The extracellular heme-binding protein HbpS from the soil bacterium Streptomyces reticuli is an aquo-cobalamin binder.

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 µm) to aquo-cobalamin (H2OCbl(+)) but not to other cobalamins. Competition experiments with the H2OCbl(+)-coordinating ligand CN(-) and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl(+) and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl(+). Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins.

Iron-mediated oxidation induces conformational changes within the redox-sensing protein HbpS.

HbpS is an extracellular oligomeric protein, which has been shown to act in concert with the two-component system SenS-SenR during the sensing of redox stress. HbpS can bind and degrade heme under oxidative stress conditions, leading to a free iron ion. The liberated iron is subsequently coordinated on the protein surface. Furthermore, HbpS has been shown to modulate the phosphorylation state of the sensor kinase SenS as, in the absence of oxidative stress conditions, HbpS inhibits SenS autophosphorylation whereas the presence of heme or iron ions and redox-stressing agents enhances it. Using HbpS wild type and mutants as well as different biochemical and biophysical approaches, we show that iron-mediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within HbpS. We demonstrate in addition that HbpS is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds. Further examination of the crystal structure and subsequent mutational analyses allowed the identification of the tyrosine residue participating in dityrosine formation, which occurs between two monomers within the octomeric assembly. Therefore, it is proposed that oxidative modifications causing structural and conformational changes are responsible for the control of SenS and hence of the HbpS-SenS-SenR signaling cascade.

The three-component signalling system HbpS-SenS-SenR as an example of a redox sensing pathway in bacteria.

The two-component system SenS-SenR and the extracellular HbpS protein of the cellulose degrader Streptomyces reticuli have been shown to act in concert as a novel system which detects redox stress. In vivo and in vitro experiments have led to the hypothesis that HbpS binds and degrades heme, communicating the extracellular presence of heme and oxidative stress to the membrane-embedded sensor histidine kinase SenS via a bound iron. The response regulator SenR would then up-regulate downstream signalling cascades, leading to the appropriate gene expression levels for bacterial survival in an oxidative environment. Sequence analysis has shown that homologs of HbpS and SenS-SenR exist in a number of ecologically and medically relevant bacterial species, suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to both Gram-negative and Gram-positive bacteria. The presented report reviews the current knowledge of the function of this novel protein family consisting of an accessory protein and its cognate two-component system, which could be more properly described as a three-component system.

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.

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli.

Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS-SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS-SenR, which regulates the expression of the catalase-peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2(1)3, with a cell edge of 152.5 A. Diffraction data were recorded to a maximal resolution of 2.25 A and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.

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.

Deciphering the Transcriptional Response Mediated by the Redox-Sensing System HbpS-SenS-SenR from Streptomycetes.

The secreted protein HbpS, the membrane-embedded sensor kinase SenS and the cytoplasmic response regulator SenR from streptomycetes have been shown to form a novel type of signaling pathway. Based on structural biology as well as different biochemical and biophysical approaches, redox stress-based post-translational modifications in the three proteins were shown to modulate the activity of this signaling pathway. In this study, we show that the homologous system, named here HbpSc-SenSc-SenRc, from the model species Streptomyces coelicolor A3(2) provides this bacterium with an efficient defense mechanism under conditions of oxidative stress. Comparative analyses of the transcriptomes of the Streptomyces coelicolor A3(2) wild-type and the generated hbpSc-senSc-senRc mutant under native and oxidative-stressing conditions allowed to identify differentially expressed genes, whose products may enhance the anti-oxidative defense of the bacterium. Amongst others, the results show an up-regulated transcription of genes for biosynthesis of cysteine and vitamin B12, transport of methionine and vitamin B12, and DNA synthesis and repair. Simultaneously, transcription of genes for degradation of an anti-oxidant compound is down-regulated in a HbpSc-SenSc-SenRc-dependent manner. It appears that HbpSc-SenSc-SenRc controls the non-enzymatic response of Streptomyces coelicolor A3(2) to counteract the hazardous effects of oxidative stress. Binding of the response regulator SenRc to regulatory regions of some of the studied genes indicates that the regulation is direct. The results additionally suggest that HbpSc-SenSc-SenRc may act in concert with other regulatory modules such as a transcriptional regulator, a two-component system and the Streptomyces B12 riboswitch. The transcriptomics data, together with our previous in vitro results, enable a profound characterization of the HbpS-SenS-SenR system from streptomycetes. Since homologues to HbpS-SenS-SenR are widespread in different actinobacteria with ecological and medical relevance, the data presented here will serve as a basis to elucidate the biological role of these homologues.

The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (K d = 1.6 × 10(-7) M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family.

Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose.

We report the complete, GC-rich genome sequence of the melanin producer Streptomyces reticuli Tü 45 (S. reticuli) that targets and degrades highly crystalline cellulose by the concerted action of a range of biochemically characterized proteins. It consists of a linear 8.3 Mb chromosome, a linear 0.8 Mb megaplasmid, a linear 94 kb plasmid and a circular 76 kb plasmid. Noteworthy, the megaplasmid is the second largest known Streptomyces plasmid. Preliminary analysis reveals, among others, 43 predicted gene clusters for the synthesis of secondary metabolites and 456 predicted genes for binding and degradation of cellulose, other polysaccharides and carbohydrate-containing compounds.

Elucidation of haem-binding sites in the actinobacterial protein HbpS.

The extracellular haem-binding protein from Streptomyces reticuli (HbpS) has been shown to be involved in redox sensing and to bind haem. However, the residues involved in haem coordination are unknown. Structural alignments to distantly related haem-binding proteins from Mycobacterium tuberculosis were used to identify a candidate haem-coordinating residue, and site-directed mutagenesis with UV/Vis spectroscopy was used to assess haem binding in vivo and in vitro. We present strong evidence that HbpS belongs to the small set of proteins, which do not use histidine to coordinate the metal in the haem group. Further spectroscopic evidence strongly indicates that threonine 113 is actively involved in coordination of haem. Subsequent protein/haem titration experiments show a 1 : 2, protein/haem stoichiometry. We also present data showing the degradation of haem by HbpS in vivo. Because HbpS is conserved in many Actinobacteria, the presented results are applicable to related species.

A Streptomyces-specific member of the metallophosphatase superfamily contributes to spore dormancy and interaction with Aspergillus proliferans.

We have identified, cloned and characterized a formerly unknown protein from Streptomyces lividans spores. The deduced protein belongs to a novel member of the metallophosphatase superfamily and contains a phosphatase domain and predicted binding sites for divalent ions. Very close relatives are encoded in the genomic DNA of many different Streptomyces species. As the deduced related homologues diverge from other known phosphatase types, we named the protein MptS (metallophosphatase type from Streptomyces). Comparative physiological and biochemical investigations and analyses by fluorescence microscopy of the progenitor strain, designed mutants carrying either a disruption of the mptS gene or the reintroduced gene as fusion with histidine codons or the egfp gene led to the following results: (i) the mptS gene is transcribed in the course of aerial mycelia formation. (ii) The MptS protein is produced during the late stages of growth, (iii) accumulates within spores, (iv) functions as an active enzyme that releases inorganic phosphate from an artificial model substrate, (v) is required for spore dormancy and (vi) MptS supports the interaction amongst Streptomyces lividans spores with conidia of the fungus Aspergillus proliferans. We discuss the possible role(s) of MptS-dependent enzymatic activity and the implications for spore biology.

Iron binding at specific sites within the octameric HbpS protects streptomycetes from iron-mediated oxidative stress.

The soil bacterium Streptomyces reticuli secretes the octameric protein HbpS that acts as a sensory component of the redox-signalling pathway HbpS-SenS-SenR. This system modulates a genetic response on iron- and haem-mediated oxidative stress. Moreover, HbpS alone provides this bacterium with a defence mechanism to the presence of high concentrations of iron ions and haem. While the protection against haem has been related to its haem-binding and haem-degrading activity, the interaction with iron has not been studied in detail. In this work, we biochemically analyzed the iron-binding activity of a set of generated HbpS mutant proteins and present evidence showing the involvement of one internal and two exposed D/EXXE motifs in binding of high quantities of ferrous iron, with the internal E78XXE81 displaying the tightest binding. We additionally show that HbpS is able to oxidize ferrous to ferric iron ions. Based on the crystal structure of both the wild-type and the mutant HbpS-D78XXD81, we conclude that the local arrangement of the side chains from the glutamates in E78XXE81 within the octameric assembly is a pre-requisite for interaction with iron. The data obtained led us to propose that the exposed and the internal motif build a highly specific route that is involved in the transport of high quantities of iron ions into the core of the HbpS octamer. Furthermore, physiological studies using Streptomyces transformants secreting either wild-type or HbpS mutant proteins and different redox-cycling compounds led us to conclude that the iron-sequestering activity of HbpS protects these soil bacteria from the hazardous side effects of peroxide- and iron-based oxidative stress.

Redox sensing: novel avenues and paradigms.

The response to changes in the redox state of the cell environment is closely coupled with the ability of living organisms to sense changing conditions. Protein-based redox sensors utilize cofactors, that is, iron-sulfur clusters, flavins, or hemes, for environmental sensing. Under oxidizing conditions a cofactor-mediated post-translational modification (i.e., thiol-oxidation, carbonylation, or dityrosine formation) accompanied by a structural change in the protein occurs that results in an appropriate reaction, mostly in terms of expression of genes involved in antioxidative stress responses. In addition to these well-studied cofactors, researchers have recently discovered and described redox-active metabolites that play a role in redox sensing. Furthermore, not only proteins but also nucleic acids are able to sense redox-stressing events and to elucidate the corresponding response. With these all sensors, organisms are well equipped to sense redox-stress signals generated extracellularly as well as cytoplasmatically. To analyze the molecular mechanisms of all these redox sensors as well as to describe the paradigms involved, a number of sophisticated tools have been applied. These include development of novel protein fluorescence resonance energy transfer probes to microscopically analyze redox signaling in cells or the application of X-ray crystallography combined with spectroscopic studies to monitor dynamics of conformational changes within redox sensors. In this Forum, novel redox-sensing systems, novel avenues, and recent technical advances in the emerging field of redox sensing are presented.

ROS-Mediated Signalling in Bacteria: Zinc-Containing Cys-X-X-Cys Redox Centres and Iron-Based Oxidative Stress.

Bacteria are permanently in contact with reactive oxygen species (ROS), both over the course of their life cycle as well that present in their environment. These species cause damage to proteins, lipids, and nucleotides, negatively impacting the organism. To detect these ROS molecules and to stimulate the expression of proteins involved in antioxidative stress response, bacteria use a number of different protein-based regulatory and sensory systems. ROS-based stress detection mechanisms induce posttranslational modifications, resulting in overall conformational and structural changes within sensory proteins. The subsequent structural rearrangements result in changes of protein activity, which lead to regulated and appropriate response on the transcriptional level. Many bacterial enzymes and regulatory proteins possess a conserved signature, the zinc-containing redox centre Cys-X-X-Cys in which a disulfide bridge is formed upon oxidative stress. Other metal-dependent oxidative modifications of amino acid side-chains (dityrosines, 2-oxo-histidines, or carbonylation) also modulate the activity of redox-sensitive proteins. Using molecular biology, biochemistry, biophysical, and structure biology tools, molecular mechanisms involved in sensing and response to oxidative stress have been elucidated in detail. In this review, we analyze some examples of bacterial redox-sensing proteins involved in antioxidative stress response and focus further on the currently known molecular mechanism of function.

Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.

AIMS: To establish conditions to study the oligomeric assembly of heme-binding protein (HbpS) in solution by applying the tools of site-directed spin labeling combined with pulse electron paramagnetic resonance (SDSL EPR) spectroscopy, as well as to analyze redox stress-based conformational changes in HbpS subunits within the oligomer in solution. In vivo elucidation of molecular mechanisms that control the downregulation of the novel redox-system HbpS-SenS-SenR. RESULTS: Using a set of specifically generated HbpS mutants, and SDSL EPR spectroscopy, we show the octomeric assembly of HbpS in solution, and demonstrate that iron-mediated stress induces conformational changes in HbpS subunits within the octamer. We further demonstrate that the catalase-peroxidase CpeB protects HbpS from hydrogen peroxide (H(2)O(2))-mediated oxidative attack in vivo. Moreover, chromosomal inactivation of cpeB results in an enhanced sensitivity of the mutant to redox-cycling compounds. INNOVATION: SDSL EPR has been used in this work for the first time to monitor redox-mediated conformational changes in a redox-sensing protein in solution. This work substantially explains redox-dependent dynamics in HbpS at the atomic level, and presents novel molecular mechanisms supporting downregulation of a signaling cascade. CONCLUSION: Iron-mediated stress induces movements of subunits within the HbpS octomeric assembly. We suggest a motion of the C-terminal α-helix toward the preceding helical segment. These events upregulate the activity of the HbpS-SenS-SenR system, in which HbpS acts as an accessory element. The mycelia-associated CpeB, under the control of HbpS-SenS-SenR, protects the extracellular HbpS from oxidation in vivo. Thus, de novo synthesized HbpS proteins downregulate the HbpS-SenS-SenR signaling cascade.

Novel redox-sensing modules: accessory protein- and nucleic acid-mediated signaling.

SIGNIFICANCE: Organisms have evolved both enzymatic and nonenzymatic pathways to prevent oxidative damage to essential macromolecules, including proteins and nucleic acids. Pathways modulated by different protein-based sensory and regulatory modules ensure a rapid and appropriate response. RECENT ADVANCES: In contrast to classical two-component systems that possess internal sensory and regulatory modules, an accessory protein-dependent redox-signaling system has been recently characterized in bacteria. This system senses extracellular iron-mediated oxidative stress signals via an extracellularly located protein (HbpS). In vivo and in vitro studies allowed the elucidation of molecular mechanisms governing this system. Moreover, recent studies show that nucleic acids may also participate in redox-signaling during antioxidative stress response. CRITICAL ISSUES: Research for novel redox-signaling systems is often focused on known types of sensory and regulatory modules. It is also often considered that the oxidative attack of macromolecules, leading to modification and degradation processes, is the final step during oxidative stress. However, recent studies have demonstrated that oxidatively modified macromolecules can be intermediary states in the process of redox-signaling. FUTURE DIRECTIONS: Analyses of adjacent regions of genes encoding for known sensory and regulatory modules can identify potential accessory modules that may increase the complexity of sensing systems. Despite the fact that the involvement of DNA-mediated signaling in the modulation of one bacterial regulator protein has been analyzed in detail, further studies are necessary to identify additional regulators. Given the role of DNA in oxidative-stress response, it is tempting to hypothesize that RNA modules may also mediate redox-signaling.

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.