Phasevarions of Bacterial Pathogens: Methylomics Sheds New Light on Old Enemies

Abstract

A wide variety of bacterial pathogens express phase-variable DNA methyltransferases that control expression of multiple genes via epigenetic mechanisms. These randomly switching regulons - phasevarions - regulate genes involved in pathogenesis, host adaptation, and antibiotic resistance. Individual phase-variable genes can be identified in silico as they contain easily recognized features such as simple sequence repeats (SSRs) or inverted repeats (IRs) that mediate the random switching of expression. Conversely, phasevarion-controlled genes do not contain any easily identifiable features. The study of DNA methyltransferase specificity using Single-Molecule, Real-Time (SMRT) sequencing and methylome analysis has rapidly advanced the analysis of phasevarions by allowing methylomics to be combined with whole-transcriptome/proteome analysis to comprehensively characterize these systems in a number of important bacterial pathogens.

Keywords: DNA methyltransferase; SMRT sequencing; methylome analysis; phase variation; phasevarion.

Figure 1. An illustration of the ways methyltransferase loci (R-M) phase-vary

Type I R-M systems can produce multiple methyltransferase variants via shuffling of variable, duplicated hsdS loci through the presence of inverted repeats (IRs) in the ORF of these hsdS genes. Type I loci can also generate multiple HsdS proteins through variation in simple sequence repeats (SSR). Variation in SSR length leads to ON/OFF switching of all three types of R-M loci.

Figure 2. Determination of phase-variable methyltransferase specificity using SMRT sequencing/methylome analysis

By using genomic DNA preparations from paired isolates that express the methyltransferase (ON variant) or do not express the methyltransferase (OFF variant or a knock-out mutant), it is possible to determine the specificity of the methyltransferase under investigation. Paired genomic DNA samples are subjected to SMRT sequencing, the resulting methylomes are compared, and the phase-variable methyltransferase specificity is identified as the motif that is absent from the OFF variant/knock-out. This example is actual SMRT data generated to decipher ModA10 specificity in NTHi [9].

Figure 3. Type III mod genes have evolved phase-variable expression multiple times

There are currently five well characterized phase-variable type III mod genes, all of which control phasevarions – modA, modB, modD, modH, and modM. Each of these mod genes is distinct, with low sequence homology between each class of mod gene. Within each class of mod gene there are multiple allelic variants, which are highly conserved except for their central DNA recognition domain (DRD). Diversity of the DRD means that each mod allele methylates a different DNA sequence, and therefore controls a different phasevarion.