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

Abstract

Periodontal disease is a common chronic inflammatory disease driven by the sub-gingival microbiota. Porphyromonas gingivalis is an important bacterium in the development of the disease that expresses a variety of virulence determinants. The environmental concentration of hemin, an essential nutrient of this organism, is a global regulator of virulence in P. gingivalis: high levels of hemin lead to increased expression of several virulence determinants. However, the mechanism through which hemin influences bacterial gene expression is poorly understood. Bacterial epigenetic processes, primarily DNA methylation, have 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 N6-methyladenine (6mA) and 5-methylcytosine (5mC) base pair modifications. Differential expression and differential 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 culture. 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 Fe-S and 4FE-4S cluster binding, and metal cluster binding. Hemin-dependent differentially methylated signals were observed for both 6mA and 5mC, with 49 and 47 signals, respectively. Coordinated genome-wide differential expression and methylation effects were observed in 6 genes, a Ppx/GppA family phosphatase, a lactate utilization protein, 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.