Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression

Abstract

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon—‘phasevarion’. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5′-CGAAT-3′. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.

Deletion of One Nucleotide within the Homonucleotide Tract Present in the hsdS Gene Alters the DNA Sequence Specificity of Type I Restriction-Modification System NgoAV

“…is SSM during replication (40). This is also the case for two type III RM systems (4,10,15), and in this study we describe such a mechanism for the NgoAV system.…”

“…The biological significance of changing the specificity of the NgoAV system remains unknown, but 916 sites are predicted for NgoAV in the N. gonorrhoeae genome, and only 340 sites would be recognized by NgoAV⌬. The genome is certainly undermethylated in NgoAV⌬-expressing bacteria, and the expression of many different genes may be affected, as has been demonstrated for type III RM systems (6,15,20,41). In addition, PV of RM systems may aid phage defense (6).…”

“…Attempts to identify functional connections between the phase variability of RM systems and other sets of genes subject to PV have been made (13). The most recently proposed mechanism was termed the "phasevarion," where switching on or off of the H. influenzae type III mod gene modulates the expression of a number of unlinked genes encoding surface and stress proteins (15). The RM systems would be part of an epigenetic mechanism of PV.…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…Pathogenic bacteria use the phase variability of gene expression to evade the host immune system. Based on sequence analysis and biochemical evidence, many type I and III enzymes of Bacteroides fragilis as well as Haemophilus, Helicobacter, Mycoplasma, and Neisseria species were found to be potentially phase variable (176)(177)(178)(179)(180)(181). The biological significance of R-M systems that exhibit phase variability is not completely understood.…”

“…It has been proposed that phase variation in these bacteria increases the diversity of R-M systems, generating up to eight different recognition subunits (176 (180,181). It was proposed that phase-variable modification systems play a role in regulating distinct set of genes, called "phase-varions" (180,181), a function independent of the defense role. Phase variability would generate different phenotypes and might confer a fitness advantage to the organism by controlling the phase-varion.…”