Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis

Periodontal disease is a common chronic inflammatory disease. Porphyromonas gingivalis is an important bacterium in the development of the disease and expresses a variety of virulence determinants. Hemin (iron [III] protopotphyrin IX), an essential nutrient of this organism, whose concentration increases with increasing inflammation, is a global regulator of virulence in P. gingivalis: high hemin levels increase expression of several virulence determinants. However, the mechanism through which hemin influences bacterial gene expression is poorly understood. Bacterial DNA methylation has the potential to fulfil this mechanistic role. Here, we characterised the methylome of P. gingivalis, and compared its variation to transcriptomic changes in response to changes in hemin concentration. Gene expression and DNA methylation profiling of P. gingivalis W50 was performed, following continuous culture in chemostats with excess or limited hemin, using Illumina RNA-Seq and Nanopore DNA sequencing. DNA methylation quantification was carried out for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC) base pair modifications. Differential expression and methylation in response to excess hemin availability are presented after multiple testing correction (FDR 5%). In excess hemin there were 161 over- and 268 under-expressed genes compared to limited hemin. Genes under-expressed in excess hemin were involved in iron recruitment (the hemophore HmuY) and transport (TonB-dependent receptors), and those over-expressed were involved in iron-sulphur cluster binding. Hemin-dependent differentially methylation was observed for the Dam ‘GATC’ motif and all-context 6mA and 5mC, with 36, 49 and 47 signals, respectively. Coordinated genome-wide differential expression and methylation effects were observed in 6 genes encoding a Ppx/GppA family phosphatase, a lactate utilization protein, a 4-alpha-glucanotransferase, two ABC transporter proteins, and a hypothetical protein HMPREF1322_RS00730. The findings indicate that altered genome methylation occurs in response to the availability of hemin and give insights into the molecular mechanisms of regulation of virulence in this bacterium. Author Summary DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the gene regulatory processes underlying these effects remain unknown. To this end, we profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin conditions that reflect conditions associated with health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam ‘GATC’ motif and both all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC) in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.