Microbial Genetics

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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

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Mutants resistant to an antibiotic
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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

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Results of point mutations
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Frame-shift mutations
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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

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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

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Recombination
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Recombination
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Genetic exchange transformation
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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)

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Avery Experiment
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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

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Mechanism of transformation
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(No Transcript)
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Transduction

• Generalizedcan be carried out by either lytic or
  temperate phage
• Specializedrequires specific integration

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Generalized transduction
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Specialized transduction
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Specialized transduction
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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

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Genetic exchange conjugation
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Conjugation early
Cell-surface structure
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Conjugation middle
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Conjugation late
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Transposons

• Rare events
• Mobile Genetic elements-jumping genes
• Carry a transposase, and flanked by inverted
  repeats
• 20bp to gt100bp
• First discovered in maize/corn
• Phase variation (invertible elements, need
  invertase)

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Transposons
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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

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Genomics

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

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Escherichia coli map
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Next Tues

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