Regulation of One-component Proteins by HaeSR in Porphyromonas gingivalis

Objectives: The black-pigmented, oral anaerobe Porphyromonas gingivalis (Pg) is associated with chronic adult periodontitis.  In the diseased gingival sulcus, Pg acquires iron or hemin from hemoglobin released from erythrocytes that are lysed by proteases and hemagglutinins.  A two-component (TCS) signal transduction system, HaeSR, has been shown to regulate the expression of some of these proteases as well as other iron acquisition genes.  Pg has four TCS and 37 one-component (OC) systems.  The output domain for these systems is typically a DNA-binding domain that binds to specific DNA sequences and alters gene. Our goal is to demonstrate how HaeR binds to several OC genes and influences their expression. 

Methods: Using an anti-HaeR antibody, we pulled down DNA bound by HaeR from Pg grown in hemin-deplete, -limited, or –replete media.  After removal of the proteins, we barcoded the DNA and sequenced the ChIP library on an Illumina HiSeq.  We used electromobility shift assays (EMSA) to validate the binding of HaeR to sequences of the OC genes.  The expression of the OC genes was analyzed in haeR- and haeR+ strains using quantitative real-time PCR (qPCR). 

Results: After the analysis of the ChIP-seq sequencing results, HaeR binds to 12 of the 37 one-component (OC) proteins in Pg.  These results are being validated by an in vitro binding assay (EMSA).  Preliminary data indicates that under hemin-deplete conditions, relative expression of OC genes PGN_0537, PGN_1224, and PGN_1300 is down in the haeR⁺ strain compared to an haeR ^- mutant. 

Conclusions: We have been able to show, through high-throughput sequencing, that HaeR can bind to the coding sequence of genes, specifically OC genes.  Furthermore, the presence HaeR has a negative effect on the expression of several of OC genes in response to the concentration of hemin supplied to Pg during growth.