21. Genetic exchange: conjugation

22. Conjugation: early Cell-surface structure

23. Conjugation: middle

24. Conjugation: late

25. Transposons • Rare events • Mobile Genetic elements-”jumping genes” • Carry a transposase, and flanked by inverted repeats • 20bp to >100bp • First discovered in maize/corn • Phase variation (invertible elements, need invertase)

26. Transposons

27. Genetic Engineering-basics • Basic steps in cloning • Restriction-modification enzymes • “shot gun” or PCR • Ligase • Recombination Plasmids (or phage)= vectors • Expression vectors • Selection of clones • Looking for a clone with a specific gene • Probes (DNA, RNA or antibody) • Wave of the future: DNA chips or “microarrays”, BAC libraries, automated sequencing etc

28. Genomics • Bioinformatics • Harvesting genes for biotech (Diversa) • Recent Science article • Comparing gene families

29. Escherichia coli map

30. Next Tues • NO quiz • Chp 12, finish up where we left of Today