Dale_Molecular Genetics of Bacteria 4th ed

Hybridize

Sequence present in both (yellow)

Sequence present in

M. tuberculosis but not BCG (red)

Figure 10.8 Genomic comparison using a microarray. The two probes are labelled with different fluorescent dyes which can be distinguished by the machine reading the microarray. Spots hybridizing only to probe 2 will be displayed as red, while spots hybridizing to both probes will be shown in yellow. Spots hybridizing only to probe 1 would be displayed as green, but are not expected in this example as probe 2 is from the same source as the array and is expected to hybridize to all spots (see also Figure 10.12)

distinguish the fluorescence of the two dyes and the results are displayed as a pattern of dots – red and green dots indicating binding of one or the other sample only, while if both labelled DNA samples bind, the result will be shown as a yellow dot. So in this example, all genes that are present in both M. tuberculosis and BCG will be shown in yellow, while those that are only present in M. tuberculosis (and missing in BCG) will appear in red. The microarray cannot show whether BCG has any extra DNA which is not present in M. tuberculosis, therefore green spots would not be expected.

This technique can therefore provide a rapid assessment of the differences between the genomes of two organisms – but it will only detect deletions. For other types of changes, in particular sequence polymorphisms, other techniques have to be used.

Sequence polymorphism

It is not (yet) possible to undertake a global analysis for all the sequence differences between two bacteria, short of complete genome sequencing. However, large numbers of strains can be rapidly surveyed for changes in specific genes. Primers derived from the sequence of a gene from one strain can be made and PCR used to amplify the corresponding gene from the other strains. The amplified product can be sequenced directly.

One example comes from studies of bacterial resistance to rifampicin which is (amongst other uses) a front-line drug in the treatment of tuberculosis. The target for rifampicin is the bacterial RNA polymerase and analysis of sequence polymorphism shows that most rifampicin-resistant strains of M. tuberculosis have base substitutions in a limited number of positions in the rpoB gene (coding for the b subunit of RNA polymerase).

Sequence polymorphisms can also be detected by specialized electrophoretic techniques which can detect differences as small as a single base change between two PCR products. Two such technologies are Single Strand Conformational Polymorphism (SSCP) in which denatured PCR products are run on acrylamide gels and Denaturing Gradient Gel Electrophoresis (DGGE) where PCR products are resolved on an acrylamide gel which contains a gradient of a denaturing agent. These techniques are often used to screen large numbers of PCR fragments to determine whether or not they contain base changes prior to nucleotide sequence determination.

10. 2. 4 Bioinformatics

The genome sequence consists only of millions of DNA bases in a defined order. Bioinformatics is the use of computers to obtain biologically interesting information from the sequence data. This starts with identifying genes, primarily by searching for open reading frames (ORFs) – although in practice it is more complex than that. The gene sequences are then automatically translated to determine the protein sequences they encode.

It is then necessary to determine the function of those genes and their products. One way of doing this is to compare each protein to all the proteins in the international databases in which all known DNA and protein sequences are stored. (There are three main databases, GenBank, EMBL and DDBJ, but all the information is exchanged between them so they are all equally informative). It is preferable to compare protein sequences rather than DNA, partly because the differences in codon usage between organisms means that the DNA can be quite different even though the proteins encoded are similar. These sequence comparisons are more complicated than simply lining up the two sequences and counting the matches, but fortunately there are programs available (one of the most widely

used being known as BLAST) which will carry out this procedure. The results may show that a gene (or protein) is similar to one in the database with a known or predicted function, so it is therefore possible to assign a putative function to that gene from your organism of interest.

The second tool for assigning function looks for the presence of shorter sequences in the protein that are characteristic of proteins with specific functions. For example there may be a specific arrangement of hydrophobic regions which is characteristic of membrane proteins, or a predicted structure that indicates a DNA-binding protein (which would be likely to be a regulatory protein). There are computer-based libraries of these signatures and software that will compare proteins of interest to those libraries. This process, which essentially labels a gene with a possible function and a unique catalogue number, is known as annotation.

However, even with the wealth of information that is available and the sophisticated software that can be used for automated screening of complete genomes, a substantial proportion of the genes in sequenced bacterial genomes give no clues at all to their function, as they are neither similar to any genes in other bacteria nor do they have any structural features which indicate their function. Determining the function of these genes remains a major challenge. The next section in this chapter includes some of the ways in which this question can be addressed.

10.3 Physical and genetic maps

The techniques described so far can be divided broadly into two categories. Analysis of recombination between mutants (by conjugation for example) produces a genetic map in which the relative positions of genes are determined. On the other hand, restriction mapping and PFGE analysis give a physical map in which the position of restriction sites or other features such as deletions and insertions are located, without any reference to the location of specific genes. Even the genome sequence, in the absence of other information, is merely a physical description of the chromosome.

How are these maps correlated? In other words, if the function of a gene and its position on a genetic map is known, how can its position on a physical map of restriction fragments or on the genome sequence be found? The question can also be reversed, i.e. if an adequate physical map (ideally the complete genome sequence) is available, how can the genes that are associated with a specific function in the cell be identified?

Some of the approaches might be obvious from the previous discussion. For example, if a gene has been cloned, it is easy to determine its position on a restriction map or within an ordered gene library by testing its ability to hybridize to the relevant DNA fragments. If the sequence of the gene is known and the genome sequence has been determined, a simple computer analysis will locate it. There are however some additional techniques that enable these questions to be

approached without such detailed information and also to refine the analysis of the relationship between gene structure and phenotype.

10.3.1 Deletions and insertions

If a mutant is selected on the basis of an altered phenotype, it may have arisen by a point mutation – a single base change in the gene concerned. Such mutations are not readily located on a physical map even if the complete genome sequence is known. It can however provide a route for cloning, and hence identifying the gene, for example by selecting a clone that complements the mutation (see Chapter 8).

On the other hand, if the loss of a gene function is due to the insertion or deletion of a larger piece of DNA, this can be visualized as a change in the size of a restriction fragment. It is then possible to clone and characterize this fragment to confirm its identity and its relationship to the observed phenotype. Alternatively, if the genome sequence has been determined, microarrays can be used as described earlier.

10. 3. 2 Transposon mutagenesis

Transposons (see Chapter 7) provide a more convenient way of doing the same job, since they are capable of inactivating genes by insertion and the inactivated genes are readily identified and recovered. A typical strategy, outlined in Figure 10.9, is to construct a plasmid carrying the transposon, using a temperature sensitive vector, i.e. a plasmid that is unable to replicate at an elevated temperature. When this plasmid is introduced into bacterial cells and the cells are grown at a temperature permissive for plasmid replication (say 308C), all the cells will carry the plasmid. If the culture is subsequently shifted to a temperature at which the plasmid is unable to replicate (e.g. 428C), the only cells to retain the transposon will be those in which the element has transposed onto the chromosome. If a transposon such as Tn5, is used, which carries a kanamycin resistance gene, these transpositions can be selected for by plating the culture at 428C on agar containing kanamycin.

This generates a transposon mutant library, with the transposon inserted at a variety of chromosomal positions and therefore causing a range of mutations. Once the mutants of interest have been selected on the basis of a specific phenotypic change, the gene(s) into which the transposon has inserted can then be identified. For this, advantage is taken of the resistance gene on the transposon. If the total DNA is cut with a restriction enzyme, the collection of fragments can be cloned to produce a gene library. If this gene library is then plated on kanamycin agar, only those clones containing Tn5 will be able to grow. These

10.3.3 Gene replacement

Having identified a gene of potential interest, for example from the genome sequence, one of the best ways of determining its function and significance is to inactivate it. This may not be easy to achieve by the classical procedures of mutagenesis and selection, especially if the phenotype associated with that gene is not known or not readily selectable. However, a cloned gene that has been inactivated in vitro can be used to replace the wild-type chromosomal gene by homologous recombination, a process also known as gene knock-out. The procedure for doing this was described in Chapter 9. Once the gene has been knocked out, it will be possible to determine what the consequences are for the phenotype of the cell which will give an indication of the function of that gene.

10.3.4 Site-directed mutagenesis

Instead of knocking out a gene completely, it may be desirable to replace it with a specifically altered gene. For this gene replacement as described above would be combined with the techniques of site-directed mutagenesis, as covered in Chapter 8, either by direct replacement or by complementing a knocked-out gene with a plasmid carrying the mutated gene.

This enables the significance of a specific amino acid in the activity of an enzyme for example, to be tested by replacing it with a series of different residues and determining the activity of the product. Or the precise requirements for the function of a regulatory sequence on the DNA, such as a promoter site, could be investigated by introducing defined mutations into that region.

10.4 Analysis of gene expression

Bacteria are capable of recognizing many different environmental stimuli and changing their pattern of gene expression to optimize survival when faced with new environments (see Chapter 3). Much can be learned about the function of a gene and our understanding of these physiological responses can be enhanced by following the changes in expression that accompany alterations in the morphological or physiological state of the cell (e.g. during sporulation or transition to stationary phase) or in different external environments (such as bacterial growth within macrophages).

The most obvious way of monitoring gene expression, by assaying enzyme activity, is not always appropriate. For a start, not all gene products are enzymes; some of the most interesting are regulatory proteins. And even if it is an enzyme, the assay procedure may be too complex to use for this purpose.

10. 4.1 Transcriptional analysis

In bacteria, gene expression is primarily regulated at the transcriptional level. It can therefore be reasonably assumed that measuring the amount of a specific mRNA in the cell is an appropriate measure of gene expression. Furthermore, in general, bacterial mRNA is short lived, so measuring the amount of a specific mRNA at a given time is likely to provide a good assessment of the current level of transcription of that gene. An in vivo method for doing just this, i.e. the use of reporter genes, has been previously encountered in Chapter 9. Here, we concentrate on the in vitro methods available for assessing mRNA levels.

Northern blots and RT-PCR

One way of looking at the production of a specific mRNA is to fractionate bacterial RNA by gel electrophoresis, and transfer the RNA to a filter by blotting, followed by hybridization of that filter with an appropriately labelled DNA probe. The intensity of the signal provides a measure of the amount of the specific mRNA in the preparation. Since this procedure is analogous to Southern blotting (see Chapter 2), it is referred to as Northern blotting.

Northern blotting is not ideal for the purpose being considered here. Firstly, many bacterial mRNA species are quite unstable, with a half-life of only 1 or 2 min. Secondly, the amount of RNA required may be more than can be readily obtained under the conditions being investigated. For example, we may want to investigate the levels of expression within different parts of a single colony or from bacteria growing within macrophages. A much more sensitive technique is provided by an adaptation of the polymerase chain reaction (PCR).

In standard PCR (see Chapter 2), a DNA polymerase is used to make copies of the template DNA. In order to obtain a sensitive method for detecting a specific mRNA, a DNA copy is made initially, using an RNA-directed DNA polymerase, reverse transcriptase (see the description of cDNA cloning in Chapter 8). Following the initial reverse transcription, a standard PCR can be used to amplify the DNA strand produced. This method, reverse transcript PCR or RT-PCR (Figure 10.10), provides a very sensitive method of detecting the presence of a specific transcript within small sections of a bacterial population.

Real-Time PCR

A serious limitation of RT-PCR, as described above, for the purpose being considered here, is that it is not easily quantifiable. It can be used to detect a transcript but not (or at least not easily) to determine how much of that transcript

Primers 1 and 2 anneal

DNA polymerase synthesises further strands

Repeated rounds of amplification

Amplified products

Figure 10.10 Reverse transcript PCR (RT-PCR)

is present. For that, another variant of PCR known as real-time PCR can be employed, since it enables continuous monitoring of the appearance of the amplified product during the reaction.

There are actually several quite different techniques for doing this, but the discussion here will be limited to the method which is conceptually the simplest. This involves including in the PCR mixture a dye which fluoresces when it binds to double-stranded DNA. This signal can be detected using a machine that not only carries out the temperature cycling but also measures the fluorescence of the sample. Initially, the DNA concentration is very low, so there will be no detectable signal. But as the reaction progresses, through one cycle after another, the amount of the amplified product increases exponentially, until eventually the machine registers a signal (Figure 10.11). The number of cycles needed to reach

To assess the expression of the genes on the microarray, cells are grown under a control condition and in the presence of a stimulus of interest (Figure 10.12) – for example, a comparison of gene expression in actively growing cells (culture A) with those in stationary phase (culture B). The mRNA is isolated from both cultures and reverse transcribed to produce a spectrum of cDNA molecules. Each cDNA preparation is tagged separately with a different fluorescent dye as described previously. The cDNA samples are then mixed and incubated with the microarray to allow them to hybridize to the complementary spots on the array. The two differentially dyed cDNAs in the mixed sample compete for binding to each spot on the array, the outcome depending on the relative amounts of the specific cDNA within the two samples. Afterwards, the microarray is scanned using a specific reader as described previously. In this example, any gene that is more strongly expressed during active growth will be represented by more cDNA labelled with the first dye. The signal from that dye will therefore be stronger which will be displayed as a red spot (spot 1). Conversely, genes that are activated during stationary phase, with cDNA labelled with the second dye, will be shown as a green spot (spot 3). If a gene is expressed equally under both conditions, there will be no difference between the signal from the two dyes and this will show as a yellow spot (spot 2).

Thus under these conditions, the relative expression of all the genes in a genome can be assessed by simply examining the colour of the spots. The spectrum of genes in a cell which are transcribed under a specific set of conditions is known as the transcriptome by analogy with the genome, which is the complete genetic content of the cell. It is worth noting that while the genome for an individual strain is more or less fixed, the transcriptome is not – it will be different according to the conditions pertaining at the time the cells were harvested.

10. 4. 2 Translational analysis

As mentioned earlier, analysis of mRNA levels assumes that gene regulation occurs at the transcriptional level – but this may not be true. Consideration should be given to the possibility that the levels of specific proteins are affected by the efficiency of translation as well as, or instead of, transcription. Furthermore, if there are several similar genes coding for different proteins, it may be quite difficult to distinguish them by transcriptional analysis, as the products may cross-hybridize.

Western blots

If specific antibodies are available which recognize the protein under study, these can be used to monitor expression of the protein under different growth