The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase.

HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 A) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.

The heme-binding protein HbpS regulates the activity of the Streptomyces reticuli iron-sensing histidine kinase SenS in a redox-dependent manner.

The SenS/SenR system of Streptomyces reticuli regulates the expression of the redox regulator FurS, the catalase-peroxidase CpeB and the heme-binding protein HbpS. SenS/SenR is also proposed to participate in sensing redox changes, mediated by HbpS. Here, we show in vitro that heme-free HbpS represses the autokinase activity of SenS; whereas hemin-treated HbpS considerably enhances SenS autophosphorylation under redox conditions using either H(2)O(2) or DTT. The presence of iron ions alone or in combination with H(2)O(2) or DTT also leads to significantly increased phosphorylation levels of SenS. Further comparative physiological studies using the S. reticuli WT, a S. reticuli hbpS mutant and a S. reticuli senS-senR mutant corroborates the importance of HbpS and the SenS/SenR system for resistance against high concentrations of iron ions and hemin in vivo. Hence SenS/SenR and HbpS act in concert as a novel three-component system which detects redox stress, mediated by iron ions and heme.

DNA-binding characteristics of the regulator SenR in response to phosphorylation by the sensor histidine autokinase SenS from Streptomyces reticuli.

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 (SenS(c)), 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, SenS(c) is autophosphorylated to SenS(c) approximately 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 approximately 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.