THE ROLE OF PHASE VARIABLE TYPE III RESTRICTION-MODIFICATION SYSTEMS IN GENE REGULATION

Phase variation, the high frequency reversible switching of gene expression, is a common feature of host-adapted bacterial pathogens and is generally associated with genes encoding surface factors. Phase variation results in genetically and phenotypically diverse populations, providing a strategy for rapid adaptation to changes within the host environment and evading immune responses. However, in a growing number of host-adapted pathogens, phase variation has been found to occur in genes encoding methyltransferases (mod genes) associated with type III restrictionmodification (R-M) systems. R-M systems traditionally confer protection against foreign DNA, and several roles have been proposed for phase variable R-M systems based on DNA restriction function. The existence of phase variable methyltransferases raises the possibility of further functions for R-M systems such as gene regulation. In this thesis the role of a phase variable methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae strain Rd was investigated. Microarray expression analysis comparing a wild-type strain expressing mod to a mod knockout mutant strain, revealed altered expression of 15 genes under Mod control, some of which were virulence associated. This key finding confirmed that this phase variable methyltransferase coordinates the random switching of expression of multiple genes. Phylogenetic studies were used to analyse phase variable mod genes associated with type III R-M systems in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae revealing that these organisms have two distinct mod genes - modA and modB. There are also distinct alleles of modA and modB that differ only in their DNA recognition domain. Phylogenetic analysis was also used to create an up-to-date list of potentially phase variable type III R-M systems present within other host-adapted organisms. To confirm whether phase variable methyltransferases controlled gene expression in other pathogens, the phase variable modA genes of Neisseria were studied. Mutant strains lacking the modA11, modA12 or modA13 genes were made and their phenotype analysed. Microarray analysis revealed that in all three modA alleles multiple genes were either up- or down-regulated, some of which were virulence associated. For example, in N. meningitidis (modA11), 80 genes were differentially expressed including the vaccine antigen candidates lactoferrin binding proteins A and B. Functional studies in N. gonorrhoeae confirmed that wild-type FA1090 modA13 ON and FA1090modA13::kan mutant strains have distinct phenotypes in antimicrobial resistance, a primary human cervical epithelial cell model of infection and biofilm formation. In summary, this thesis provides experimental confirmation that in three important human pathogens, H. influenzae, N. meningitidis and N. gonorrhoeae, alteration of expression of a type III DNA-methyltransferase causes switching of multiple genes. This novel genetic system has been termed the phasevarion (phase variable regulon). The wide distribution of phase variable mod genes indicates that this may be a common strategy used by host-adapted bacterial pathogens to randomly switch between distinct differentiated cell types