Topic_9 - Topic 9: Principles of Bacterial Genetics...

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Unformatted text preview: Topic 9: Principles of Bacterial Genetics I Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld Learning Objectives: To learn some of the terminology of microbial genetics, and also to begin to understand the tremendous power of the genetic approach. Microbial Genetics is of fundamental importance, for many reasons. First, cell function is determined by gene function and gene regulation. Also, since genetics of prokaryotic microorganisms is generally much more accessible than genetics of eukaryotic organisms (with the exception, perhaps, of yeast), microbial systems are very useful as experimental systems. Microbial Genetics gave rise to Molecular Biology, and most of the tools of molecular cloning are of prokaryotic origin. Terminology • • • • Mutant: a strain carrying a mutation Genotype: genetic description of a strain (e.g. hisC) Phenotype: observable properties of a strain (e.g. His ­) Wild ­type strain: the original isolate, from which mutant strains are derived Depending on their phenotype, mutations may be selectable or nonselectable. Selectable mutations confer on the strain the ability to grow under conditions that do not allow growth of a strain that does not carry the mutation. For example, an antibiotic resistance phenotype would be selectable, while an antibiotic sensitivity phenotype would be nonselectable. It is possible to select directly for the antibiotic resistance phenotype on antibiotic containing medium. In contrast, the antibiotic sensitivity phenotype cannot be selected for, but could be screened for. In Microbial Genetics, inappropriate use of the terms selection and screening is not tolerated. Replica plating (Figure 11.5) is a technique for screening a large number of colonies for a nonselectable phenotype. This technique is especially useful for the isolation of auxotrophs, which are mutants that have a nutritional requirement (e.g. amino acid, vitamin). The non ­mutant, parental strain is referred to as a prototroph. Penicillin selection is a method that can be very useful as a negative selection to enrich for nonselectable mutants. The key to the success of this method lies in the fact that penicillin only kills growing cells. The addition of penicillin to a population of mutated cells growing in a particular growth medium will result in the death of only cells that are able to grow in that growth medium. The mutants that are unable to grow because they require a growth factor, which is absent from the growth medium, will not be killed and will become enriched in the population. Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld Mutations arise spontaneously. During DNA replication, the DNA polymerase will incorporate errors at a low frequency (10 ­7  ­ 10 ­11 per base ­pair replicated). Point mutations are those mutations that involve the change of a single base pair by substitution, insertion or deletion (Figures 11.6 and 11.7). The effect of a substitution mutation within a protein ­coding region depends on the resulting codon change. In some cases, especially if the substitution is at the third base of a codon, the new codon might encode the same amino acid as the original codon, and the result will be no change in protein. This is called a silent mutation. A missense mutation is when the new codon encodes a different amino acid than the original codon. A nonsense mutation is when the new codon is a stop codon. Insertion and deletion mutations in protein coding sequence result in a reading frame shift (Figure 11.7). They are thus frameshift mutations. The correct reading frame can be restored by a second insertion or deletion mutation near the first mutation, and sometimes this will fully or partially restore activity of the protein. A revertant is a strain that has regained wild ­type phenotype from mutant phenotype. It is easy to envision how a point mutation could revert back to the wild ­type sequence, or at least a sequence that restores activity to the encoded protein. Mutations that restore the wild ­type phenotype also sometimes arise at a different site from the original mutation, and these are called suppressor mutations. Some suppressor mutations arise in the same gene, such as the ones described above that restore the correct reading frame in a frameshift mutation. Suppressor mutations can also occur in other genes resulting in compensation for the loss of the protein activity caused by the original mutation. Other types of mutations include deletions, in which large segments of DNA can be removed, and insertions of large segments within the DNA. Some insertions are caused by insertion sequences, which are genetic elements that are able to transpose (="hop") from one part of the genome to another part of the genome. Although mutations arise spontaneously, there are many mutagens that increase the rate of mutation. Major chemical mutagens are listed in Table 11.3. Base analogs (Figure 11.8) can be incorporated in place of the correct nucleotide but do not base ­pair properly. Some other chemical mutagens are able to chemically alter nucleotides within a DNA strand, resulting in changes to their base ­pairing properties. Yet another class of chemical mutagens interferes with the spacing between adjacent base ­pairs, causing errors during replication. Another important type of mutagen is radiation (Figure 11.9). Ultraviolet light is strongly absorbed by the nucleotide bases, with a peak at 260 nm. The UV light can cause the Fundamentals of Microbiology (Biology 140) Course notes Dr. Josh D. Neufeld formation of pyrimidine dimers between adjacent pyrimidine bases on the same DNA strand, and this can disrupt replication, increasing the incorporation of errors. UV light is a common tool used for mutagenesis in the microbial genetics laboratory. The cell has mechanisms to repair certain types of DNA damage. However sometimes the DNA damage cannot be repaired to give the original sequence, and the cell must repair the DNA however it can, often incorrectly, or else die because the damage is too severe. Some of the DNA repair systems that are used as the last resort are error ­prone. The genes of some of the DNA repair systems are expressed in response to DNA damage, regulated by the SOS regulatory system (Figure 11.10). The Ames Test is a measure of the mutagenicity of a compound, which is often correlated with carcinogenicity. It is based on the ability of the compound to cause reversion of an auxotrophic point mutation in Salmonella typhimurium or Escherichia coli, allowing the strain to grow in the absence of the nutritional supplement (Figure 11.11). This test has been standardized, and is an important first screen in determining the potential carcinogenicity of a compound. ...
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