The ChrA-ChrS and HrrA-HrrS Signal Transduction Systems are Required for Activation of the hmuO Promoter and Repression of the hemA Promoter in Corynebacterium diphtheriae

Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892

^* To whom correspondence should be addressed. Email: michael.schmitt{at}fda.hhs.gov.

	Abstract

Transcription of the Corynebacterium diphtheriae hmuO gene, which encodes a heme oxygenase involved in heme iron utilization, is activated in a heme or hemoglobin-dependent manner in part by the two-component system, ChrA-ChrS. Mutations in either chrA or chrS resulted in a marked reduction in hemoglobin-dependent activation at the hmuO promoter in C. diphtheriae; however, it was observed that significant levels of hemoglobin-dependent expression were maintained in the mutants, suggesting that an additional activator is involved in regulation. A BLAST search of the C. diphtheriae genome sequence revealed a second two-component system, encoded by dip2268 and dip2267, that shares similarity with ChrS and ChrA, respectively: we have designated these genes hrrS (dip2268) and hrrA (dip2267). Analysis of hmuO promoter expression demonstrated that hemoglobin-dependent activity was fully abolished in strains that were deleted for both the chrA-chrS and hrrA-hrrS two-component systems. Similarly, deletion of the sensor kinase genes, chrS and hrrS, or the genes encoding both of the response regulators, chrA and hrrA, also eliminated hemoglobin-dependent activation at the hmuO promoter. We also show that the ChrA-ChrS and HrrA-HrrS regulators are involved in the hemoglobin-dependent repression of the promoter upstream of hemA, which encodes a heme biosynthetic enzyme. Evidence for cross-talk between the ChrA-ChrS and HrrA-HrrS systems is presented. In conclusion, these findings demonstrate that the ChrA-ChrS and HrrA-HrrS regulatory systems are critical for full hemoglobin-dependent activation at the hmuO promoter, and also suggest that these two-component systems are involved in a complex mechanism in the regulation of heme homeostasis in C. diphtheriae.