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

Mutants resistant to an antibiotic

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

Results of point mutations

Frame-shift mutations

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

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

Recombination

Genetic exchange: transformation

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 kb 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)

Avery Experiment

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

Mechanism of transformation

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

Generalized transduction

Specialized transduction

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

Genetic exchange: conjugation

Conjugation: early Cell-surface structure

Conjugation: middle

Conjugation: late

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)

Transposons

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

Genomics Bioinformatics Harvesting genes for biotech (Diversa) –Recent Science article Comparing gene families

Escherichia coli map

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