Download Microbial Genetics

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: 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
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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