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

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. Mutation of either the chrA or the chrS gene resulted in a marked reduction of 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 from which both the chrA-chrS and the hrrA-hrrS two-component systems were deleted. 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 regulators ChrA-ChrS and HrrA-HrrS are involved in the hemoglobin-dependent repression of the promoter upstream of hemA, which encodes a heme biosynthesis 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 the complex mechanism of the regulation of heme homeostasis in C. diphtheriae.

FIG. 1.

Schematic of hrrSA genes in C7(−)wt (wild-type) and mutant strains. The sensor kinase (hrrS) and response regulator (hrrA) genes of the HrrA-HrrS two-component system are flanked by two ORFs, DIP2269 and DIP2266. The regions deleted from mutant strains C7hrrSΔ and C7hrrSAΔ are indicated in black. Strain C7hrrA⁻ is a vector integration mutant with two stop codons (black diamonds) inserted into the hrrA coding region. The putative promoters upstream of hrrS and hrrA are shown in the C7(−) genome as arrows labeled P_SA and P_A, respectively.

FIG. 2.

Heme iron utilization defect of the double mutant chrSA hrrSA. Wild-type C7(−)wt and mutants C7chrSΔ, C7hrrSΔ, C7chrSΔ/hrrSΔ, and C7hmuOΔ were grown in semidefined low-iron medium (mPGT + 3.6 μg/ml EDDA) with 10 μg/ml (A) or 50 μg/ml hemoglobin (B). The OD₆₀₀ was measured after overnight incubation (18 to 24 h). The data shown are averages of three experiments, and the error bars represent standard deviations.

FIG. 3.

Mutant strain C7hrrSΔ requires a heme source for optimal growth. (A) Wild type C7(−)wt (black symbols with solid line) and mutant C7hrrSΔ (open symbol with dotted line) strains were inoculated into HIBTW alone (diamonds) or with 140 μg/ml hemoglobin (squares), and optical density was measured at the time points indicated. (B) The C7hrrSΔ heme source requirement can be complemented with a plasmid carrying hrrS (pKN-hrrS). The wild-type strain with vector alone (pKN2.6) (black symbols with solid line) and C7hrrSΔ, with either vector alone (open symbol with dotted line) or a plasmid with hrrS (pKN-hrrS) (gray symbols and line), were inoculated into HIBTW medium alone (diamonds) or with 140 μg/ml hemoglobin (squares), and optical density was measured at the time points indicated. The data points shown are the averages of three experiments.

FIG. 4.

hrrA is expressed from a hemoglobin-repressed promoter. Expression from the hrrS (phrrS-PO) and hrrA (phrrA-PO) promoters was examined in medium without (open bars) and with 140 μg/ml hemoglobin (crosshatched bars) in C7wt and in C7hrrA⁻. Expression is reported as β-galactosidase activity in Miller units (29). Results shown are the averages of three experiments with the standard deviations indicated by the error bars.

FIG. 5.

Proposed mechanism for hmuO and hemA promoter regulation by ChrA-ChrS and HrrA-HrrS (see Discussion for a detailed description). (A and B) Regulation of the hmuO promoter. LacZ assay results from Table 3 are shown. In the absence of a heme source [(minus) heme], the ChrA-ChrS and HrrA-HrrS systems are inactive at the hmuO promoter (A). In high-iron (+Fe) medium, hmuO transcription is repressed by DtxR, while in low-iron (−Fe) medium, hmuO is expressed at a low level. In the presence of a heme source (+ heme), the ChrS and HrrS sensor kinases phosphorylate (P) and activate the response regulators ChrA and HrrA, respectively (B). In low-iron medium, expression of hmuO is fully activated by direct binding of ChrA and HrrA upstream of the promoter. In high-iron medium, DtxR-mediated repression of hmuO appears to be partially reversed by the ChrA-ChrS system. (C and D) Regulation of the hemA promoter. LacZ data from Table 5 are shown for strains C7wt, C7chrSAΔ, C7hrrSAΔ, and C7chrSAΔ/hrrSAΔ. Expression in the absence of heme (C) is partially repressed by HrrA, since deletion of hrrSA results in an increase in expression. In the presence of a heme source (D), both the ChrA-ChrS and the HrrA-HrrS systems can repress expression, since deletion of both systems abolishes heme-dependent repression. However, only one system is required since a mutation in either system alone has a minimal effect on expression.