Chapter 8: Bacterial Genetics

1
Chapter 8Bacterial Genetics
2
Important Point
If you are having trouble understanding lecture
material Try reading your text before
attending lectures. And take the time to read it
well!
3
Bacterial Genetics

• Acquiring genes through gene transfer provides
  new genetic information to microorganisms, which
  may allow them to survive changing environments.
• The major source of variation within a bacterial
  species is mutation.
• In mutations, usually only a single gene changes
  at any one time.
• In contrast, gene transfer results in many genes
  being transferred simultaneously, giving the
  recipient cell much more additional genetic
  information.

4
Bacterial Genetics Overview

• Most bacteria are haploid which means that there
  is no such thing as dominance-recessive
  relationships among bacterial alleles.
• Bacteria dont have sex in the animal/plant sense
  of sex (i.e., mating followed by recombination of
  whole genomes).
• Instead, bacteria acquire DNA from other bacteria
  through three distinct mechanisms
• Transformation
• Transduction
• Conjugation
• This DNA may or may not then recombine into the
  recipients genome.
• We use phrases like Lateral or Horizontal
  Gene Transfer to describe these sexual
  interactions.
• Bacterial DNA is also subject to mutation, damage
  (not the same thing as mutation), and natural
  selection.

5
Mutation Terms Concepts

• Wild Type refers to the microorganism as isolated
  from the wild.
• A mutated microorganism that has lost a metabolic
  function, particularly an ability to synthesize a
  specific growth factor, is called an Auxotroph.
• The wild-type parent to an auxotroph is called a
  Prototroph.
• A Mutation is found in a gene a mutant is an
  organism harboring a Mutation.
• We designate mutant phenotypes using three-letter
  abbreviations the phenotype of a
  tryptophan-requiring auxotroph would be described
  as Trp-.
• A bacterium that has mutated to resistance to an
  antibiotic (or other substance) is given the
  superscript R thus, the phenotype ampicillin
  resistance is indicated as AmpR.
• Mutants can be spontaneous or induced by a
  Mutagen an agent that causes DNA to mutate.

6
Types of Mutations

• Base Substitution
• Point mutation single base is substituted.
• Missense mutation base change changes single
  amino acid to different amino acid.
• Nonsense mutation base change changes single
  amino acid to stop codon.
• Null or Knockout mutation mutation that totally
  inactivates a gene.
• Deletion or insertion mutation change in number
  of bases making up a gene.
• Frameshift mutation insertion or deletion of
  something other than multiples of three bases.
• Frameshifts typically radically change downstream
  codons, generating stop codons, and typically
  knocking out gene function.
• Reversion mutation mutated change back to that
  of wild type.

7
Rates of Mutation

• The mutation rate of different genes usually
  varies between 10-4 and 10-12 mutations per cell
  division (essentially equivalent to per cell).
• 10-4 one in 10,000 10-12 one in one
  trillion.
• To calculate the probability of two independent
  mutations we multiple the two mutation rates.
• Thus, if streptomycin resistance occurs at a rate
  of 10-6 mutations per cell division and the rate
  of mutation to resistance to penicillin is 10-8
  then the rate of mutation to both antibiotics is
  10-6 10-8 10-14 (note that the exponents are
  added).
• That is, we would have to have a population of
  one-hundred trillion cells to have one double
  mutant, which even for bacteria is a lot of
  cells.
• This is the basis for Combination Therapy, e.g.,
  the use of more than one chemotherapeutic against
  tuberculosis, HIV, cancer, etc.
• The odds of sufficiently multiply resistant
  mutants drops with each new chemotherapeutic
  added to the mix.

8
Direct Selection for Mutants
9
Indirect Selection Replica Plating
10
Indirect SelectionPenicillin Enrichment
11
Indirect SelectionIsolation of ts Mutants
This is one example of isolation of mutants
carrying conditionally lethal mutations found in
essential genes.
12
Ames Salmonella Test
13
DNA-Mediated Transformation
Note that DNA is taken up naked from the
environment.
14
DNA-Mediated Transformation
15
Original Transformation Exp.F. Griffith (1928)
using pneumococci
16
Artificial Competenceby Electroporation
Competence denotes the ability to take up DNA
naked from the environment.
Most bacteria are not naturally competent but
many can be made artificially so.
Artificially induced competence is very important
to gene cloning.
17
Generalized Transduction
Bacteriophages are viruses that only infect (and
can kill) bacteria.
18
Generalized Transduction
19
Conjugation Sex or F Pilus
20
Conjugation F Plasmid Transfer
21
F and Other Plasmids

• F plasmids encode genes that allow both their
  replication and transfer.
• They are thus known as Self-Transmissible
  Plasmids.
• There are other plasmids that can take advantage
  of conjugation but dont encode the the necessary
  genes. These are non-self transmissible plasmids.
• Transduction is also capable of transferring
  smaller plasmids.
• R plasmids are named not for their mode of
  transmission but instead for the resistance genes
  that they encode such as to antibiotics.
• Some plasmids are present in bacteria in low copy
  numbers (1 or 2/bacterium) whereas other plasmids
  are present in high copy numbers (such
  100s/bact.).
• Plasmids, R and otherwise, can have very wide
  host ranges allowing easy transfer of already
  evolved genes between bacterial species.

22
Self-Transmissible R Plasmid
Note multiple resistance genes.
Resistance Transfer Factor (conjugation genes)
23
Transfer of non-R Virulence Factors

• Genes that can make bacteria more virulent (able
  to cause disease) are called Virulence Factor
  genes.
• Virulence factors include fimbriae that allow
  attachment to host tissues, exotoxins, etc.
• Virulence factor genes may be transferred by
  transformation, transduction, or conjugation.
• Virulence factor genes tend to congregate on
  bacterial chromosomes in regions known as
  Pathogenicity Islands.
• New bacterial pathogens can emerge via the uptake
  of entire pathogenicity islands transferred
  intact from unrelated bacteria.

24
Transfer Protection R-M Systems

• Not all incoming DNA is necessarily good for the
  receiving bacterium (i.e., DNA can be parasitic).
• Bacteria employ Restriction Enzymes to protect
  themselves from the foreign DNA.
• Restriction enzymes recognize specific,
  palindromic (same spelling backward and forward)
  DNA sequences of 4 to 8 base pairs in length that
  are known as Recognition Sequences.
• Bacteria also employ Modification Enzymes that
  modify DNA to protect it from Restriction
  Enzymes.
• Together these are called Restriction-Modification
  Systems.
• Restriction enzymes are crucial components of
  genetic engineering.

25
Restriction Endonuclease Action
26
Restriction Endonuclease Action
Note in particular that DNA is cut at palindromic
regions.
27
DNA Modification RE Protection
28
Link to Next Presentation