Bacterial Genetics and Genetic Changeability

Bacterial Genetics

  • Bacteria exhibit unique changeability compared to eukaryotes.

Barbara McClintock and Transposons

  • Barbara McClintock (1902–1992) observed unpredictable kernel color inheritance in corn.
  • She determined that DNA pieces moved in and out of color-related genes.
  • This idea was initially met with skepticism due to the belief in DNA stability.
  • By the 1970s, her idea gained acceptance, and she was awarded the Nobel Prize in 1983.
  • The existence of transposons, or "jumping genes," explains the maze-like corn kernel patterns.

Staphylococcus aureus and Antibiotic Resistance

  • Staphylococcus aureus is a common cause of skin and wound infections.
  • Since the 1970s, it has been treated with penicillin-like antibiotics like methicillin.
  • In 2004, over 60% of S. aureus strains from hospitalized patients were methicillin-resistant (MRSA).
  • Millions of healthy people in the U.S. harbor MRSA.
  • Healthcare-associated MRSA (HA-MRSA) is resistant to other antibiotics, including drugs of "last resort."
  • Antibiotic resistance in bacteria is a significant problem due to the ability of bacteria to pass resistance genes horizontally.

Adaptation and Genetic Studies

  • Organisms adapt to changing environments, with natural selection favoring those with greater fitness.
  • Bacteria are an excellent system for genetic studies due to their rapid growth and large numbers.
  • More is known about E. coli genetics than any other organism.
  • Even a couple of cells can grow into millions of cells quickly.
  • E. coli was the first organism to have its entire DNA sequenced.

Genetic Change in Bacteria

  • Genetic change in bacteria occurs through mutation and horizontal gene transfer.
  • Mutation involves spontaneous changes in the genome.
  • Horizontal gene transfer involves the exchange of genetic information between members of the same population, such as plasmid transfer.
  • Vertical gene transfer is parent to offspring.

Mutations and Phenotype

  • Mutations can change an organism’s phenotype.
  • Some mutants require something that other bacteria don’t need.
  • If a growth factor is now required, the mutant is termed an auxotroph.
  • Auxo = “increase”, troph = “nourishment”
  • A prototroph does not require growth factors.
  • Proto = “first”
  • Geneticists compare mutants to wild type.
  • New strains are designated by three-letter abbreviations, such as Trp– (cannot make tryptophan) or StrR (streptomycin resistance).
  • If a bacteria can't make something anymore, it's lost the ability to make its original product.

Spontaneous Mutations

  • Normal processes yield spontaneous mutations that occur randomly but infrequently.
  • Mutations are passed to progeny.
  • Large populations contain mutants (e.g., cells in a colony).
  • A colony contains millions/billions of identical bacteria where couple mutants are similar to other bacteria.

Base Substitution

  • Base substitution is the most common mutation.
  • It involves the incorporation of an incorrect nucleotide during DNA synthesis.
  • A point mutation is a change of a single base pair.
  • This can happen because of a mistake during replication causing a complimentary change in DNA which is now stable.

Outcomes of Base Substitution

  • Silent mutation: wild-type amino acid
  • Missense mutation: different amino acid; resulting protein may only partially function.
  • Nonsense mutation: Specifies stop codon; yields shorter protein
  • Any mutation that inactivates a gene is called a knockout mutation.

Genetic Code

  • A codon consists of three nucleotides read in sequence.
  • Many amino acids are specified by more than one codon.

Deletion or Addition of Nucleotides

  • Impact depends on number of nucleotides.
  • One or two pairs yields frameshift mutation (different set of codons translated).
  • Often results in premature stop codon.
  • Three pairs is the same size as one codon, so it will not cause a frameshift.
  • Impact depends on location within protein.
  • A reading shift completely changes codons, unlike a base substitution.

Transposons (Jumping Genes)

  • Can move from one location to another.
  • Gene insertionally inactivated – function destroyed.
  • Terminator was dance red corn-pigment gene is working.
  • In others a transposon gene jumps in so it doesn't work anymore.

Transposons and Color Variation in Corn Kernels

  • Classic studies carried out by Barbara McClintock observed color variation in corn kernels resulting from transposons moving into and out of genes controlling pigment synthesis.

Induced Mutations

  • Result from outside influence.
  • Agent that induces change is mutagen.
  • Geneticists may use mutagens to increase mutation rate.
  • Two general types: chemical, radiation
  • Chemical or physical agents encourage mutations by 100X, 1000X.

Chemical Mutagens

  • Some chemicals modify bases.
  • Alkylating agents add alkyl groups onto nucleobases.
  • This disrupts a bond by making a base look like another base.
  • Strands of DNA look at for something side it of can.

Base Analogs

  • Base analogs resemble bases.
  • Can be mistakenly incorporated by DNA polymerase.
  • Give bacteria something that looks like base analog.

Intercalating Agents

  • Intercalating agents cause frameshift mutations.
  • Flat molecules that intercalate (insert) between adjacent base pairs in DNA strand.
  • Pushes nucleotides apart, produces space.
  • EB showed itself between nucleotides, a mutagen that could cause error in replication.

Radiation

  • Ultraviolet irradiation forms thymine dimers (covalent bonds between adjacent thymines).
  • Cannot fit into double helix; distorts molecule
  • Replication and transcription stall at distortion.
  • Cell will die if damage not repaired.
  • Mutations result from cell’s SOS repair mechanism.
  • X-rays cause single- and double-strand breaks in DNA.
  • Double-strand breaks often produce lethal deletions.
  • Bacteria can break those T=T bonds with enzymes.
  • Eukaryotes can only go in & cut out damage and fix it.
  • Exposing DNA to Thymine binders: Can't repair mistakes.
  • Errors = cancer.
  • Break sugar phosphate backbone holding DNA when trying to repair that encourage mutations.

DNA Repair

  • There is an enormous amount of spontaneous and mutagen-induced damage to DNA.
  • We have mechanisms of repair so we can survive.
  • If not repaired, can lead to cell death; cancer in animals.
  • Spontaneous or induced mutations may be repaired by enzymes removing this.

Proofreading

  • During replication, DNA polymerase sometimes incorporates wrong nucleotide.
  • Mispairing slightly distorts DNA helix
  • Recognized by enzymes
  • Mutation prevented by repairing before DNA replication
  • Two mechanisms: proofreading, mismatch repair
  • Performed by DNA polymerase, verifies accuracy and can back up, excise nucleotide and incorporates correct nucleotide.
  • Very efficient but not flawless

Mismatch Repair

  • Fixes errors missed by DNA polymerase.
  • Enzyme cuts sugar-phosphate backbone
  • Another enzyme degrades short region of DNA strand
  • DNA polymerase, DNA ligase make repairs

Repair of Damage from UV Light

  • Photoreactivation: light repair (only found in bacteria).
  • Enzyme uses energy from light to break covalent bonds of thymine dimer.
  • Excision repair: dark repair (bacteria and eukaryotes).
  • Enzyme removes damage; DNA polymerase and DNA ligase repair.

Mutant Selection

  • Mutants are rare and difficult to isolate.
  • Two main approaches:
    • Direct selection: cells inoculated onto medium that supports growth of mutant but not parent (e.g., antibiotic-resistant mutants exposed to antibiotic).
    • Indirect selection: isolates auxotroph from prototrophic parent strain. More difficult since parents will grow on any media on which auxotroph can grow. Replica plating allows.

Horizontal Gene Transfer

  • Microorganisms commonly acquire genes from other cells through horizontal gene transfer.
  • Genes naturally transferred by three mechanisms:
    • Transformation: naked DNA uptake by bacteria
    • Transduction: bacterial DNA transfer by viruses
    • Conjugation: DNA transfer between bacterial cells

DNA Replication and Integration

  • DNA replicated only if is a replicon
  • Has origin of replication
  • Plasmids, chromosomes
  • DNA fragments added to chromosome via homologous recombination
  • Only if sequence similar to region of recipient’s genome

DNA-Mediated Transformation

  • Cells release DNA when they are lysed
  • Addition of DNase prevents transformation

Transduction

  • Transfer of genes by bacteriophages
  • Specialized transduction: specific genes
  • Generalized transduction: any genes of donor cell
  • Rare error during phage assembly
  • Transfer of DNA to new bacterial host

Conjugation

  • DNA transfer between bacterial cells requiring contact between donor and recipient cells
  • Conjugative plasmids direct their own transfer
  • F plasmid (fertility) of E. coli most studied

F Plasmids

  • Encode proteins including F pilus (sex pilus) that brings cells into contact
  • Enzyme cuts plasmid transferred single strand
  • Complementary strands synthesized
  • Both cells are now F+

Hfr Cells

  • High frequency of recombination cells arise when F factor integrates into chromosome
  • When an Hfr donor passes a portion of its chromosome into an F– recipient, a recombinant F– cell results.

The Mobile Gene Pool

  • Genomics reveals surprising variation in gene pool of even a single species
  • Perhaps 75% of E. coli genes found in all strains (core genome of species)
  • Remaining make up mobile gene pool (Plasmids, transposons, genomic islands, phage DNA)

Plasmids

  • Found in most Bacteria, Archaea
  • Usually dsDNA with origin of replication
  • Generally nonessential; cells can be cured
  • Can contain few to thousands of genes
  • Low or high-copy-number
  • Most have narrow host range (single species); some broad host range (includes Gram– and Gram+)

Resistance Plasmids (R Plasmids)

  • Resistance to antimicrobial medications, heavy metals (mercury, arsenic) compounds found in hospital environments
  • Often broad host range that normal microbiota can transfer to pathogens

Transposons

  • Provide mechanism for moving DNA
  • Composite transposons include one or more genes
  • Integrate via non-homologous recombination

VRSA

  • Transposons yielded vancomycin resistant Staphylococcus aureus strain
  • Patient infected with S. aureus susceptible to vancomycin
  • Also had vancomycin resistant strain of Enterococcus faecalis
  • Transferred transposon-containing plasmid to S. aureus
  • Transposon jumped to plasmid in S. aureus