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.

Figure 1.

Phylogeny of the 176 representative phase-variable mod genes Sequences were aligned using Muscle (28), and phylogeny analyzed by RAxML (29). Groups highlighted with colored circles are new mod gene groups discussed in the main text, with the bacterial genera or strain, and the SSR unit, also stated. Groups with colored text represent currently described phase-variable mod genes/mod gene groups, e.g.modA, modB, etc., with the SSR unit also stated. The phase-variable mod gene from STEC strain DG131/3 is highlighted with a *; the phase-variable mod gene from S. suis strain T15 is highlighted with a # in the pentanucleotide Gram +ve (2) group.

Figure 2.

Phase-variable mod gene present in a subset of Shiga Toxin producing Escherichia coli. (A) schematic representation of the phase-variable mod gene from STEC, with location of the CAGCGAC[n] repeat tract shown, and probable location of the central TRD (red), and location of PCR primers used for fragment length analysis of the CAGCGAC[n] repeat tract. The green hexagon on the forward primer depicts a 6-Fluoresceine (FAM) fluorescent label to allow analysis of PCR products using the GeneScan system (Applied Biosystems International); (B) alignment of representative phase-variable mod genes from five STEC strains showing variable repeat tract length contained within the conserved genomic sequence. SSR tract is highlighted in red, with actual number of CAGCGAC repeats for each strain in red. Putative start codons are highlighted in green. The start codon immediately upstream of the CAGCGAC[n] repeat tract contains a putative Shine-Dalgarno ribosome binding site immediately upstream (GAGGGAA) and (C) fragment length analysis of the mod repeat tract from three strains of STEC showing variable repeat tract lengths are present within the population of these individual strains.

Figure 3.

A comparison of the frequency of occurrence of SSR tracts containing different repeat unit lengths. All phase-variable repeat tracts from the 662 individual mod gene set (Supplementary Table S4) were analyzed for the composition of the SSR unit (one to nine nucleotide long repeating units) and this plotted against the frequency by which tracts containing those repeat units occur.

Figure 4.

Summary of a selection of representative phase-variable mod genes found in a variety of bacterial species. Individual mod genes are represented by colored arrows, with different colors indicating different, distinct mod genes/mod gene groups. The location of the repeat tract is shown by a grey box underscored with the repeating unit found in these tracts if just a single example is present. An underscore [n] indicates that multiple examples of this mod gene are present (e.g. there are 21 different alleles in the modA group, 7 alleles in the modB group), with a range of repeat tract lengths present in these examples.