Acknowledgements: We thank Amanda Nouwens and Peter Josh in SCMB MS facility, University of Queensland for conducting SWATH-MS proteomic analysis. We thank Allison Banse and Eric Johnson at SNPsaurus (University of Oregon, USA) for conducting PacBio SMRT sequencing and methylome analysis. We thank the Australian Genome Research Facility (AGRF, Brisbane) and Nicole Hogg at the Griffith University DNA Sequencing Facility (GUDSF) for fragment length separation and analysis.Funder: Wellcome Trust; DOI: https://doi.org/10.13039/100004440Funder: John Atack strategic fundsFunder: Griffith University; DOI: https://doi.org/10.13039/501100001791Actinobacillus 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
