Microbial Genetics

Bacteria of the same species are not genetically homogeneous - there is immense genetic diversity. In some cases more diversity than what is found between man and other mammals. Bacteria are good organisms to study molecular evolution and genetics.
Mutants - differ from their parents at the DNA sequence level. By definition they differ in their genotypes - the DNA sequence. Mutants may also differ from their parents in their phenotypes - their observable characteristics. Mutants may or may not have different phenotypes from their parents - there are many mutations that do not lead to observable differences between the wild-type and the mutant.
Mutants can be selectable or non-selectable. Selectable mutants have an advantage over the parental strain under certain environmental conditions. For example a spontaneous antibiotic resistant mutant would have a growth advantage over a wild-type sensitive strain in the presence of an antibiotic. Another example of mutants are formations of nutritional mutants called auxotrophs. There wild-type parents are called prototrophs. Auxotrophs are non-selectable mutants. Non-selectable mutants have no advantage over their parental strain and must be screened for by some sort of assay.
How would you select for auxotrophs? Recall they do not grow on minimal medium where they may have to make a specific amino acid. Neat little trick - add penicillin to a minimal medium minus the growth factor (e.g., amino acid). Penicillin will kill only the dividing cells such as the prototrophs, but will not kill the non-dividing cells. Now wash the cells free of the penicillin and spread the cells on minimal medium plus the growth factor. You will have both auxotrophs and a few prototrophs that escape the penicillin treatment.
Molecular basis of mutations
What are mutations? A heritable changes in base sequence of nucleic acid.
How do these heritable changes occur?
Point mutations - involve one or few bases.
Base-pair substitutions - can lead to (i) silent mutations, where the mutation doesn't lead to a different amino acid; (ii) nonsense mutations, where the mutation results in a stop codon and premature stopping translation; and (iii) missense mutations, where there is a change in the amino acid at that position. Some missense mutations lead to what are called temperature sensitive mutations. Temperature sensitive mutants (or conditionally lethal) are able to grow at a low temperature but are unable to grow at a higher temperature. Why? Because the protein effected is unable to fold properly at the high temperature and therefore cannot function properly whereas it is functional at the cool temperature. These are valuable for research purposes.

Microdeletion and Microinsertions
Both of these lead to frameshift mutations potentially. This could lead to a faulty protein depending on what region of the protein is effected.
Remember point mutations and micro deletions and insertions only affect the protein if they occur in the protein coding region of the gene.
Revertants
Defined as mutants that return to wildtype phenotype.
Two types of revertants: Same site revertants and second site revertants
Same site revertants is where a second mutation restores the site of the first mutation.
Second site revertants is where a mutation occurs at a different site in the DNA. These suppressor mutations restore the wild-type phenotype by compensating for the first mutation.
Deletions and insertions
large deletions and insertions may occur. Deletions are not revertable which makes them distinguishable from point mutations. Insertions may be due to insertion sequences such as transposons or insertion elements.
Interestingly, RNA genomes, such as found in viruses, may have a 1000 fold higher mutation rate than DNA genomes. This may be important in the evolution of new virulent forms of viruses.
Mutagens
agents that cause or induce mutations.
Chemical mutagen
base analogs resemble bases but cause errors in replication - examples inclue 5-bromouracil which represents thymine.
alkylating agents modify bases which leads to mutations - example nitrosoguanidine.
Both of these lead to basepair substitutions frequently.
Intercalating agents insert themselves between bases of the DNA and spread them apart causing microdeletions and insertions that lead to frameshift mutations - a common example is acridine orange.
Radiation
Nonionizing radiation - visible light is roughly between 400 and 700 nm wavelenghts. Below 400 nm down to 150 nm is ultraviolet wavelengths. The bases of DNA absorb these wavelengths because of the chemical nature of the nitrogenous bases. 260 nm ultraviolet is an excellent wavelength to kill bacteria because of the light absorbing properties of the bases. Pyrimidine dimers are formed by ultraviolet. The pyrimidine dimers interfere with DNA replication and can lead to basepair substitutions.
Ionizing radiation is defined by electromagnetic wavelengths that cause water and other substances to ionize. These wavelengths are in the region of xrays and gamma rays and are high energy. They produce hydroxyl radicals which react with macromolecules such as DNA and cause damage.
DNA repair induced mutations - Damaged DNA can induce a regulon that itself may cause mutations. The regulon is called SOS regulatory system.
RecA is activated by damaged DNA. RecA inactivates a repressor called LexA which allows an error-prone DNA repair mechanism to function.
Biological mutagens - transposons, insert elements, and viruses. These may insert themselves into random locations in the chromosome thus inactivating specific genes.
Ames test - a test to determine if a chemical is mutagenic, which may also be potentially carcinogenic. The Ames test makes use of various auxotrophs, such as histidine auxotrophs of Salmonella typhimurium with simple point mutation that use the error-prone pathways to repair DNA.
Test - the chemical is usually treated with an enzyme preparation of rat liver which modify the chemical much like your liver does. Many chemicals are not directly mutagenic/carcinogenic until they pass through the liver and are modified by liver enzymes.
The modified chemical is then absorbed onto a disc of filter paper much like you did when you looked at antimicrobial agents in the lab. This disc is laid on a plate with a lawn of the appropriate auxotroph and incubated overnight. Mutagens will increase the rate of back mutation from auxotroph to prototroph which will result in a high number of colonies growing around the disc compared to the proper controls.
Mutations are the primary source of genetic variation, but homologous or general recombination is another source. In recombination, one bacterium's DNA is integrated into another bacterium's genome. Remember that the only way this DNA is propagated is if it is integrated into the recipient's genome since the donor's DNA probably will not have an origin of replication.
How does one bacterium's DNA enter into another bacterium's cell?
Transformation - requires naked DNA in the environment
Transduction - requires virus mediated transfer
Conjugation - requires cell-cell contact and a conjugative-plasmid to mediate transfer
Transformation - one of the first microbial genetic experiments. Griffith showed that rough, avirulent forms of Streptococcus pneumoniae could be transformed into smooth, virulent forms of S. pneumoniae. Avery, MacLeod and McCarty showed that it was DNA that transformed the avirulent cells into virulent cells.
Requires free, naked DNA in the environment and competent cells which are cells that are capable of taking up DNA.
Naturally transformable cells (i.e., competent cells) are found in nature as well as in the laboratory which suggests that this is an important process in the evolution of new strains in nature. Azotobacter, Bacillus, Streptococcus, Haemophilus and Neisseria are naturally transformable. We can induce competence in the lab based on the treatment of cells at a certain growth phase that are treated with divalent cations and other chemicals.
Transduction - DNA is transferred from cell to cell by viruses.
Generalized transduction - random segments of the host bacterium's genome becomes part of the viruses genome. How? During the lytic cycle parts of the host bacterium's genome are accidentally packaged into the virus particle producing a transducing particle. The transducing particle may infect a new cell and genetic recombination occur to produce a new strain.
Specialized transduction - a specific region of the host chromosome becomes part of the viruses genome. The virus is usually a temperate virus - a virus that may integrate into the recipient's genome and replicate with it before causing lysis of the cell.
Phage integrates into a specific site in the host bacterium's genome, e.g., near the galactose genes. Upon induction of the lytic cycle, UV light perhaps, the virus packages either only its DNA or bits of its DNA and the host's DNA.
Many different species of bacteria are transducible including Escherichia, Pseudomonas, Salmonella, Staphylococcus, and Rhodobacter. Again, this process is probably important in nature.
Conjugation - cell to cell contact required to transfer DNA from one cell into another.
Before we talk about conjugation, we need to look at plasmids. We have mentioned them previously.
Plasmids are usually double stranded circular DNA that replicate independently of the host chromosome. They range in size from 1 kilobase to greater than 1000 of kilobases. They exist in cells as supercoiled DNA. There may be only a few copies of the plasmid to 100s of copies of a plasmid in a cell. The copy number is genetically controlled. Some cells contain several typse of plasmids. The coexistence of plasmids is genetically controlled by inc genes or the incompatibility genes. Plasmids of the same incompatibility group cannot reside in the same bacterium. Plasmids of a incompatibility group are genetically related and ones of different incompatibility groups are unrelated.
There are 1000s of plasmids that have been identified. They encode many different functions including (i) antibiotic resistance - so called R factors or plasmids, (ii) toxins and virulence factors such as colonization factors, hemolysins, enterotoxins (diarrhea), (iii) bacteriocins - agents that inhibit or kill closely related species or strains of the same species of bacteria. Generally bacteriocins are polypeptides, (iv) catabolic genes (e.g., naphthalene on the Nah plasmid) and (v) cryptic plasmids that have no known function.
Conjugative plasmids - plasmids that are transferred from one cell to another cell in replicative fashion - that is both cells contain the plasmid in the end. Not all plasmids are conjugative; the plasmid codes for the conjugation functions. A set of genes in a regions called the tra region code for the transmissibility of the plasmid.
Some conjugative plasmids integrate into the chromosome and mobilize the chromosome into recipient cells. Large segments of the chromosome may be mobilized from the donor cell to the recipient cell. The donor cell is called Hfr for high frequency of recombination.
Some plasmids move between a wide variety gram-negative bacteria - so called broad host plasmids. Some move between bacteria and other higher organisms such as plants.
Classic model - the F plasmid - F for fertility - in Escherichia coli.
Requires a donor cell or male and a recipient cell or female.
Genes on the conjugative plasmid in the donor cell code for a sex pili and if we are talking about the F plasmid the pili is called a F pili. Again sex pili of the male attaches to a specific receptor on the female cell and then begins to retract to bring the two cells into contact.
DNA transfer - (see Figure 9.23)
Rolling circle replication - a nick is introduced into the plasmid at OriT - origin of transfer. The 5´ end of the nick is passed into the female cell. At this point it is not real clear what happens. In the rolling circle replication, a primer is laid down on the plasmid and replication proceeds in the 5´ to 3´ fashion as usual and one strand is passed to the female which acts as a template to synthesize the complementary strand.
A second model suggests that a single strand is passed to the recipient and upon completion of the transfer a complementary strand is made to each of the strands in the donor and recipient.
Chromosome mobilization - movement of bits of chromosome by conjugation can occur with the aid of plasmids such as the F plasmid.
F-plasmid is an episome - a plasmid that can occur independent of the chromosome or integrated in the chromosome.
F⁺ cells are cells with the F plasmid unintegrated.
F^- cells are without the F plasmid and make good recipients and are referred to as female cells.
Hfr cells are cells with the F plasmid integrated into the chromosome. The plasmid integrates at specific sites called insertion sequences which represent sites of homology between the plasmid and the chromosome (See figure 9.25). Transfer of the DNA from an Hfr to a F^- cell is similar to the transfer of the unintegrated plasmid. Different Hfrs occur because there are a number of insertion sites in the chromosome.
F-prime are F plasmids that have excised from the chromosome and carry a portion of the chromosome with them during excision.
Results of transfer involving F plasmids
F⁺ mating with a F^- results in two F⁺.
Hfr mating with a F^- results in the donor being Hfr still but the female is usually still F^- since the entire F plasmid is not transferred to the female cell. In this mating usually the mating pair is broken before complete transfer of the donor chromosome and the F plasmid. Therefore the donor genes will not be detected unless they recombine with the recipient genes. More on this later.
F-prime mating with a F^- cell results in a conversion of the F^- cell to a F⁺ , F-prime or an Hfr.
Microbial geneticist can use the F plasmid to order genes on the chromosome. Using different, independent Hfrs with their different sites of integration of the F plasmid, the gene order and a genetic map can be determined. (See Figure 9.27).
How? Antibiotic sensitive Hfrs that are prototrophs are mated with antibiotic resistant recipients that are auxotrophs. Matings are allowed to occur for different periods of time. The mix is sheared to disrupt the mating pairs and this is plated out on appropriate minimal medium with the antibiotic. The recombinants are scored. Genes close to the origin of transfer are transfered to the recipient cell more frequently than more distal genes. (See Figure 9.28). The chromosome requires about 100 minutes for complete transfer.
Conjugative plasmids occur in other gram negative and positive cells. Conjugative transposons occur in in different species of gram positive bacteria. They may move between different species of gram positive bacteria. Leads to potential rapid evolution of new strains, e.g., antibiotic resistant strains.
Using mainly conjugation and transduction and some transformation, the gene order and position of over 1400 genes has been determined. Furthermore, several species of bacteria have had their genome sequenced to the bases.
Transposable elements: Transposons and insertion sequences
Transposable elements first found in maize by Barbara McClintock.
Transposons and IS elements both code for a transposase, which is essential for transposition, and terminal inverted sequences.
Insertion sequences are the simplest elements and only code for genes necessary for transposition.
Transposons- are more complex and code for other properties including antibiotic resistance. They may also be conjugative as well.