Two-component signal transduction systems provide a mechanism by which bacteria can rapidly adapt to changing environmental conditions. Environmental signals are integrated through the action of a histidine kinase and a partnered response regulator, which is often a transcription factor; thus, a common effect of the environmental stimulus is the up or down regulation of one or more genes. C. diphtheriae
encodes 11 predicted two-component signal transduction systems (10
), and until now, only the ChrA-ChrS system has been characterized. Although ChrA-ChrS was the first bacterial heme-responsive two-component system described, several heme-responsive transcriptional regulators have been reported (6
). Moreover, two additional signal transduction systems with amino acid sequence similarities to ChrA-ChrS have been described recently, CgtR11-CgtS11 in C. glutamicum
and SenR-SenS in Streptomyces reticuli
). An environmental signal involved in the activation of these systems has yet to be identified.
In this study, we have identified an additional C. diphtheriae two-component signal transduction system encoded by hrrA and hrrS, which is involved in the hemoglobin-dependent activation at the hmuO promoter and in the hemoglobin-dependent repression at putative promoters upstream of hemA and hrrA. While the contribution of HrrA-HrrS to hemoglobin activation at hmuO is severalfold lower than that of ChrA-ChrS, the HrrA-HrrS system is critical for wild-type levels of hemoglobin-activated expression under low-iron conditions.
An unusual finding from this study was that disparate results were observed among the hrrSA
mutants, which suggests that the HrrA-HrrS system does not conform to the model of the typical two-component system, such as the ChrA-ChrS system. Observations similar to what we have shown in the HrrA-HrrS system, in which a mutation in a gene encoding a sensor kinase results in a different phenotype than that from a mutant that carries a defective cognate response regulator, have been reported previously (25
). In some of these cases, the disparate characteristics are attributed to cross talk with a second two-component system, and examples of cross talk between noncognate sensor kinase-response regulator pairs have been described (14
). In the present study, evidence of cross talk was observed between the ChrA-ChrS and HrrA-HrrS systems at the hmuO
promoters. Hemoglobin-dependent activation of the hmuO
promoter was observed for C7chrAΔ/hrrSΔ, a strain with functional ChrS and HrrA proteins (Table ). Cross talk between ChrS and HrrA provides a possible explanation for the wild-type levels of hmuO
expression observed for the hrrS
mutant C7hrrSΔ (Table ); this could occur through activation of both ChrA and HrrA (via phosphorylation) by ChrS to promote wild-type levels of expression of hmuO
We also provided evidence that cross talk between the HrrA-HrrS and ChrA-ChrS two-component systems occurred at the hemA promoter, and this phenomenon may account for some of the phenotypes observed for the hrrSA mutants, although alternative explanations are possible. The physiological relevance of the cross talk reported in this study is not known, and we have no direct evidence that cross talk occurs in the wild-type strain. Nevertheless, cross-regulation between these two-component systems could facilitate or “fine tune” the control of heme homeostasis by the bacteria. The hmuO gene encodes a heme oxygenase, an enzyme that is involved in heme degradation, while the hemA operon controls heme biosynthesis; it is perhaps not surprising that these two opposing activities (i.e., heme degradation and heme synthesis) exhibit some level of coordinated regulation.
The findings in this study suggest that HrrA and ChrA function as activators at the hmuO
promoter and as repressors of hemA
expression, and examples of two-component systems possessing this type of dual function have been described previously (7
). While DNA binding sites for ChrA and HrrA have not been identified, it was previously shown that hemoglobin-dependent regulation of hmuO
requires a 50-bp sequence upstream of the promoter, a region which may harbor binding sites for ChrA and/or HrrA (42
). A previous study showed that the cloned chrA
gene is able to activate hmuO
expression in E. coli
, suggesting direct activation by ChrA at the hmuO
). However, similar studies with the cloned hrrA
gene in E. coli
failed to demonstrate activation of hmuO
expression (data not shown), which suggests that HrrA does not act directly at the hmuO
promoter, although alternative explanations are possible, such as poor expression of hrrA
or a requirement for additional activating factors or environmental signals.
A model that describes the transcriptional regulation of the hmuO and hemA promoters by the HrrA-HrrS and ChrA-ChrS systems is presented in Fig. . The model does not account for all the mutant phenotypes but rather focuses on the most significant results from this study to provide a description of the complex regulation observed at the hmuO and hemA promoters. The model predicts that in the absence of hemoglobin or any heme source, the HrrA-HrrS and ChrA-ChrS systems are inactive at the hmuO promoter (Fig. ). Under high-iron conditions in the absence of heme, hmuO transcription is fully inhibited due to DtxR-mediated repression, while in low-iron medium, hmuO is expressed only at a low level, since ChrA and HrrA are not activated (Fig. ). In the presence of a heme source, it is predicted that the detection of heme or hemoglobin (possibly at the cell surface) by the ChrS and HrrS sensor kinases results in the phosphorylation and subsequent activation of the response regulators ChrA and HrrA, respectively, such that under low-iron conditions, expression of hmuO is fully activated by ChrA and HrrA, either by direct binding of these activators upstream of the promoter (as shown in Fig. ) or through an indirect mechanism that may involve additional, but as-yet-unknown, regulatory factors. The mutant studies indicated that the ChrA-ChrS systems provides >80% of the activity under low-iron conditions in the presence of hemoglobin (Table and ). In high-iron medium containing hemoglobin, DtxR-mediated repression of hmuO appears to be partially reversed by the ChrA-ChrS system (Fig. ). HrrA-HrrS appears to have no affect on expression under these conditions, since no activity is observed in high-iron medium in the chrSA mutants (Table , HrrAS+).
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 are shown. In the absence of a heme (more ...)
Since iron regulation was not observed at the hemA promoter in the wild-type strain, the model shown in Fig. only considers hemA expression in the presence and absence of hemoglobin. This model predicts that both the HrrA-HrrS and the ChrA-ChrS systems contribute to the hemoglobin-dependent repression of hemA expression. This proposal is based on the analysis of the various mutants which shows that the deletion of either system alone had only a minimal effect on repression, while deletion of both systems abolished hemoglobin-dependent repression (Fig. and Table ). Although in the absence of hemoglobin disparate results were observed between hrrS and hrrA hrrSA mutants, the data overall suggest that the HrrA-HrrS system is involved in the repression of hemA in the absence of a heme source (Fig. ). It is unclear what role, if any, the ChrA-ChrS system has in regulating hemA expression in the absence of hemoglobin, since mutations in the chrSA genes had no effect on expression (Fig. and Table ).
The findings from this study suggest that the ChrA-ChrS and HrrA-HrrS two-component systems of C. diphtheriae regulate heme homeostasis through the activation and repression of genes involved in heme degradation and heme biosynthesis. An understanding of the mechanism for ChrA-ChrS and HrrA-HrrS activation and repression at the various promoter regions will require additional studies that focus on putative interactions among the various purified activators and repressors (DtxR) and on the identification of putative binding sites for these response regulators.