A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions

Abstract

Many bacteria utilize simple DNA sequence repeats as a mechanism to randomly switch genes on and off. This process is called phase variation. Several phase-variable N6-adenine DNA-methyltransferases from Type III restriction-modification systems have been reported in bacterial pathogens. Random switching of DNA methyltransferases changes the global DNA methylation pattern, leading to changes in gene expression. These epigenetic regulatory systems are called phasevarions — phase-variable regulons. The extent of these phase-variable genes in the bacterial kingdom is unknown. Here, we interrogated a database of restriction-modification systems, REBASE, by searching for all simple DNA sequence repeats in mod genes that encode Type III N6-adenine DNA-methyltransferases. We report that 17.4% of Type III mod genes (662/3805) contain simple sequence repeats. Of these, only one-fifth have been previously identified. The newly discovered examples are widely distributed and include many examples in opportunistic pathogens as well as in environmental species. In many cases, multiple phasevarions exist in one genome, with examples of up to 4 independent phasevarions in some species. We found several new types of phase-variable mod genes, including the first example of a phase-variable methyltransferase in pathogenic Escherichia coli. Phasevarions are a common epigenetic regulation contingency strategy used by both pathogenic and non-pathogenic bacteria.

Systematic analysis of REBASE identifies numerous Type I restriction-modification systems that contain duplicated, variablehsdSspecificity genes that randomly switch methyltransferase specificity by recombination

“…Our systematic analysis of REBASE identified Type I loci containing multiple hsdS genes where we detect IRs in a range of commensal organisms such as Bacteroides fragilis and multiple Ruminococcus species, in environmental bacterial species such as Leuconostoc mesenteroides , and in a number of Lactobacillus species that are important to the biotechnology and food production imdustries (Supplementary Data 3). This reflects our previous studies where we observed simple sequence repeats that mediate phase-variation in multiple Type I (38) and Type III methyltransferase genes (9) present in a variety of commensal and environmental organisms. One obvious reason for generating diversity in methyltransferase specificity is that it will increase resistance to bacteriophage.…”

“…We cloned and over-expressed two hsdS alleles, alleles A and B, of the Type I inverting system that we found in S. suis (26) in order to solve the methyltransferase specificity of the Type I methyltransferases containing these HsdS proteins. We have used this approach extensively with Type III mod genes in order to solve specificity (5, 9), with the same site observed using the native protein using genomic DNA from the actual species and the over-expressed protein in E. coli (26). We only expressed HsdS alleles A and B as we do not observe any strains of S. suis with annotated genomes where either allele C or allele D (Figure 3B) is present in the hsdS expressed locus immediately downstream of the hsdM (26).…”

“…Many of the veterinary pathogens that we show contain inverting Type I loci also contain separate, distinct Type III or Type I R-M systems that are capable of phase-varying via changes in locus located simple sequence repeats. These species include Actinobacillus pleuropneumoniae, Mannheimia haemolytica, Streptococcus suis, Haemophilus (Glasserella) parasuis , and multiple Mycoplasma species (9, 38). This means all these veterinary pathogens have evolved phase-variation of both Type I and Type III methyltransferases, and in the case of Type I systems, by both SSR tract length changes (38) and by recombination between variable hsdS genes containing IRs (this study).…”

“…Several bacterial pathogens also contain well characterised cytoplasmic N 6 -adenine DNA methyltransferases, that are part of restriction-modification (R-M) systems, that exhibit phase-variable expression. We recently characterised the distribution of SSR tracts in Type III mod genes and Type I hsdS, hsdM , and hsdR genes in the REBASE database of restriction-modification (R-M) systems, and demonstrated that 17.4% of all Type III mod genes (9), and 10% of all Type I R-M systems contain SSRs that are capable of undergoing phase-variable expression. Phase variation of methyltransferase expression leads to genome-wide methylation differences, which can result in differential regulation of multiple genes in systems known as phasevarions ( phase-vari able regul on ).…”

Phase-variable bacterial loci: how bacteria gamble to maximise fitness in changing environments

“…In these systems, the methyltransferase (Mod) phase-varies between two states (ON or OFF) by variation in the number of SSRs in the encoding mod gene [98]. A recent survey of all Type III methyltransferases in REBASE showed that nearly 20% of Type III mod genes contain SSRs, are therefore able to phase-vary, and potentially able to control a phasevarion [99]. mod genes are highly conserved (>95% DNA sequence identity) in their 5 0 and 3 0 regions, but contain a highly divergent central domain, the TRD (for Target Recognition Domain).…”