Microbial Genetics

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BACTERIAL GENETICS
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GENETICS

• Genetics is the study of the transmission of
  things from one generation to the next
• These things can be
• Traits / characteristics
• Chromosomes
• Genes
• Genetics can be studied at various levels
• Subcellular (molecular) level
• Individual
• Population

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GENETICS

• Genetic characteristics of a population can
  change over time
• Evolution
• The speed and magnitude of this genetic change is
  profound in many microorganisms

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BACTERIAL GENETICS

• Bacteria have only one set of genes
• Haploid
• Possess no homologous chromosome with a second
  copy of the gene
• Recessive alleles are not masked by dominant
  counterparts
• Changes in the bacterial genome can make big
  differences very quickly

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BACTERIAL ADAPTATION

• Bacteria are able to quickly adapt to a changing
  environment
• Evolution
• This adaptive evolution requires
• Genetic variation
• Natural selection

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GENETIC VARIATION

• Genetic variation is produced in two ways
• Mutation
• Heritable changes in DNA sequence
• Gene transfer
• Acquiring genes from another species

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MUTATION

• Heritable change in DNA sequence
• Relatively rare
• Generally occur during DNA replication or repair
• May also occur in response to mobile DNA elements
• Transposons and viruses
• May affect gene expression

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MUTATIONS

• There are several different types of mutations
• Chromosome mutation
• Significant change in chromosome structure
• Genome mutation
• Change in number of chromosomes
• Single gene mutation
• Mutation affecting a single gene
• We will focus (almost) exclusively on single gene
  mutations

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SPONTANEOUS MUTATIONS

• Occur without effects of outside agents
• Radiation, chemical mutagens, etc.
• Various types
• Base substitutions
• One or more base pairs changed
• Insertions and deletions
• Sometimes caused by transposable elements
• Jumping genes
• Insertion of one or more bases
• Deletion of one or more bases

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MUTATIONS

• Base substitution mutations
• One base pair altered
• Various effects
• Silent mutation
• Missense mutation
• Neutral mutation
• Nonsense mutation
• Frameshift mutation

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LEAKY NULL MUTANTS

• Leaky mutants
• Mutation may still function, but not as well
• Function may be slow, but not stopped
• Partially functioning protein
• Null mutant
• Knockout mutation
• Total inactivation of gene

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MUTATIONS

• Insertions Deletion Mutations
• Frameshift mutations
• Insertion or deletion of base pair(s)
• e.g., GGA ? GAGA (gly ? glu)
• Generally alter reading frame
• Frameshift
• All downstream amino acids altered
• What if three nucleotides are added?
• Protein function generally affected
• Typically knockout mutants

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TRANSPOSABLE ELEMENTS

• Transposons / Jumping Genes
• DNA segments spontaneously entering or exiting
  chromosomes
• Transposition into a gene constitutes a large
  insertion
• Gene is generally inactivated
• Transposition out of a gene may restore gene
  function
• Imperfect excision leaves small insertion, etc.

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TRANSPOSABLE ELEMENTS

• First discovered by Barbara McClintock in the
  1940s
• Worked with maize (corn)
• Kernel color varied as sequences entered and
  exited pigment genes
• Insertion with complete inactivation ? no pigment
• Insertion with partial inactivation ? partial
  pigment
• Full excision restored full function ? normal
  pigment
• If this sounds crazy now, how do you think it
  sounded when a female scientist suggested it half
  a century ago?

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TRANSPOSABLE ELEMENTS

• Discovered them in 1940s, but nobody paid
  attention
• Ideas finally accepted in 1970s
• Nobel prize in 1983
• She was 81 years old!!
• Some of Barbaras historically significant
  research plots were destroyed by nutcases
  protesting genetic engineering
• None of the plants were engineered

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INDUCED MUTATIONS

• Some mutations occur spontaneously
• Rare
• Certain chemicals or radiation can cause
  mutations
• Mutagens
• Greatly increase the frequency of mutations
• e.g., 1,000X or more

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INDUCED MUTATIONS

• Chemical Mutagens
• Alkylating agents
• Add short carbon chains to bases, changing
  H-bonding properties ? changing pairing
• Base analogs
• Resemble base, and incorporated instead
• Different H-bonding properties
• Intercalating agents
• Interfere with spacing between base pairs on DNA
  strand
• Polymerase adds extra bases to compensate
• Results in insertion ? frameshift
• e.g. ethidium bromide

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INDUCED MUTATIONS

• Induced transposition
• Introduce isolated transposon
• Create knockout insertion mutants
• Radiation
• Ultraviolet (uv)
• Forms thymine dimers (T-T)
• Damage during imperfect repair
• Ionizing radiation(X-rays, gamma rays)
• Breaks in single strands
• Breaks in double strands
• May result in deletions, insertions,
  translocations

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DNA REPAIR

• Mutations are rare
• Many errors are enzymatically corrected
• Mutations in genes for DNA repair enzymes are
  particularly problematic
• Increase mutations

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NORMAL DNA REPAIR

• Proofreading
• DNA polymerase detects error during synthesis
• Backs up, excises wrong base, and continues
• Relatively accurate
• Mismatch repair
• Endonuclease recognizes mismatch if DNA
  polymerase misses it
• Excises incorrect segment
• DNA polymerase fills in gap correctly
• DNA ligase joins the segments
• Relatively accurate

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T-T DNA REPAIR

• Light repair by photolyase
• Breaks thymine dimer in presence of light
• Relatively accurate

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T-T DNA REPAIR

• Excision repair
• Cuts out damaged sequence and replaces
• Light not required
• Relatively accurate

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T-T DNA REPAIR

• SOS repair
• Error Prone Repair
• Used when many thymine dimers are present
• Synthesis occurs across damaged region, ignoring
  damaged region
• Eliminates gaps due to thymine dimers
• Results in many errors ? mutations

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MUTATIONS

• Consequences of Mutations
• Daughter cells are not always identical to mother
  cell
• Mutations increase variation within a population
• Random mutations may result in differences even
  in cells of a single colony
• 106 or more cells
• May offer population of cells options for
  adapting to changing environment

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BACTERIAL ADAPTATION

• Genetic variation exists within populations
• Natural selection can increase the frequency of
  desirable genetic elements
• Individuals possessing these elements are more
  likely to survive and reproduce
• Individuals lacking these elements are less
  likely to survive and reproduce
• Differential reproductive success

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BACTERIAL ADAPTATION

• Genetic variation exists within populations
• Natural selection can decrease the frequency of
  undesirable genetic elements
• Individuals possessing these elements are less
  likely to survive and reproduce
• Differential reproductive success

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BACTERIAL EVOLUTION

• Mutations that persist are passed on to next
  generation
• Positive selection for mutant phenotype ?
  evolution of bacterial populations
• Antibiotic kills wild-type cells
• Resistance mutant capable of surviving antibiotic
• Grows competitively in presence of antibiotic
• Even if it was non-competitive in absence of
  antibiotic
• Selection pressure (e.g. antibiotic presence)
  maintains resistance genes in populations (e.g.
  in hospital)
• Prevalence of gene increased

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MULTIPLE MUTATIONS

• Genes mutate independently of one another
• Probability of two different mutations in same
  cell is product of prob. of each
• P (streptomycin res.) 10-6
• P (penicillin res.) 10-8
• P (both in same cell) 10-6 X 10-8 10-14
• Therefore, if you treat simultaneously with both
  drugs, will have very good chance of killing all
  bacterial cells in population
• What if you treat sequentially?

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MUTANT SELECTION

• Direct Selection
• Variation exists in initial population
• Grow on media supporting mutant but not wild-type
• Only rare mutants will survive
• e.g., antibiotic resistance

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MUTANT SELECTION

• Indirect Selection
• Use when there is no medium on which the mutant
  can grow and the parent cell cannot
• Used when mutant requires growth factor that is
  not normally present
• (Auxotrophic mutants)
• Remember Beadle Tatum?
• Auxotrophic arg- mutants?
• Procedure requires
• Replica plating
• Minimal media
• Various additions to minimal media

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MUTANT SELECTION

• Indirect Selection
• Replica plating to select auxotrophic mutants
• Master plate with enriched media
• Blot with sterile velvet acts like Velcro
• Transfer to minimal media and to enriched complex
  media
• Prototrophic wild-type grow on both plates
• Cells from auxotrophic mutant colonies grow only
  on enriched
• Continue to blot and test on minimal media with
  supplements to identify specific mutation

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MUTANT SELECTION

• Penicillin Enrichment
• Treat bacterial population with mutagen
• Grow on minimal medium (glucose salts) with
  penicillin
• Penicillin kills growing cells (actively making
  cell walls)
• Non-growing cells survive
• Add penicillinase to break down penicillin
• Plate onto enriched medium
• Only works on penicillin sensitive cells
  (gram-positive)

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MUTANT SELECTION

• Selecting Conditional Lethals
• Defects in important genes are normally lethal
• e.g., gene for DNA polymerase, ribosomal
  proteins, etc.
• Mutant enzyme may function at low temperatures
• Temperature sensitive mutants
• e.g., May survive at 25C, but not at 37C
• Incubate at 25C to grow all cells
• Replica plate and incubate at different
  temperatures

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MUTANT SELECTION

• Phenotypic Screening
• Recognizable differences in colony morphology
  not selection, just screening
• pH indicators
• Differential breakdown of materials in media
• e.g. sugars
• e.g. blood

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GENE TRANSFER

• Transfer of genes from one cell to another
• e.g. antibiotic resistance genes
• Three mechanisms
• Transformation
• Transduction
• Conjugation
• Characteristics of all three
• Part of chromosome transferred
• Homologous genes are replaced in recipient
• Very few recipient cells receive transfer

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BACTERIAL TRANSFORMATION

• Remember Griffith?
• Donor cell lyses, fragmented chromosome released
• Naked DNA picked up and integrated by recipient
  cells
• Recipients must be competent
• Near end of log phase
• Cell wall changes
• Receptor proteins made
• Fragment integrates into recipient chromosome by
  breakage and reunion
• Mismatch repair
• Growth of transformed cells

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TRANSDUCTION

• Bacterial DNA may be packaged into phage heads
• Viral DNAse degrades all host DNA
• Some bacterial DNA inadvertently packaged into
  viral coat
• Generalized transduction
• Any genes transferred
• Imperfect excision of prophage from host
  chromosome
• Host DNA attached to packaged viral DNA
• Specialized transduction few specific genes
  transferred
• Transducing virus infects a new bacterial cell
• Bacterial genes transferred

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CONJUGATION

• Requires contact between donor and recipient
• Contact through sex pilus
• F cells possess sex pilus
• F- cells lack sex pilus
• Production of sex pilus encoded by a
  small,non-essential plasmid
• F-factor / Fertility factor
• Self transmissible
• F factor is transferred during conjugation

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CONJUGATION

• Generally only the F (fertility) factor is
  transferred
• Unidirectional transfer F ? F-
• Contact by sex pilus
• Plasmid nicked at origin of transfer
• Transferred as single strand
• Becomes double stranded in recipient
• Recipient becomes F
• Sometimes other plasmids also transferred

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CONJUGATION

• F factor sometimes integrates into host
  chromosome
• Hfr cell (High Frequency of Recombination)
• Integrated DNA nicked at origin of transfer
• Transfers portion of F factor
• Transfers portion of chromosomal DNA
• Takes too long for entire chromosome to transfer
• Recipient remains F-
• Homologous genes replaced

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CONJUGATION

• F factor sometimes integrates into host
  chromosome
• Hfr cell (High Frequency of Recombination)
• Integrated F factor can spontaneously excise
• Hfr cell converted to F cell
• Excision is sometimes imperfect
• Hfr cell converted to F cell
• F plasmid easily transferred to F- cells
• Fragment of host DNA also readily transferred
• F plasmid remains extrachromosomal

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OTHER PLASMIDS

• Plasmids usually have useful, but not
  indispensable genes
• May be present in
• Bacteria
• Archaea
• Some Fungi
• Some Algae
• Some Protozoa
• Vary in size
• All have replicons
• DNA coding for replication
• Some have other genes
• e.g., Resistance to various substances

• Examples of plasmids
• Pseudomonas spp. may grow on unusual substrates
  (e.g., camphor) with certain plasmids
• Ti (tumor inducing) plasmid of Agrobacterium
  tumefasciens
• Allows transfer of tumor-inducing genes to plants
• Useful to engineer for gene transfer into plants
• Remove tumor inducing genes and add YFG (Your
  Favorite Gene)
• R plasmids

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R PLASMIDS

• Resistance plasmids
• Carry genes that confer resistance to
  antimicrobials or heavy metals
• Two regions
• R genes code for resistance
• May be multiple genes
• RTF codes for resistance transfer factor
• Pilus synthesis
• Origin of transfer
• Mobilization genes

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R PLASMIDS

• May be transferred to sensitive bacteria
• Within species
• Between species
• Most common where antimicrobial selection
  pressure is high
• Hospitals
• Where antimicrobials are freely available
• Non-pathogens may harbor R plasmids and transfer
  to pathogenic organisms
• May have arisen by homologous recombination
  between plasmids
• One carrying R genes
• One carrying plasmid transfer genes
• May have arisen by transposons carrying R genes