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
