The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions

Abstract

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wildtype ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

“…The highest binding affinity of purified ChrA was observed in the presence of the phosphate donor phosphoramidate, indicating that ChrA follows the classical model and is active in its phosphorylated state (Gao et al, 2007;Stock et al, 2000). This is consistent with recent studies where the phosphotransfer from the soluble kinase domain of ChrS to the response regulator ChrA was described for the C. diphtheriae ChrSA system (Burgos & Schmitt, 2012). The autophosphorylation of ChrS was shown to occur in the presence of haemin in purified E. coli proteoliposomes, indicating a direct interaction of ChrS with haem (Ito et al, 2009).…”

“…anthracis (Bibb & Schmitt, 2010;Burgos & Schmitt, 2012;Stauff & Skaar, 2009b). However, our data reveal differences in the network composition of C. glutamicum ChrSA and HrrSA in comparison to what is known for the C. diphtheriae systems.…”

The ChrSA and HrrSA Two-Component Systems Are Required for Transcriptional Regulation of the hemA Promoter in Corynebacterium diphtheriae

“…The transcriptional expression of the hmuO promoter is under dual regulation; in high-iron environments, DtxR represses transcription of hmuO, while the presence of heme or Hb activates transcription (14). Heme-dependent activation of hmuO is mediated by the ChrSA and HrrSA two-component signal transduction systems (TCSs) (14,15). Two-component signal transduction systems are ubiquitous in bacteria and are critical for enabling bacteria to sense and rapidly adapt to environmental stimuli through modulation of gene expression (16).…”

Structure of the response regulator ChrA in the haem-sensing two-component system ofCorynebacterium diphtheriae

“…Sequence analysis of ChrA shows that it belongs to the NarL/FixL subfamily of RRs consisting of N-terminal regulatory and C-terminal DNA-binding domains (Burgos & Schmitt, 2012). Several full-length crystal structures of NarL/ FixJ family members have been determined and showed conserved folds in each domain, but with various relative orientations of the two domains (Baikalov et al, 1996;Leonard et al, 2013;Milani et al, 2005;Park et al, 2013).…”

“…ChrS is a sensor HK (Ito et al, 2009) and ChrA is a cognate RR. The activated ChrA specifically binds to either the hmuO or hrtAB promoter regions to promote the transcription of the genes that encode the haem oxygenase and the ABC-type haem exporter, respectively (Bibb et al, 2005;Burgos & Schmitt, 2012).…”