Dale_Molecular Genetics of Bacteria 4th ed

7.4.1 Variation mediated by simple DNA inversion

One of the simplest phase variation systems is that which controls the expression of type 1 fimbriae in E. coli. These are external filamentous appendages which enable adherence to host cells. The bacteria switch between expression and nonexpression of this structure at a significant frequency such that any culture of the strain, although predominantly in one phase, will contain some cells in the opposite phase. This ensures that, if the host mounts an immune response to the fimbriae, at least some cells survive – or conversely that if the structure is required to initiate infection then at least some cells are able to do this. The promoter for the fimA gene, which encodes the fimbrial structural subunit, is located upstream of fimA in a 314-bp region of DNA which is bordered by two 9-bp inverted repeats (Figure 7.15). In the ‘ON’ position the promoter is directed towards fimA and expression of this gene occurs. Two other genes in this system encode integrases, FimE and FimB, which can act upon the inverted repeats to flip or invert this DNA region. If this occurs then the promoter is no longer directed towards fimA and expression of this gene no longer occurs. The inversion of this sequence is carefully controlled and at 378C, a temperature appropriate for growth in humans, the ON state is favoured. Nevertheless, because of the inversion mechanism, there will always be a small population of cells that do not produce fimbriae even at this temperature.

Figure 7.15 Phase variation of type I fimbriae expression in E. coli. fimA codes for the structural subunit of the fimbriae and is expressed from a promoter located on an invertible DNA region. In the first phase expression is on, but when the control region is inverted the promoter faces in the wrong direction and expression is off. The products of fimB and fimE carry out the inversion

Whilst this system results in the simple ‘ON/OFF’ expression of a gene, other more complicated systems of phase variation are present in bacteria. For example, most species of Salmonella are capable of producing two different, and antigenically distinct, types of flagellum (H-antigens) in a phase-variable manner. Whilst this mechanism is controlled by the inversion of a DNA region, as is the E. coli fim system, the result of the inversion is more complicated. Expression of the H2 flagella is mediated by a promoter that is located in a 996-bp region of DNA bounded by two 14-bp inverted repeats (Figure 7.16). In the H2 phase, this promoter drives the expression of the H2 flagellin gene and also another gene called rh1, which encodes a repressor that inhibits transcription of the alternative flagellin gene H1. So, in this orientation, H2 flagellin expression is promoted whilst that of H1 is repressed. Inversion of the region containing the promoter is mediated by a site-specific recombinase (Hin, standing for H inversion) which is encoded within the invertible region and which acts upon the inverted repeats. When inversion occurs, the promoter faces in the opposite direction and consequently the H2 flagellin gene is no longer expressed. Since the rh1 repressor is also no longer produced, the H1 flagellin is expressed instead.

7.4.2 Variation mediated by nested DNA inversion

Campylobacter fetus is an economically important pathogen of farm animals. One of its defences against the host response is a surface layer (the S-layer) of a protein called SapA. Although this renders cells resistant to serum killing, it is also antigenic and so provides a target for antibody attack. C. fetus is able to cope with this problem because it possesses up to nine different sapA genes that encode antigenically-distinct proteins. Each cell expresses only one of these genes, as the others lack a promoter and are therefore silent. However, rearrangement of the DNA can move the promoter to a site adjacent to a different sapA gene, thus allowing a switch of antigen expression. It can be seen from Figure 7.17 that the promoter is found between two sapA genes which are in different orientations. Looping of the chromosome allows recombination between conserved regions at the 50 end of each gene, thus delivering the promoter to a different sapA gene by inversion of the promoter-containing region. As can be seen from the figure, if the inverted region contains one or more sapA genes in addition to the promoter, then the promoter can be moved to any of the previously silent genes. This switch of gene expression results in antigenic variation that allows the bacterium to escape from the immune response.

7.4.3 Antigenic variation in the gonococcus

The Gram-negative bacterium Neisseria gonorrhoeae, familiarly known as the gonococcus, which is the causative agent of gonorrhoea, also evades the host

(a) Expression of phase 2 antigen

Figure 7.16 Salmonella phase change by inversion of a control element. Salmonella can express two types of flagella (H antigens). (a) In phase 2 the H2 gene is expressed and the H1 gene is repressed by the product of the gene rh1. (b) Inversion of the control region switches off both H2 and rh1 allowing production of phase 1 flagella

immune response by antigenic variation. This bacterium produces pili (or fimbriae) which are thin, proteinaceous structures that protrude from the cell surface. Pili are important virulence determinants since they enable the bacterium to attach to the mucosal surface during the initial stages of infection. Gonococcal pili are polymers of a polypeptide (pilin) of Mr about 20 kDa. The 50 or so amino

Figure 7.17 Antigenic variation in Campylobacter fetus. C. fetus has a number of different versions of the sapA gene, only one of which is expressed at a time, as the others lack a promoter. Recombination between the conserved regions at the 50 end of inverted copies of sapA causes inversion of the control region, putting the promoter adjacent to a different copy of sapA. , conserved region

acids at the N-terminus of pilin are the same in all pili, but the remainder of the molecule (about 100 amino acids) varies considerably.

As with the C. fetus sapA system described above, the gonococcus contains a number of pil genes, but only one is expressed at a time. However the mechanism is different from that of sapA. A simplified model is presented in Figure 7.18. Silent copies of the pilin genes are maintained at a locus known as pilS, while one pil gene is found at an expression locus, pilE. The switch in gene expression that causes antigenic variation arises by replacement of the pil gene at the

Figure 7.18 Antigenic variation of gonococcal pili. The pilS locus contains several different copies of the gene, all lacking a promoter; hence they are not expressed. Antigenic change occurs by replacement of part of the expressed gene (at pilE) with a copy of the equivalent part of a previously silent gene. This is a highly simplified representation of part of a complex system

expression locus with a copy of a different pil gene, an event known as gene conversion.

The variation arising from this system is actually much more extensive than simply switching expression between alternative genes. Since the gene conversion may involve not a whole gene but only part of one, the newly expressed pil gene will include sequences from two different copies. It has been calculated that this can give rise to 107 antigenically-distinct varieties of the pilus. In addition, since N. gonorrhoeae is naturally transformable (see Chapter 6), transfer between cells of DNA containing pil genes adds to the versatility of this organism in evading the immune response.

7.4.4 Phase variation by slipped strand mispairing

Runs of a single nucleotide, e.g. AAAAAAAAAAAA (homopolymeric tracts), or repeated units of more than one nucleotide, called multimeric repeats (e.g. ATATATATATATAT or GCCGCCGCCGCCGCC), provide another mechanism for phase variation through errors during DNA replication which result in the loss or gain of one or more of the repeat units. This process is called slipped strandmispairing (Figure 7.19). When the chromosome replicates, whilst in many cases the sequence remains unchanged, a small population of cells arises which have a different number of repeat units. This can affect gene expression at either the translational or transcriptional level. If the repeat is within a coding sequence, the reading frame of the gene will be altered, leading to premature termination of translation. This mechanism of phase variation controls the expression of many

RNA polymerase

Expressed

Methylated

sites

Figure 7.20 Antigen 43 phase variation by differential DNA methylation. OxyR can block transcription of the agn43 gene. However if the three GATC sequences within the OxyR binding site are methylated, OxyR cannot bind and agn43 will be expressed

spacing is optimal for RNA polymerase recognition and expression is fully on. It should be noted that this form of phase variation is very unusual in that it not only gives ON or OFF regulation but also exhibits control over the amount of protein produced.

8

Genetic Modification:

Exploiting the Potential of Bacteria

The term ‘genetic modification’ has become associated specifically with in vitro genetic manipulation technology (‘gene cloning’), but it should be considered alongside older, in vivo, methods of altering the genetic composition of bacteria. In this chapter, we will review the methods available for producing useful bacterial strains. These include enhanced formation of natural products such as antibiotics, making non-bacterial products such as human growth hormone and the development of vaccines against infectious diseases. In Chapter 9 the role of genetics in developing our understanding of the biochemical and physiological processes that are at work within the cell will be discussed, while Chapter 10 continues the story of genome sequencing and bioinformatics. There is a considerable degree of interaction between the concepts and techniques in these three chapters and the distribution of material between them is to some extent arbitrary.

8.1 Strain development

Natural evolution is based on three processes: the generation of variants, the selection of variants with desirable properties and the re-assortment of characteristics between strains by genetic exchange. The application of strain improvement programmes to commercially useful microorganisms, mostly for increased product formation, has mainly involved the first two processes.

8.1.1 Generation of variation

In earlier chapters, it has been shown that variation occurs naturally but at a low frequency. For practical purposes the frequency needs to be increased by using