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Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA - PubMed

Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA

Julia Frunzke et al. J Bacteriol. 2011 Mar.

Abstract

The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed that C. glutamicum is able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of an hrrA deletion mutant and C. glutamicum wild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely, ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa(3) oxidase and three subunits of the cytochrome bc(1) complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with an hrrA deletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.

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Figures

FIG. 1.
FIG. 1.

Growth of the C. glutamicum ATCC 13032 wild-type (WT) strain and Δhmu, ΔhrrA, and ΔhmuO deletion mutants on agar plates (A and B) or in liquid culture (C and D). For panels A and B, serial dilutions of the indicated strains were spotted on CGXII minimal medium plates containing either 2.5 μM FeSO4 (A) or 2.5 μM hemin (B) as the iron source. The plates were incubated at 30°C for 48 h. For panels C and D, the C. glutamicum wild-type strain (filled squares) and ΔhrrA (triangles), ΔhmuO (diamonds), and Δhmu (circles) mutants were cultivated in CGXII minimal medium with 4% (wt/vol) glucose and either 2.5 μM FeSO4 (C) or 2.5 μM hemin (D) as the iron source. When CGXII minimal medium without an iron source was inoculated with wild-type cells, growth stopped at an OD600 of about 3 (data not shown).

FIG. 2.
FIG. 2.

Autophosphorylation of the kinase domain MBP-HrrSΔ1-248 and phosphoryl group transfer to the response regulator HrrA. Purified MBP-HrrSΔ1-248 (12 μM final concentration) was incubated with [γ-33P]ATP for 20 min (A). Then the purified response regulators HrrA was added (12 μM final concentration), resulting in a 2-fold dilution of MBP-HrrSΔ1-248 and the ATP. The samples were incubated at room temperature for a further 60 min. At the indicated time points, samples were taken, mixed with SDS loading buffer, and stored on ice (see Materials and Methods). Finally, these samples were separated by SDS-PAGE and the dried gels were analyzed with a phosphorimager.

FIG. 3.
FIG. 3.

Identification of direct target genes of HrrA in C. glutamicum. (A) DNA fragments (500 bp) covering the promoter region of hmuO, ctaE, cgtS8, hemE, hemH, and hemA were incubated without or with a 5, 10, and 25 molar excess of (partially) phosphorylated HrrA protein (0 to 380 nM), as described in Materials and Methods. After incubation, samples were separated on a 10% nondenaturating polyacrylamide gel and stained with Sybr green I. DNA fragments covering the promoter region of cytP or pck served as a negative control. (B) DNA fragments containing the promoter region of a gene postulated to be activated by HrrA (ctaE) and one repressed by HrrA (hemH) were incubated with equal amounts of unphosphorylated HrrA (upper gels) or HrrA that had been phosphorylated by preincubation with MBP-HrrSΔ1-248 (HrrS-K) and ATP. These examples clearly demonstrate increased binding affinity of the phosphorylated response regulator to its target promoters.

FIG. 4.
FIG. 4.

Model for the regulation of heme homeostasis by the two-component system HrrSA in C. glutamicum. Under conditions of sufficient iron supply, the global iron regulator DtxR represses expression of the response regulator hrrA, heme uptake systems (htaA and hmuTUV), and heme oxygenase (hmuO), an enzyme involved in the release of heme iron. Under iron limitation, expression of hrrA increases. In this study, we could show that the two-component system HrrSA of C. glutamicum is involved in the heme-dependent activation of hmuO and genes encoding heme-containing protein complexes of the respiratory chain. Furthermore, HrrA directly represses transcription of genes coding for heme biosynthesis enzymes (for operon prediction, see

http://coryneregnet.cebitec.uni-bielefeld.de

). The signal sensed by the sensor kinase HrrS is not yet known, but heme itself or a heme-related metabolite is likely. Not included in this model is the postulated repression of the CgtSR8 two-component system by HrrSA, which is currently under investigation and might disclose a further level of complexity in this regulatory network.

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