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The two-component system SenS–SenR from Streptomyces reticuli has been shown to influence the production of the redox regulator FurS, the mycelium-associated enzyme CpeB, which displays heme-dependent catalase and peroxidase activity as well as heme-independent manganese peroxidase activity, and the extracellular heme-binding protein HbpS. In addition, it was suggested to participate in the sensing of redox changes. In this work, the tagged cytoplasmic domain of SenS (SenSc), as well as the full-length differently tagged SenR, and corresponding mutant proteins carrying specific amino acid exchanges were purified after heterologous expression in Escherichia coli. In vitro, SenSc is autophosphorylated to SenSc∼P at the histidine residue at position 199, transfers the phosphate group to the aspartic acid residue at position 65 in SenR, and acts as a phosphatase for SenR∼P. Bandshift and footprinting assays in combination with competition and mutational analyses revealed that only unphosphorylated SenR binds to specific sites upstream of the furS–cpeB operon. Further specific sites within the regulatory region, common to the oppositely orientated senS and hbpS genes, were recognized by SenR. Upon its phosphorylation, the DNA-binding affinity of this area was enhanced. These data, together with previous in vivo studies using mutants lacking functional senS and senR, indicate that the two-component SenS–SenR system governs the transcription of the furS–cpeB operon, senS–senR and the hbpS gene. Comparative analyses reveal that only the genomes of a few actinobacteria encode two-component systems that are closely related to SenS–SenR.
One of the major signal transduction systems governing bacterial responses and adaptation to environmental changes is the two-component system (TCS). A typical TCS consists of an autophosphorylating sensor histidine kinase (SK) and a cognate response regulator (RR) . SKs detect stimuli via an extracellular input domain or intracellular signals via cytoplasmic regions, or use transmembrane regions and sometimes additional short extracellular loops for sensing . In addition to the N-terminal input domain, SKs contain a C-terminal portion representing the transmitter module, with several blocks of amino acid residues being conserved among these kinase types. Phosphorylation within a typical SK usually takes place at a conserved histidine residue; the phosphoryl group of the SK is subsequently transferred to a conserved aspartic acid residue within the receiver domain of the RR. As a result, its C-terminally located output domain has an altered DNA-binding capacity for the regulatory region of target gene(s) or operons [3,4]. The well-studied receiver domain within the nitrogen regulatory protein C − controlling the transcription of genes involved in nitrogen metabolism − has been shown to change its topology upon activation by phosphorylation . Generally, the signaling pathway includes a phosphatase that returns the RR to the nonphosphorylated state. The phosphatase can exist as an individual protein, or reside on a module, which is linked either to the RR or to the kinase. A combination of kinase and phosphatase activity ensures rapid coordination of the cell response .
Streptomycetes are Gram-positive and G + C-rich bacteria with a complex developmental life cycle. Germination of spores and subsequent elongation of germ tubes lead to a network of vegetative hyphae. In response to nutritional stress and extracellular signaling, aerial hyphae develop, in which spores mature . As soil-dwelling organisms, streptomycetes need to respond to highly variable conditions. The range of environmental stimuli to which a bacterium can respond is expected to correlate with the number of functional SKs and RRs. These are assumed to have evolved by selection pressure for different ecophysiologic properties of the different strains . The complete genome sequence of Streptomyces coelicolor A3(2) comprises 84 SK genes and 80 RR genes . The physiologic roles of only a few of them have been investigated experimentally. For instance, the AbsA1–AbsA2 system negatively regulates the production of several antibiotics [10,11], and the VanR–VanS system activates the expression of vancomycin resistance [12,13]. Phosphate control of the production of actinorhodin and undecylprodigiosin in S. lividans and S. coelicolor A3(2) is mediated by the two-component PhoR–PhoP system, which also controls the alkaline phosphatase gene (phoA) and other phoA-related genes [14,15]. To date, however, the phosphorylation cascade between a Streptomyces SK and its cognate RR leading to altered DNA-binding affinity of the RR has not been analyzed in detail.
The cellulose degrader S. reticuli has been reported to contain the neighboring genes senS and senR, which encode an SK and an RR, respectively. SenS (42.2 kDa) comprises five predicted membrane-spanning portions. SenR (23.2 kDa) has a C-terminal region with a predicted helix–turn–helix motif, which is characteristic for different DNA-binding proteins . It was concluded that SenR is the cognate RR for the SK SenS. Comparative transcriptional and biochemical studies with a designed S. reticuli senS–senR chromosomal disruption mutant showed that the presence of SenS–SenR influences the transcription of the furS–cpeB operon encoding the redox regulator FurS and the catalase-peroxidase CpeB, and the hbpS gene for the secreted HbpS, representing a novel type of heme-binding protein . Physiologic studies showed that the production of HbpS is positively influenced by hemin in S. reticuli; this correlated with increased hemin resistance. Interestingly, the presence of HbpS leads to enhanced synthesis of the heme-containing CpeB .
In this study, we describe the in vitro phosphorylation cascade between the purified cytoplasmic domain of SenS (SenSc) and SenR. Using designed mutant proteins, the phosphorylation sites within SenSc and SenR have been investigated. Bandshift and footprinting analyses have allowed the characterization of the DNA-binding properties in response to phosphorylation by the sensorkinase SenS.
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The designed cloning procedures allowed us to obtain the cytoplasmic domain of SenS carrying a Strep-tag (SenSc) and the full-length SenR protein with a His-tag (SenR) at high purity as a basis for in vitro studies. SenSc was found to function as an efficient autokinase. Chemical stability assays revealed that the ligand within the phosphorylated SenSc (SenSc∼P) must be a phosphoamidate, which is extremely acid-labile but relatively base-stable. This feature discriminates all phosphorylated amino acid residues (except arginine) from phosphoamidates . Additional mutational investigations demonstrated that SenSc requires the histidine residue at position 199 for autokinase activity. The in vitro transfer of the phosphate group from SenSc to SenR (dephosphorylated SenR) occurred very rapidly, but did not occur in a designed mutant SenR protein carrying a substitution of the aspartic acid residue at position 65 (D65). The kinase SenSc was found to act additionally as a phosphatase for SenR∼P (phosphorylated SenR).
Bandshifts revealed that SenR, but not SenR∼P, binds specifically to a region (site I) upstream of the furS–cpeB operon encoding the redox regulator FurS and the catalase-peroxidase CpeB. The deletion of site I abolishes the interaction with SenR. Interestingly, this site is located within the previously determined FurS operator  and overlaps with its central region. The data imply that, in addition to FurS, SenR participates in regulating the transcription of the furS–cpeB operon. This conclusion is supported by the earlier finding that the absence of a functional furS or senR gene provokes enhanced transcription of the furS–cpeB operon [16,20]. Overlapping DNA-binding sites have also been described for other known regulators. Depending on the physiologic condition, either the activator NhaR or the RR RcsB from E. coli interacts with overlapping motifs within the upstream region of osmC. This gene encodes a predicted envelope protein that is required for resistance to organic peroxides and also for long-term survival in the stationary phase [21,22]. The regulator PutR and the activator CRP from Vibrio vulnificus bind simultaneously to overlapping sites but probably to opposite faces. This process leads to activation of the transcription of the operon encoding a proline dehydrogenase and a proline permease . PutR has been suggested to facilitate the DNA binding of CRP by direct protein–protein interaction or to induce a change in DNA topology that allows more efficient recruitment of CRP.
Comparative transcriptional and biochemical studies have revealed that SenS–SenR modulates the transcription of the furS–cpeB operon as well as the hbpS gene encoding a novel heme-binding protein. Interestingly, SenR has a high affinity for the intergenic region between hbpS and senS, spanning 21 bp (site II) and 19 bp (site III). Both became hypersensitive to DNaseI treatment at their ends after incubation with SenR∼P, indicating altered DNA topology. The phosphorylated form of RRs has been shown to provoke oligomerization and to bind cooperatively to target DNA sequences [24,25]. Altered DNA binding upon phosphorylation was observed, for example, for the RR RegR of the RegS–RegR system from Bradyrhizobium japonicum, controlling the expression of numerous genes, the products of which are either directly involved in nitrogen fixation or in functions associated with the microaerobic lifestyle of this symbiont . A corresponding observation was also made for MisR of the TCS MisR–MisS from Neisseria meningitides, which is required for its pathogenicity , and for NtrC of the NtrB–NtrC system, which controls the expression of genes involved in nitrogen metabolism in Rhodobacter capsulatus.
The two SenR-binding sites II and III share a common motif CNTCCNGT in the same orientation. Additionally, binding site III is localized within a region (CGGCCCGGACCGGGCCG) representing a perfect inverted repeat (Fig. 7). The use of DNA fragments lacking either binding site II, binding site III or both showed that each of them is necessary for specific targeting by SenR. Further single replacements within each binding site (I, II or III) may reveal the essential role of single nucleotides in the specific interaction with SenR. The position of the SenR operator (sites II and III) indicates that the transcription of senS–senR is autoregulated. As reported previously , SenS–SenR shows similarity to the ChrS–ChrA system from Corynebacterium diphtheriae. ChrA has so far not been purified, but it has been predicted to modulate the transcription of the heme oxygenase gene (hmuO) [28,29]. On the basis of our data, we identified a DNA region upstream of hmuO with high similarity to SenR-binding site II and an additional shared sequence (GGGCGTCGG) near to its 3′-end (data not shown). This is in accordance with the fact that the helix–turn–helix DNA-binding domains of SenR and ChrA share 61% amino acid identity (data not shown).
The designed SenRD60 and SenRD65A proteins showed reduced DNA-binding affinity for up-furS as well as for up-hbpS. D60 and D65, together with other aspartic acid residues (in positions 19 and 20), in SenR correspond to those that have been predicted to form an acidic pocket within RRs containing a CheY-like receiver domain . Mutations at any of the acidic pocket aspartates result in loss of functionality . SDS/PAGE analysis of purified SenR proteins revealed that SenRD60A appeared to be partially degraded and aggregated (Fig. 1). Both SenRD60A and SenRD65A seem to be perturbed in their conformation and hence show altered DNA-binding abilities.
Our previous data revealed that the presence of SenS–SenR considerably enhances the resistance of S. reticuli to hemin or the redox cycling compound plumbagin, suggesting its relevance in sensing of redox changes . Further preliminary comparative analysis (data not shown) revealed that under different redox-stress conditions, the presence of SenS–SenR is required for the production of additional extracellular proteins, whose characteristics remain to be clarified. One key part of the sensing processes is expected to be orchestrated by the heme-binding protein HbpS . How it participates in delivering signals to SenS will be explored.
Sequence comparisons showed that the relative organization of senS and senR is identical to those of homologous genes within the S. coelicolor A3(2) genome ; these genes are also preceded by an uncharacterized gene that is closely related to hbpS. Further sequence alignments revealed the presence of other predicted TCSs showing high amino acid identity with SenS–SenR within Rhodococcus sp. RHA1  and Arthrobacter aurescens TC1 . Interestingly, each of the corresponding homologous genes is also preceded by a close homolog of hbpS, the organization of which is identical to that within the S. reticuli genome. The fact that each corresponding intergenic region comprises motifs that are related to the SenR-binding sites (II and III) (data not shown) indicates that these homologous systems are also autoregulated. According to these findings, it could be assumed that HbpS and SenS–SenR, and probably also the corresponding homologs from the other mentioned actinobacteria, interact together to mediate an appropriate response to environmental changes. Such a mode of interaction has been recently postulated for the lipoprotein LpqB and the TCS MtrA–MtrB, which together might form an actinobacterial three-component system . The elucidation of the exact role of accessory proteins for the modulation of bacterial TCSs might give new insights into the complex network of signaling processes.
Taking the presented and previous data into account, the TCS SenS–SenR from S. reticuli is a model that is well suited to elucidate the role of other related TCSs from different actinobacteria.