Phase variation is an adaptive process by which bacteria undergo frequent and reversible phenotypic changes resulting from genetic alterations in specific loci of their genomes. This helps in colonization of the host, adaptation to host environments, and evasion of immune responses. The main mechanism of phase variation involves insertion or deletion of polyG or polyC repeat tracts repeat units in ORFs during genome replication. While phase variation is typically associated with genes encoding surface antigens, several host-adapted bacterial pathogens have MTases (mod gene) associated with Type III R–M systems, which contain single tandem repeats that are prone to phase variation.
In every case studied to date, phase-variable ON/OFF switching of the Type III DNA MTase, mediated by SSRs, results in differential regulation of multiple genes. Currently, the well characterized “phase-varions” and mod genes include modA in non typeable H. influenzae (NTHi), N. meningitidis, and N. gonorrhoeae; modB in N. meningitidis and N. gonorrhoeae, modD in N. meningitides, modH in H. pylori and modM in M. catarrhalis. The list of phase-variable DNA MTases controlling phase varions is ever expanding, with many new systems characterized within the past few years. The identification of a variety of phase-variable MTase, that switch their expression via distinct mechanisms, implies that phase varions have evolved independently in different species and suggests that this type of variable epigenetic regulation provides a strong selective advantage. The random and reversible switching of phase-variable DNA MTase leads to multiple distinct phenotypes in a population that are subject to periodic selection and counter selection in different environments.
What is the biological role of these phase variable MTases? Phase variation of surface-associated proteins leads to generation of a heterogeneous population that expresses a different set of genes allowing rapid adaptation to different environmental conditions. The reversible ON/OFF switching of MTase genes may result in analogous heterogeneity in the methylation patterns. Thus, phase variation might provide subpopulations with a barrier to phage infection or represent a mechanism to regulate genetic exchange between unrelated strains.
Type III systems are also encoded by two genes, mod and res. The R-M enzyme is composed of two subunits of MTase (Mod) and a restriction subunit (Res) having an ATPase and an endonuclease domain. The dimeric Mod or the trimeric Res-Mod complex catalyzes hemi-methylation of the DNA at 4–6 bp asymmetric recognition sites. Type III REases cut DNA 25–27 bp away from the recognition site and show a strong preference for cleavage to occur when there are two copies of their asymmetric unmethylated recognition sites in an indirectly repeated, head to head orientation within a distance up to 3.5 kb. DNA cleavage occurs at one of the two sites, 25 or 26 nt downstream in the top strand and 27 or 28 nt downstream in the bottom strand. Interestingly, after cleavage, the enzyme remains bound to the still intact recognition site.
Genes encoding rglA and rglB (restricts glucoseless phage) that restrict non-glucosylated hydroxymethylcytosine in T-even phages led to the discovery of the new class of R-M systems. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction) and were classified as Type IV containing modification-dependent REases. Escherichia coli K12 strains code for atleast three REases selectively directed against DNA containing modified bases. These enzymes are encoded by mcrA, mcrBC, and mrr genes. REases that cleave DNA containing modifications such as m5C, hm5C, ghm5C, m6A, m4C and more recently phosphorothioate DNA are loosely grouped as Type IV REases. Type IV enzymes recognize modified DNA with low sequence selectivity and exhibit separation of DNA binding and cleavage into different domains on the same protein, or even into different polypeptide chains.
More than 90% of the sequenced genomes posses at least one R-M system with a direct correlation on their occurrence to the size of the genome. Helicobacter pylori, Neisseria and Campylobacter species are exceptions in that they have unusually large number of R-M systems compared to their genome size, possibly evolved to take care of additional cellular functions. Analysis of the genome sequences of more than 75 H. pylori strains identified an extraordinary number of genes (~45) with homology to R-M genes in other bacterial species. Recent advances in the application of single-molecule real-time DNA sequencing to analyse DNA methylation have revealed for the first time comprehensive pictures of the genome-wide distribution of methylation sites in various strains of H. pylori. The methylomic data published so far have not only confirmed the significant inter-strain diversity of H. pylori MTases and their DNA methylation profiles, but also identified numerous novel MTase target recognition sites. Methylomic studies hold promise for providing a deeper understanding into the roles of H. pylori MTase and R-M systems in the physiology, epigenetics and possibly also pathogenesis of this important human pathogen.