Genetic Concepts in Microbial Genetics: Mutation and Genetic Analysis
Genetic Concepts in Microbial Genetics: Mutation and Genetic Analysis
Lecture Introduction
This lecture marks a transition from DNA replication, transcription, and translation to other crucial genetic concepts in microbial genetics.
The focus will be on mutation and genetic analysis, primarily from Chapter 3.
These concepts represent the fundamental tools geneticists employ to solve biological problems, emphasizing the process of how genetics is applied and understood.
Nomenclature in Genetics
Proper nomenclature is essential for communicating effectively in genetics.
Genes:
Designated in lowercase and italics.
Bacterial genes typically consist of three small letters followed by a large letter (e.g., rpoA, dnaA).
In the genomic era, genes with unknown functions are often given genomic designations (e.g., BC017A).
Upon discovery of function, these genes can be renamed (e.g., BC017A was renamed capB for c AMP activated phospholipase in Vibrio).
Anecdote: The discovery of a gene's function for the first time is highlighted as an exciting aspect of scientific research, contributing a new piece of information to human knowledge.
RNA:
Messenger RNA (mRNA): Typically follows the gene nomenclature (e.g., catB messenger RNA, italics).
Small RNAs (sRNAs): Follow protein nomenclature, meaning no italics and an uppercase first letter (e.g., SgrS small RNA, where SgrS is a small regulatory RNA that controls gene expression).
Proteins:
Follow the same nomenclature as genomic designations or renamed genes: no italics and uppercase letters (e.g., RPOA, DNAA, BC017A, CAPB).
Phenotypes:
Can be designated even when the underlying gene is unknown.
Examples:
Motility: Wild-type (swimming) is designated Mot+; non-motile is designated Mot- or simply Mot (implying a defect as wild-type phenotypes are generally assumed to be +).
Histidine biosynthesis: Cells able to make histidine are designated His+; cells unable to make histidine are designated His- or simply His.
Specific Mutations in Naming:
Specific amino acid changes in proteins can be indicated within the name (e.g., LuxO D47E).
Here, LuxO D47E indicates that the aspartate (D) amino acid at position 47 in the LuxO protein has been mutated to glutamate (E).
Similar designations can be used for nucleotide changes in genes.
Auxotrophic Mutants
Importance: These are crucial mutants in microbiology and make excellent test questions due to the logic of genetic circuits involved.
Definition: Mutants that are deficient in making or using a particular substance.
Two Classes:
Anabolic Auxotrophic Mutants (Synthesis Defective):
Cells cannot synthesize a particular metabolite.
Example: His- (cannot make histidine), Bio- (cannot make biotin, a cofactor).
Growth Requirement: These cells must be supplied with the missing metabolite to grow (e.g., a His- mutant cannot grow without external histidine).
Catabolic Auxotrophic Mutants (Degradation or Use Defective):
Cells cannot utilize a specific substrate (e.g., for carbon, nitrogen, or phosphorus).
Often involves sugars, which typically end in "-ose" (e.g., glucose, lactose, arabinose, galactose, fructose).
Example: Lac- mutant (cannot grow if lactose is the only carbon source), Arabinose- mutant (cannot grow if arabinose is the only carbon source).
Growth Requirement: These cells cannot grow if the specific substrate they are defective in utilizing is the only nutrient source.
Generating Auxotrophic Mutants
Basic Method: Patching Colonies:
Procedure:
Start with a complete plate containing all necessary nutrients (e.g., glucose, histidine, biotin) and growth will be observed for all colonies.
Take individual colonies and "patch" them (rearrange them) onto two selective plates.
Selective Plate 1: Lacks a specific nutrient (e.g., glucose + histidine - biotin).
Selective Plate 2: Lacks a different specific nutrient (e.g., glucose - histidine + biotin).
Track which colony is which across the plates.
Interpretation:
A wild-type colony will grow on all plates.
An auxotrophic mutant (e.g., Bio-) will grow on the complete plate and the plate containing biotin, but will fail to grow on Plate 1 (lacking biotin).
Similarly, a His- mutant will grow on the complete plate and the plate containing histidine, but will fail to grow on Plate 2 (lacking histidine).
Challenge: Due to the low natural mutation rate of E. coli (10^{-9} mutations per base pair per generation), finding mutants this way by chance is highly improbable.
Increasing the Chance of Finding Mutants
Mutator Strains:
Use bacterial strains with defective proofreading or DNA repair systems.
Examples:
dnaQ- mutant: Defective proofreading. dnaQ encodes the 3^{'} to 5^{'} exonuclease activity of DNA polymerase III, which corrects errors during replication.
mutS- mutant: Defective methyl-directed mismatch repair system.
Effect: Both types of mutator strains significantly increase the mutation rate, thereby increasing the number of mutants in a population and reducing the number of colonies that need to be screened.
Mutagens:
Compounds that increase the mutation rate by causing damage to DNA.
Definition of Mutation: A change in the base sequence of the genome.
Examples: Ethidium bromide, UV light (discussed more in DNA repair lectures).
Example: Isolating Ampicillin Resistance (AmpR) using a Mutagen (Selection):
Scenario: To isolate ampicillin-resistant mutants.
Normal plating: On an LB + ampicillin plate, very few (if any) colonies would grow from a normal E. coli culture due to the low natural mutation rate.
Using a mutagen:
Spread E. coli on an LB + ampicillin plate.
Place a filter disc soaked in a mutagen (e.g., ethidium bromide) onto the center of the plate.
The mutagen diffuses outwards, creating a concentration gradient.
Observations:
High mutagen concentration (near disc): No growth occurs because the mutation rate is too high, leading to too many detrimental mutations that kill the cells.
Intermediate mutagen concentration: A high number of ampicillin-resistant mutants will grow, forming colonies, as the mutation rate is elevated enough to produce resistance but not lethal.
Low mutagen concentration (far from disc): Few (or no) resistant mutants will grow, similar to a plate without mutagen.
Selection: This method represents a selection, where only the desired mutants (ampicillin-resistant cells) can grow, while wild-type cells are killed.
Trade-offs:
High mutation rate (with mutagen): Yields many mutants, but each colony may have numerous mutations, making it difficult to pinpoint the exact mutation responsible for the desired phenotype.
Low mutation rate (without mutagen): Yields few mutants, but these likely have very few mutations, simplifying the identification of the causal mutation through genome sequencing.
Enriching for "No Growth" Mutants (Penicillin Enrichment)
Problem: How to enrich for mutants (like auxotrophs) whose desired phenotype is no growth under specific conditions, as selections generally rely on growth.
Key Principle: Penicillin's Mechanism:
Penicillin inhibits cell wall synthesis.
Only growing, actively dividing cells (that need to synthesize new cell wall material) are killed by penicillin.
Non-growing cells (which do not need to synthesize new cell wall) are resistant to penicillin and survive.
Procedure (Example: Enrichment for Bio- auxotrophs):
Start with a population of mutagenized E. coli containing both wild-type (Bio+) cells and desired auxotrophic (Bio-) mutants.
Place the cells in a flask containing a medium with glucose (carbon source) and penicillin, but without biotin.
Wild-type (Bio+) cells:
Can grow because they have glucose and can synthesize their own biotin.
As they grow and attempt to divide, penicillin inhibits their cell wall synthesis, leading to their death.
Bio- auxotrophic mutants:
Cannot grow because biotin is absent from the medium, which they cannot synthesize.
Since they are not growing (and thus not synthesizing new cell wall), penicillin has no effect on them; they survive.
Result: The wild-type cells are killed, while the Bio- auxotrophs survive, effectively enriching the population for the desired mutants. This is an enrichment process, not a selection, as non-mutants are killed rather than only mutants growing.