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LECTURE 14. MICROBIAL GENETICS - OVERVIEW OF

MUTATION, RECOMBINATION, COMPLEMENTATION, SELECTIONS AND SCREENS


I. Microbial genetics addresses:

A major goal of microbial genetics is to connect genes to their in vivo functions.

The approaches and the tools - an overview.

A. APPROACH

1. Selection, screen. A frequently used approach is to devise a selection or a screen to identify individuals with unique characteristics. This is a first step in identifying genes that are involved in some process under defined physiological conditions. A selection essentially involves killing undesirable individuals by manipulating the conditions so that only desirable clones can grow. This technique is very powerful because one only has to deal with the survivors of the selective conditions. A screen is a more tedious process in which each clone is individually examined (often by visual inspection) for some characteristic.		 B. TOOLS

1. Selection, screen. The task is to set up the appropriate physiological conditions in which the selection or screen will distinguish individuals. For example:
  • One can select for individuals resistant to some poison by growing a population on media containing that substance. Individuals sensitive to the poison will not form colonies while those resistant will grow.
  • One can screen for changes in metabolism that will cause a visible change in the colony phenotype of some individuals, e.g. acidification of the media that leads to a color change in a pH indicator.
  • One can screen for individuals deficient in synthesizing some nutrient. Individuals are replica plated on media having the nutrient (permissive media) vs. media lacking the nutrient (selective media). Growth on permissive but not on selective media indicates that those individuals cannot synthesize the nutrient and require it to grow.

Note the difference between the selection and the screens - in the first example, resistant clones are selected for (everyone else dies) while in the other examples, each clone is individually examined.
2. Mutagenesis. Often, the approach is to mutagenize randomly the genomes of a population of cells before doing the selection or screen, in order to destroy the function of some gene required for the process under investigation. 2. Mutagenesis. The tools employed to mutagenize cells include transposons, irradiation (e.g. with UV), and chemicals. In addition, mutations may be produced via mistakes in error-prone replication and repair of DNA.
3. Complementation. Complementation allows one to identify the gene whose mutagenesis resulted in a physiological deficiency. The approach is to reintroduce a "correct" copy of the gene into the mutant cells and demonstrate restored performance by "complementing" the defective copy of the gene. 3. Complementation. The tools most often employed to introduce a "good" copy of a gene into cells are conjugation and transformation. Plasmid vectors are used so that the gene can be stably maintained in the cells.
4. Recombination. In this case, the defective copy of a gene is actually swapped with a wild-type (i.e. "good") copy within the mutant cell. 4. Recombination. When recombination is desired, the genes are not placed on stable genetic elements as was the case in complementation.The tools used to recombine genes are:
  1. Transduction - this involves the transfer of fragments of DNA between cells that is mediated by viruses. These DNA fragments may contain genes that can potentially recombine with the bacterial chromosome.
  2. Hfr-mediated transfer of chromosomal fragments by conjugative plasmids may be followed by recombination of genes introduced into the recipient cell.
  3. Transformation using fragments of DNA or "suicide plasmids" that cannot be maintained stably within cells due to the absence of a functional origin of replication. Such fragments may recombine with the bacterial chromosome.

NOTE - Complementation and recombination are invariably followed by a selection or a screen to identify the cells that have acquired the desired genes.


C. Details of selection.

Selection allows the isolation of extremely rare individuals - up to 1 cell among 1011. For comparison, screens can detect 1 cell among 104. This is actually a "practical" limit; the value can be higher depending upon how many colonies you are willing to examine individually! Two commonly used types of selection are:

1. Positive selection - involves the identification and isolation of individuals whose growth is favored under a defined set of conditions. For example: 2. Negative selection - involves the identification and isolation of individuals that are dormant under a defined set of conditions. "Dormant" means that the cells are not growing (or growing very slowly), but they are not dead.
  • Isolation of antibiotic-resistant cells by growth in the presence of the antibiotic. Sensitive cells will not be able to grow in the presence of the antibiotic.
  • Isolation of cells lysogenic for a certain phage by growth in the presence of the phage (due to immunity to superinfection). Cells not containing a prophage will be infected and lysed by the added phage.
  • Isolation of individuals that can degrade a pollutant as a source of energy by growing the cells in the presence of the pollutant (e.g. as their sole carbon source). Clones that cannot use the pollutants will not grow.
    		•  A commonly used strategy to isolate such cells is called penicillin enrichment. The idea is that since penicillin only kills actively growing cells, dormant cells are by definition immune to the action of the antibiotic. After penicillin destroys actively growing cells, dormant cells can be isolated. The penicillin is removed, and the clones are amplified under permissive conditions. Unambiguous identification of relevant clones by replica plating (in permissive vs. selective conditions) usually follows.
    e.g. Penicillin enrichment is frequently used to isolate nutritional auxotrophs. An auxotroph is an organism that requires a nutritional supplement to grow. This is often due to a defect in a gene involved in the synthesis of the nutrient. The strategy is to grow a population of cells on media lacking the nutrient but containing penicillin. Normal cells will attempt to multiply, but the penicillin will kill them. Mutants that are unable to grow will not be hurt by the penicillin and can be subsequently recovered.

    C. Details of screens.

    Screens involve the direct examination of each clone individually to distinguish mutants from normal cells. In screens, we examine the phenotype of each clone. A phenotype is an observable characteristic of an organism . In contrast, the genotype is a characteristic of the genomic sequence of an organism. We can directly see a phenotype but not a genotype.

    1. A screen can be used to detect the ability of individuals to grow under different conditions. Replica plating (Figure 7.2) is commonly used for this purpose. Cells are taken from a master plate and placed (in the same orientation) onto two test plates containing different media. Usually, one plate is "permissive," allowing the growth of all cells; the other is "restrictive," not allowing the growth of the mutants of interest. Each clone is individually scored for its ability to grow on the restrictive media and mutants can be recovered from the permissive plates for further analysis.

    2. Screens involve the detection of various phenotypic characteristics: e.g. smooth vs. rough Pneumococci in Griffith's classic transformation experiments; e.g. small vs. large colonies; e.g. colony color when pH indicators or chromogenic substrates (like X-gal) are included in the media.