Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions

Abstract

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Figure 1.

An illustration of the mechanisms of phase variation that occur in bacterial DNA methyltransferases. (A) Type III mod genes can contain variable length simple DNA sequence repeat (SSR) tracts in their open-reading frame. During DNA replication, polymerase slippage can occur when replicating these tracts, leading to loss or gain of repeat units. This variation in length results in the gene being in-frame downstream of the SSR tract, and expressed (ON), or a change in the reading frame leading to a frame-shift, a premature stop codon, and no expression of the encoded Mod protein (OFF). This means DNA is methylated (Mod ON) or not (Mod OFF). (B) Type I R-M systems that phase-vary often do so by shuffling between multiple variable hsdS specificity genes at the Type I R-M locus via recombination between inverted repeats (IRs) encoded in each of these multiple variable hsdS genes. This results in multiple different HsdS specificity proteins being expressed in a bacterial population, meaning multiple different DNA sequences are methylated in the bacterial population by the M₂S trimer formed, dependent on the HsdS variant present in each individual bacterial cell of the population (four variants in the example in the illustration).

Figure 2.

The distribution of phase-variable Type I and Type III methyltransferases in A. pleuropneumoniae. A neighbour joining tree of A. pleuropneumoniae showing the genetic distance between isolates based on all sites in a core genome. Serovars are represented by coloured clades and the presence of phase variable Type I and Type III methyltransferases are indicated by different shapes. The scale bar indicates the genetic distance (number of substitutions per site).

Figure 3.

Demonstration of phase variation of the Type I R-M system in A. pleuropneumoniae. (A) Illustration of the Type I R-M system containing duplicate, variable hsdS specificity loci that contain IRs. Gene shuffling between variable 5′ and 3′ TRDs produces four unique hsdS genes at the hsdS expressed locus downstream of the hsdM gene, resulting in the expression of four unique HsdS allelic variants. (B) Alignment of the four 5′ and four 3′ TRDs present in 15 A. pleuropneumoniae strains available in REBASE (see full sequences and analysis in Supplementary Data 1). Alignments were carried out using Muscle and visualized in JalView overview feature. (C, D) Semi-quantitative RT-PCR was carried out to demonstrate the presence of mRNA for each of the four encoded hsdS alleles determined to be encoded in each strain, indicating that all four alleles are expressed in the population of AP76 and JL03. Expected sizes are: allele A = 587 bp, allele B = 557 bp, allele C = 492 bp, allele D = 522 bp, allele E = 626 bp, allele F = 821 bp, allele G = 717 bp. ± annotation refers to the presence (+) or absence (–) of reverse transcriptase in the reverse transcription (cDNA synthesis) reaction.

Figure 4.

Phase-variable expression of modP. (A) Illustration of the Type III modP gene present in A. pleuropneumoniae. The modP gene contains a variable number of GCACA_(n) repeats immediately downstream of the ATG start codon, and a highly variable target recognition domain (TRD) that dictates DNA sequence specificity of the encoded protein, represented by the hatched box. The green star on the forward primer depicts a 6-carboxyfluoresceine (FAM) fluorescent label which allows analysis of PCR products using GeneScan technology (Applied Biosystems International). (B) Full-length DNA sequences of modP1, modP2, modP3 and modP4 were aligned using Muscle and visualized in JalView overview feature. Each blue line represents one nucleotide. (C) Fragment length analysis PCR over the GCACA_(n) repeat tracts from enriched modP1 and modP2 strains with three consecutive SSR tract lengths in strains AP76 (modP1) and JL03 (modP2). (D) Western blot analysis using anti-ModP sera confirmed phase-variable expression of ModP, which was only present in a population of A. pleuropneumoniae enriched for 10 repeats (ModP1) or 25 repeats (ModP2). MW marker shown represents 90 kDa band (NEB color-plus).

Figure 5.

Phase-variable expression of modQ. (A) Illustration of the Type III modQ gene present in A. pleuropneumoniae. The modQ gene is present as single allelic variant (a single TRD, represented by the purple box), and like modP, also contains a variable number of GCACA_(n) repeats immediately downstream of the ATG start codon. The green star on the forward primer depicts a 6-Carboxyfluoresceine (FAM) fluorescent label. (B) Fragment length analysis PCR over the GCACA_(n) repeat tract from enriched modQ strains with three consecutive SSR tract lengths in strain JL03. (C) Western blot analysis using anti-ModQ sera confirmed phase-variable expression of ModQ, which was only present in a population of A. pleuropneumoniae enriched for nine repeats. MW marker shown represents 90 kDa band (blue) and 72 kDa band (red) (NEB color-plus)

Figure 6.

The impact of modP and modQ phase-variation on growth rate in BHI-NAD broth (right) and biofilm formation (left) was investigated with enriched modP and modQ ON/OFF variants. (A) The ModP1 ON variant showed a higher growth rate and formed a statistically larger biofilm (determined with Student's t-test; **P ≤ 0.01) than ModP1 OFF. (B, C) Phase variation of modP2 and modQ genes had no impact on growth rate and biofilm formation (no significant difference, NSD; assessed using Student's t-test).