Bacterial Genetics

• Bacteria studied in genetic research

  • Escherichia coli
  • Salmonella
  • Streptomyces
  • Bacillus
  • Pseudomonas

  
• The study of bacterial genetics has come a long way
• Advantages of bacteria for genetic research

  • Each cell is a complete organism
  • Bacterial growth is measured by duplication of cells, leading to short generation time and rapid increase in biomass
  • Cellular differentiation is much simpler than in eukaryotes, making it easier to study
  • Bacteria have a very simple genome organization, with only one complete copy of their genome and most genes as a single copy (unlike eukaryotes)
  • Gene mutation will often produce a readily observable phenotype
• Nutritional mutants


  Exhibit altered metabolic requirements
• Lederberg hypothesised that genetic information can be exchanged between bacterial species with different phenotypes
• Auxotrophs


  Have mutations that disrupt specific metabolic pathways, making them unable to produce a pathway product
• Prototrophs


  Able to produce a pathway product
• Genetic exchange experiment


  1. Grow Met-/Pro+ & Met+/Pro- strains in defined media lacking Met/Pro

  2. Growth indicated presence of Met+/Pro+ (Exchange of genetic information)
• Reversion

  Spontaneous mutation that "corrects" a metabolic abnormality back to the wild-type form
• Reversion was a problem as it could mask desired results
• Double and triple auxotroph mutant strains

  Decreases the possibility that a spontaneous reversion mutation might occur, masking desired results
• Bacterial genomes


  • Small and genetically compact, with most of the DNA coding for functional proteins and no introns
  • Single chromosome and plasmid(s)
  • Chromosomal genes: basic metabolic processes
  • Genes on plasmid: not essential, but contribute to pathogenesis, antibiotic resistance, virulence etc.
• Replicon

  DNA molecule that replicates from a single origin of replication (ori)

  
• Plasmid copy number

  Controlled by initiation of DNA replication by DNA polymerase, using mechanisms like antisense RNA and inhibitory proteins

  
• Plasmids with the same origin of replication (ori) cannot be inherited together in the same cell (plasmid incompatibility)

  
• Mutant


  Cell/strain possessing a mutation (change in DNA sequence) relative to wild-type
• Mutation

  Can result in loss of function, regaining of function, modification of gene function, or no change (silent)
• Genotype

  The collection of alleles of a given set of genes in an organism
• Phenotypes used to investigate mutants

  • Auxotrophy
  • Antibiotic resistance
  • Biofilm formation
  • Capsule formation
  • Pigment production
  • Motility

  
• Phenotypic selection


  Method for detection of rare mutants, using selective medium that allows only strains with particular combination of phenotypic traits to grow

  
• Phenotypic screening


  Method for detecting mutants, where all cells form colonies and the desired phenotype must be identified by the researcher

  
• Replica plating

  1. Allows identification of mutants that cannot be identified by phenotypic selection

  2. Colonies are lifted from one plate using a piece of sterile velvet cloth and deposited onto a fresh plate

  
• Mutation can have detrimental effects, no effects or be beneficial
• Environmental pressure constantly selects organisms with the most successful life strategy (survival of the fittest)
• Lederberg demonstrated that streptomycin-resistant mutations arise spontaneously in the absence of selective pressure

  
• Luria & Delbruck showed that variable resistance to phage infection arises in bacteria without selective pressure

  
• Restriction enzymes


  Molecular scissors that cut DNA at specific, short palindromic sequences (restriction sites)

  
• Sticky ends


  Asymmetrical cuts made by restriction enzymes, resulting in overhanging ends that can anneal with each other
• Ligation


  Phosphodiester bond formed between 5' phosphate and 3' hydroxyl groups by DNA ligase, joining the sticky ends
• Mutations can arise spontaneously
• Restriction enzymes (RE)

  Molecular scissors that allow cutting of specific DNA pieces (a required prerequisite to clone DNA fragments)

  
• Restriction enzymes


  • They recognize a specific, short DNA sequence (restriction site, palindromic) and cut it
  • Cuts are often asymmetrical: sticky overhang

  
• Restriction enzyme cleavage and DNA ligation

  1. Similar ends of cut DNA can anneal with one another

  2. Paired ends can be ligated by DNA ligase: Phosphodiester bond between 5' PO4 & 3' OH

  
• Cloning vectors


  Scaffolds used to insert a recombinant DNA molecule into a recipient host bacterial cell

  
• Cloning vectors

  • Plasmids
  • Phages
  • Cosmids
• Recombinant molecules can be used to "clone," or make many copies, of a bacterial gene of interest
• Cloning a gene into a plasmid vector

  1. Choose appropriate RE and digest cloning vector and genomic DNA

  2. Mix fragments and ligate

  3. Transform into E. coli and select on AmpR Agar plates

  4. Replica Plate Screen for TetS

  5. Patch TetS colonies in agar with starch

  
• Blue-white screen

  Combines antibiotic resistance selection and differential screening to determine cloning efficiency

  
• Origin of replication (ori)

  Determines the applicability of a cloning vector by restricting the host cell type
• Homologous recombination


  Occurs when two identical (or nearly identical) DNA fragments line up and exchange pieces, mediated by enzymes RecBCD and RecA