Control of Heme Homeostasis inCorynebacterium glutamicumby the Two-Component System HrrSA

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.

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

“…1.5-fold) in the DchrSA mutant. Together with the observed derepression of chrSA in a DhrrA mutant (Frunzke et al, 2011) these data hint at a cross-regulation of both systems at the level of transcription. Further genes exhibiting an altered mRNA level include a regulator of unknown function (cg3303) and the redox-sensing regulator qorR, whose DNA-binding activity was reported to be affected by oxidants (Ehira et al, 2009).…”

“…The TCS ChrSA (previously CgtSR8): sequence similarities and genomic organization In a previous study the TCS HrrSA was reported to play a central role in the control of haem homeostasis in C. glutamicum (Frunzke et al, 2011). In vitro DNA binding studies with purified HrrA protein provided evidence that the response regulator HrrA binds to the upstream promoter region of an operon encoding another TCS, chrSA (cg2201-cg2200) (Kocan et al, 2006).…”

“…The cell debris was removed by centrifugation (6900 g, 4 uC, 20 min), followed by ultracentrifugation of the cell-free extract for 1 h (150 000 g, 4 uC). ChrA was purified from the supernatant via Ni 2+ -NTA (nickel-nitriloacetic acid) affinity chromatography as described for C. glutamicum HrrA (Frunzke et al, 2011). ChrA was eluted from the column with TNI100 buffer (containing 100 mM imidazole) and analysed on a 12 % SDS-polyacrylamide gel.…”

“…In a recent study, we demonstrated that the TCS HrrSA exhibits a central function in the control of haem homeostasis and haem utilization in C. glutamicum. In the presence of haem, the response regulator HrrA directly represses haem biosynthesis genes and activates haem oxygenase (hmuO) as well as genes encoding haemcontaining components of the respiratory chain (Frunzke et al, 2011). Expression of hrrA itself underlies control by the global iron regulator DtxR, which represses transcription from the promoter P hrrA , downstream of hrrS, under conditions of sufficient iron supply (Wennerhold & Bott, 2006).…”

“…For haem utilization, C. glutamicum, as well as its pathogenic relative Corynebacterium diphtheriae, depends on a haem uptake apparatus composed of the ABC transporter HmuTUV, several cell surface haem-binding proteins (Allen & Schmitt, 2009Drazek et al, 2000;Frunzke et al, 2011) and a haem oxygenase (HmuO), which catalyses the intracellular degradation of the tetrapyrrol ring to abiliverdin, free iron (Fe 3+ ) and carbon monoxide Schmitt, 1997;Wilks & Schmitt, 1998). Acquisition of haem, however, exposes the respective organism to the toxicity associated with high levels of haem.…”