Dale_Molecular Genetics of Bacteria 4th ed

(e.g. by wrapping the bottles in foil) while the cells are recovering from the UV treatment.

SOS repair

Photoreactivation is an error-free process, and therefore does not lead to mutation. If light is excluded, and photoreactivation prevented, the cell still has recourse to other error-free repair processes such as excision repair and recombination repair (post-replication repair), as described in Chapter 1. It is only when these processes are overwhelmed (or if we use mutant strains that lack these errorfree repair mechanisms) that significant numbers of mutations result. This is due to yet another repair mechanism that is error-prone which is part of the so-called SOS response. (Although we are considering this effect in relation to UV irradiation, it is also involved in repair of other types of DNA damage and is related to the more general stress response systems).

In the presence of damaged DNA, the expression of a number of genes involved in DNA repair is induced; these genes include the excision repair genes uvrA and uvrB, the recA gene and the genes involved in error-prone repair. This inducibility of the repair pathways can be demonstrated by irradiating lambda bacteriophage and testing its ability to form plaques on irradiated and non-irradiated host cells. More plaques are obtained if the host cells are also subjected to a low dose of UV irradiation before infection by the phage due to the induction of the repair enzymes in the host by the pre-existing DNA damage.

The mechanism of induction involves the products of two genes: recA and lexA. The LexA protein acts as a repressor of the genes of the SOS response, including both recA and lexA itself. The LexA protein also has the ability to cleave itself but only after the RecA protein binds to it. The RecA protein has a co-protease function in stimulating the self-proteolysis of LexA. This activity of RecA arises after it binds to single-stranded DNA which arises as a consequence of DNA damage. This causes a conformational change in the protein that enables it to bind to LexA, resulting in cleavage of LexA and expression of the SOS genes.

Two of the SOS genes in particular, umuC and umuD, are involved in mutagenesis, since strains that are defective in these genes not only have increased UV sensitivity but also are not susceptible to UV-induced mutagenesis. Certain plasmids carry analogous genes (mucA and mucB); the presence of these genes increases resistance to UV and also results in an increased level of mutagenesis by UV and many other mutagenic agents. The UmuCD complex is able to act as a DNA polymerase, taking over from the normal polymerase (DNA polymerase III) when that enzyme stalls due to the presence of DNA damage. The low specificity of the UmuCD polymerase enables it to continue synthesis

of DNA past the lesion, but at the expense of producing errors in the new DNA strand.

2.7 Isolation and identification of mutants

2.7.1 Mutation and selection

With the techniques described above, it is usually easy enough to alter the DNA randomly; the real art comes in devising means of isolating mutants in which specific genes have been altered. If possible, this is most effectively done using growth conditions in which either the original strain or the mutant is not able to grow. The choice of a method for selection of a mutant depends on the nature of the mutation and its consequences for the cell. We can consider mutants of the three types referred to earlier:

(1)mutants that are resistant to antibiotics or to specific bacteriophages, toxic chemicals or any other agents that are usually lethal or inhibitory to the parent cell;

(2)auxotrophs, i.e. mutants that require some additional growth factor, such as an amino acid;

(3)mutants that are unable to use a particular growth substrate (usually a sugar).

Resistant mutants can be obtained for any type of bacterium (provided it can be grown in the laboratory). Mutants of the second and third types cannot be isolated easily unless the bacterium can grow on a simple defined medium. This is a major reason why bacterial geneticists concentrated initially on E. coli and a limited range of other bacteria such as Salmonella typhimurium and Bacillus subtilis, although the range of organisms studied has now expanded substantially.

Antibiotic resistant mutants are, in principle, easy to isolate. The procedure simply involves plating the culture on agar containing the antibiotic at a concentration that will inhibit the parent strain. These selective conditions are extremely powerful so it is often not necessary to use any mutagenic agent. Very large numbers of bacteria can be used (108–109 cells or 0.1–1 ml of an overnight culture of E. coli), so mutations that occur at a very low frequency can be readily detected. If the mutation frequency is 10 8 and 1 ml of culture containing 109 cells is plated out, then (on average) 10 resistant mutants on each plate would be expected.

Auxotrophic mutants cannot usually be selected directly in this way, since it is not possible to devise conditions where the mutant will grow but the

parent will not. For example, wild type E. coli will grow on a minimal medium consisting of a buffered solution of inorganic salts plus glucose as a carbon/ energy source and ammonium ions as a source of nitrogen. A histidine auxotroph will not grow on this medium but will require the addition of histidine, since it is defective in one of the enzymes of the histidine biosynthetic pathway. The parental strain will also grow quite happily in the presence of histidine. So, although it is not possible to devise a medium on which only the auxotrophic mutant will grow, the converse (a medium on which the auxotroph will not grow) is easy.

If the mutation rate was high enough, it would be possible to pick colonies one by one and test each one to see if it has lost the ability to grow on the unsupplemented minimal medium. Such a test procedure is indeed common practice for many purposes in a microbial genetics laboratory. Each colony is sampled by stabbing it with a sterile toothpick and its ability to grow on a different medium (or a range of media) is tested by stabbing or touching the surface of each test plate in turn. A number of colonies (usually up to 100) can be tested on each plate, provided they are arranged systematically on a numbered grid so that one plate can be compared with another. This procedure becomes extremely tedious with more than a few hundred colonies (although automated procedures involving robotic workstations are now available). The frequency with which a desired mutation occurs in a culture, even after treatment with a mutagenic agent, is very low, so that recovering the required mutant would usually involve testing thousands (or even millions) of colonies.

2.7. 2 Replica plating

Replica plating (Figure 2.13) provides a method for testing large numbers of colonies more rapidly. In this procedure, the mutagenized culture is plated out to obtain single colonies on a nutrient medium on which mutants and parents will grow. After incubation, a sterile velvet pad is pressed lightly onto the surface of the plate so that a tiny impression of each colony is transferred to the velvet. This is used to inoculate first a minimal agar plate and then a similar plate to which the appropriate supplement (in this case, histidine, since we are looking specifically for histidine auxotrophs) has been added. Histidine-requiring auxotrophs will be unable to grow on the first plate, but will grow on the second one, from which they can be picked off for further work.

The parent strain will grow on both plates, while other types of auxotrophic mutant will not grow on either. The second plate thus serves to eliminate other types of auxotrophs that are not wanted and also serves as a control to ensure that the absence of growth on the first plate was not due to failure to pick that colony up on the velvet pad initially.

Failure to grow indicates possible auxotrophs

Minimal medium

Original plate (nutrient medium)

Supplemented minimal medium

Figure 2.13 Replica plating to isolate auxotrophic mutants

Even with replica plating, the number of colonies that can be handled is still limited. If the original plate contains more than a few hundred colonies it is very difficult to identify colonies that have failed to grow. If the required mutation occurs at a frequency of 10 6 (a high frequency for a spontaneous mutation in E. coli) it would be necessary to replicate over 1000 plates to find it. Pre-treatment with a mutagenic agent will increase the proportion of mutants in the population so that they can be isolated from a manageable number of plates.

Replica plating can also be used to test whether mutation occurs at random, without exposure to the selective agent, rather than being induced by selection (see the earlier discussion in this chapter). For example a master plate containing a very large number of colonies can be used to replicate colonies onto an agar plate containing streptomycin. This will indicate which regions of the master plate contain streptomycin-resistant mutants. A pool of colonies can be recovered from those parts of the plate and subjected to further rounds of replica plating, until eventually a pure culture of streptomycin-resistant bacteria which have

2.7.4 Isolation of other mutants

Mutants that are not able to utilize a particular carbon source (lactose, for example) can be isolated in a similar way, i.e. by comparing the ability of colonies to grow on plates containing lactose or glucose as the carbon/energy source. Alternatively, with E. coli for example, advantage can be taken of the fact that sugar utilization often leads to acid production which can be detected by incorporating a pH indicator in the agar. E. coli mutants that are not able to ferment lactose (Lac mutants) can be detected using a medium with a nutrient base (so that both mutants and parents can grow), which also contains lactose and a pH indicator. Bacteriologists often use MacConkey agar for this purpose. This medium contains lactose and a pH indicator so that Lacþ colonies will be red and Lac colonies will remain a pale colour that can be easily distinguished.

A more convenient and sensitive test for distinguishing Lacþ from Lac colonies is to use a chromogenic substrate, i.e. a substrate that shows an easily detectable colour change when acted on by the enzyme concerned. In this case the enzyme is b-galactosidase, which catalyses the hydrolysis of lactose into its constituent sugars glucose and galactose. A commonly used chromogenic substrate for b-galactosidase is 5-bromo-4-chloro-3-indolyl-b-d-galactoside, more popularly known as X-gal. This is a synthetic analogue of the natural substrate, containing a dye linked to galactose. X-gal itself is colourless; the colour of the dye is only manifest when it is released by hydrolysis of the linkage by b-galactosidase. Lacþ colonies will be blue on a medium containing X-gal while colonies that do not produce b-galactosidase will be white.

2.7.5 Molecular methods

The concepts and techniques described so far in this chapter relate to the behaviour of the bacteria themselves. However, some of the molecular methods that will be increasingly important in later chapters, especially the polymerase chain reaction (PCR), gene probes and Southern blotting, and the sequencing of DNA, also have a role in the characterization of mutants, so it is appropriate to introduce them briefly here. In later chapters, we will consider the impact of gene cloning and related techniques which provide highly specific and versatile ways of manipulating the genetic composition of a bacterial cell.

Gel electrophoresis

Central to many of these techniques is the ability to separate DNA fragments on the basis of their size by electrophoresis through an agarose gel (or for very small fragments, an acrylamide gel) – see Box 2.2. At the pH used (commonly 8.3),

DNA is negatively charged, and will therefore move towards the positive electrode. Since smaller fragments move faster than larger ones, gel electrophoresis can be used to estimate the sizes of specific DNA fragments. In Chapter 9 we will consider further how this can be used to examine variation between bacterial strains.

Polymerase chain reaction

The principle of the polymerase chain reaction is outlined in Box 2.3. It depends on the fact that DNA synthesis requires primers base-paired to the template DNA. So using synthetic oligonucleotides that are complementary to the DNA on either side of the gene being studied, specific synthesis of the target gene can be achieved. Repeated cycles of heating the mixture (to dissociate the DNA strands), and then allowing the primers to associate to the new strands followed by further DNA synthesis, result in an exponential increase in the amount of the target DNA. Twenty cycles give a 1 million-fold increase in the amount of the specific target. If the mutation being studied has caused a significant change in the size of a specific gene (such as an insertion or a deletion) this can be detected by a change in the size of the PCR product which can be determined by using gel electrophoresis as described above.

Although conventional gel electrophoresis can only be used to detect changes in the size of a DNA fragment, rather than sequence changes, there are some specialized electrophoretic techniques that can detect small differences (even single base changes) between two PCR products. These are considered in Chapter 10.

Gene probes and Southern blotting

The use of a labelled DNA fragment (a gene probe) to detect similar sequences by hybridization was introduced in Chapter 1. If we use a short oligonucleotide as a probe, under conditions of high stringency (high temperature and/or low ionic strength), small changes in the sequence of a gene, even single nucleotide substitutions, can be detected.

A common method of using gene probes for looking at similarities, or differences, in the structure of a gene, or in a PCR product, is the technique known as Southern blotting. As shown in Box 2.4, this involves separating fragments of DNA by electrophoresis in an agarose gel (see above) and transferring them to a filter which can then be hybridized with the labelled probe. Not only can we confirm that we have amplified the correct PCR product, but by using highly specific probes we can detect differences in the sequence. Furthermore, Southern

Box 2.4 Southern blotting

Southern blotting enables the discriminatory power of gel electrophoresis to be combined with the specificity of DNA hybridization. The name has no geographical connotations, but is named after Ed Southern who first developed it.

In the original version of this technique (as shown in the diagram below), a filter is laid on top of the gel in which the DNA fragments have been separated by electrophoresis and a stack of dry blotting paper is used to draw buffer through the gel and the filter by capillary action. The DNA fragments travel with the buffer and are trapped on the filter where they can be detected by hybridization with a labelled DNA probe followed by autoradiography.

Dry blotting paper

Filter paper wick

Filter

Gel

Buffer

This forms the basis of widely-used procedures for analysing variation between bacteria, as considered in more detail in Chapter 9.

DNA sequencing

In order to establish exactly what changes have occurred in the gene under study, it will be necessary determine the DNA sequence of that gene. This is now a routine procedure in molecular biology laboratories, extending not just to individual genes or fragments but to the complete sequence of the DNA (the genome) of the organism. The methods for doing this and the impact this has had on the study of bacteria are described in Chapter 10.