Characterization of the Arc two-component signal transduction system of the capnophilic rumen bacterium Mannheimia succiniciproducens

The ArcB/A two-component signal transduction system of Escherichia coli modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the putative arcA and arcB genes of Mannheimia succiniciproducens MBEL55E, a capnophilic (CO2-loving) rumen bacterium, encode functional proteins that specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro, and the arcA and arcB genes of M. succiniciproducens confer toluidine blue resistance to E. coli arcA and arcB mutants. However, neither the quinone analogs (ubiquinone 0 and menadione) nor the cytosolic effectors (d-lactate, acetate, and pyruvate) affect the net phosphorylation of M. succiniciproducens ArcB. Our results indicate that different types of signaling molecules and distinct modes of kinase regulation are used by the ArcB proteins of E. coli and M. succiniciproducens.     

The ArcB sensor kinase of Escherichia coli: genetic exploration of the transmembrane region

The Arc two-component signal transduction system of Escherichia coli regulates the expression of numerous operons in response to respiratory growth conditions. Cellular redox state or proton motive force (Delta(H(+))) has been proposed to be the signal for the membrane-associated ArcB sensor kinase. This study provided evidence for a short ArcB periplasmic bridge that contains a His47. The dispensability of this amino acid, the only amino acid with a pK in the physiological range, renders the Delta(H(+)) model unlikely. Furthermore, results from substituting membrane segments of ArcB with counterparts of MalF indicate that the region does not play a stereospecific role in signal reception.     

Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphos-phorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.     

Effect of D-lactate on the physiological activity of the ArcB sensor kinase in Escherichia coli

The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory growth conditions. Under anoxic growth conditions ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons. The anaerobic metabolite D-lactate has been shown to stimulate the in vitro autophosphorylating activity of ArcB. In this study, the in vivo effect of D-lactate on the kinase activity of ArcB was assessed. The results demonstrate that D-lactate does not act as a direct signal for activation of ArcB, as previously proposed, but acts as a physiologically significant effector that amplifies ArcB kinase activity.     

Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites. An in vitro study with different protein modules

In Escherichia coli, changes in redox condition of growth are sensed and signaled by the Arc two-component system. This system consists of ArcB as the membrane-associated sensor kinase and ArcA as the cytoplasmic response regulator. ArcB is a tripartite kinase, possessing a primary transmitter, a receiver, and a secondary transmitter domain that catalyzes the phosphorylation of ArcA via a His --> Asp --> His --> Asp phosphorelay, as well as the dephosphorylation of ArcA-P by a reverse phosphorelay. When ArcA and ArcB were incubated with ATP, the peak levels of phosphorylated proteins increased in the presence of the fermentation metabolites D-lactate, acetate, or pyruvate. In this study, we report that these effectors accelerate the autophosphorylation activity of ArcB and enhance the transphosphorylation of ArcA, but have no effect on the dephosphorylation of ArcA-P. Moreover, the presence of the receiver domain of ArcB is essential for the effectors to influence the autophosphorylation rate of the primary transmitter domain of ArcB.     

Anaerobic regulation by an atypical Arc system in Shewanella oneidensis

Shewanella oneidensis strain MR-1 is well known for its respiratory versatility, yet little is understood about how it regulates genes involved in anaerobic respiration. The Arc two-component system plays an important role in this process in Escherichia coli; therefore, we determined its function in S. oneidensis. arcA from S. oneidensis complements an E. coli arcA mutant, but the Arc regulon in S. oneidensis constitutes a different suite of genes. For example, one of the strongest ArcA-regulated gene clusters in E. coli, sdh, is not regulated by the Arc system in S. oneidensis, and the cyd locus, which is induced by ArcA in E. coli under microaerobic conditions, is repressed by ArcA in S. oneidensis under anaerobic conditions. One locus that we identified as being potentially regulated by ArcA in S. oneidensis contains genes predicted to encode subunits of a dimethyl sulphoxide (DMSO) reductase. We demonstrate that these genes encode a functional DMSO reductase, and that an arcA mutant cannot fully induce their expression and is defective in growing on DMSO under anaerobic conditions. While S. oneidensis lacks a highly conserved full-length ArcB homologue, ArcA is partially activated by a small protein homologous to the histidine phosphotransfer domain of ArcB from E. coli, HptA. This protein alone is unable to compensate for the lack of arcB in E. coli, indicating that another protein is required in addition to HptA to activate ArcA in S. oneidensis.     

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Bioprocesses conducted under conditions with restricted O₂ supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O₂ and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of d-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, d-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD⁺ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.     

Induction and function of the phage shock protein extracytoplasmic stress response in Escherichia coli

The phage shock protein (Psp) F regulon response in Escherichia coli is thought to be induced by impaired inner membrane integrity and an associated decrease in proton motive force (pmf). Mechanisms by which the Psp system detects the stress signal and responds have so far remained undetermined. Here we demonstrate that PspA and PspG directly confront a variety of inducing stimuli by switching the cell to anaerobic respiration and fermentation and by down-regulating motility, thereby subtly adjusting and maintaining energy usage and pmf. Additionally, PspG controls iron usage. We show that the Psp-inducing protein IV secretin stress, in the absence of Psp proteins, decreases the pmf in an ArcB-dependent manner and that ArcB is required for amplifying and transducing the stress signal to the PspF regulon. The requirement of the ArcB signal transduction protein for induction of psp provides clear evidence for a direct link between the physiological redox state of the cell, the electron transport chain, and induction of the Psp response. Under normal growth conditions PspA and PspD control the level of activity of ArcB/ArcA system that senses the redox/metabolic state of the cell, whereas under stress conditions PspA, PspD, and PspG deliver their effector functions at least in part by activating ArcB/ArcA through positive feedback.     

Domain Analysis of ArcS,the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System,Reveals Functional Differentiation of Its Two Receiver Domains

In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli.     

The ArcB leucine zipper domain is required for proper ArcB signaling

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70-121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling.     

Phosphorelay as the sole physiological route of signal transmission by the arc two-component system of Escherichia coli

The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutant arcB alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.     

Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli.

In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation.     

Analysis of HI0220 protein from Haemophilus influenzae, a novel structural and functional analog of ArcB protein from Escherichia coli

A Haemophilus influenzae gene encoding a protein with high homology to ArcB receptor protein from Escherichia coli has been cloned. An error in the previously reported sequence of this gene has been found, thus increasing its open reading frame. The cloned gene comprising the entire open reading frame restores oxygen-dependent regulation of succinate dehydrogenase in an ArcB-deficient E. coli strain. Thus, this gene is a functional analog of ArcB from E. coli. By screening partially sequenced bacterial genomes using the BLAST program, proteins with high homology to ArcB protein from E. coli were found in Salmonella typhi, Yersinia pestis, Vibrio cholerae, and Pasteurella multocida. Comparison of these proteins with ArcB protein from E. coli and H. influenzae revealed conserved amino acid regions. Transmembrane helix II was shown to be highly homologous in all the ArcB-type proteins. The involvement of this region in ArcB-mediated oxygen-dependent regulation is suggested.