microbial genetics why? terms –genotype refers to genetic makeup –phenotype refers to expression...

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

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 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)

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

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: earlyCell-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