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

Abstract

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.

Fig 1

Simplified comparative model of the domain architectures of and phosphoflow in the Arc systems of E. coli and S. oneidensis. The E. coli system (upper panel) consists of the membrane-integrated histidine sensor kinase (HK) ArcB and the response regulator (RR) ArcA. A reduced state of the quinone pool preserves the sulfhydryl groups of two highly conserved cysteine residues located within the PAS sensor domain. Under these conditions, the histidine kinase is highly active and a phosphorelay to the receiver domain of the RR ArcA occurs. Phosphorylated ArcA then binds to its target DNA sequences as indicated. Under aerobic conditions, the oxidized state of the quinone pool allows formation of interprotein disulfide bonds between two ArcB monomers, resulting in a rotational switch that decreases ArcB kinase activity. Under these conditions, reverse phosphotransfer from the RR to the HK occurs, resulting in signal decay. In Shewanella (lower panel), the Arc system is formed by the HK ArcS, the phosphotransfer protein HptA, and the RR ArcA. ArcB and ArcS have fundamentally different domain architectures. In previous studies, we and others have established that autophosphorylation of ArcS occurs and that a phosphoryl group can be transferred from the HK to the RR and back. However, the mechanisms of signal perception and the exact route of the phosphotransfer between the components remain elusive so far. Transmembrane regions are represented by black bars. CaChe, CaChe-sensing core domain; PAS, PAS sensing domain; HisKA, histidine kinase dimerization protein; HATPase, histidine kinase ATPase domain; Hpt, histidine transfer domain; Rec, receiver domain; Q_red, reduced quinone pool; Q_ox, oxidized quinone pool. In ArcS, a putative signaling helix is located between the transmembrane and the PAS-I domains.

Fig 2

ArcS Western blot analysis of localization and production. Membrane (M) and cytosolic (C) fractions of S. oneidensis MR 1 cells bearing a copy of plasmid-encoded His₆-ArcS (ΔarcS + pBAD33-HisA-ArcS, left lanes), lacking ArcS (ΔarcS, middle lanes), or with a chromosomally encoded His₆-ArcS version (KI-His6-arcS; right lanes) were analyzed for the presence of ArcS by SDS-PAGE using either Coomassie stain (upper panel) or His₆-specific antibodies (lower panel). The exposure time for chemiluminescence development is given in minutes.

Fig 3

In vitro studies of phosphotransfer from ArcA to ArcS. (A) Autoradiographic analysis of phosphotransfer between the indicated variants of ArcS_aa694-1236, GST-HptA, and ArcA. Phosphorylated ArcA was incubated for 1 min with the indicated components, and the samples were then separated by SDS-PAGE and analyzed for their radioactive signal intensities. (B) Corresponding signal intensities were calculated using ImageJ and are given as normalized numbers, setting each lane as 100%.

Fig 4

In vivo analysis of the effect of ArcS substitution mutants on csgB expression. (A) Overproduction of His₆-ArcS and its mutated variants (indicated at the top). Extracts of cells in which plasmid-borne expression was induced or not induced (as indicated at the bottom) were separated by SDS-PAGE. The proteins were specifically detected using antibodies raised against the N-terminal His tag. (B) csgB promoter activity depending on various levels of ArcS and its mutated variants under aerobic conditions. Promoter activity was determined by a chromosomally integrated P_csgB-lux fusion as light emission (RLU) relative to the OD₆₀₀. Wild-type expression levels were almost below background levels (not shown). The white bar displays the activity in the absence of arcS. The black bars display the activity when arcS and corresponding mutants were expressed at chromosomal levels, and the gray bars display the activity when the corresponding proteins were overproduced. The error bars display the standard deviations from three independent experiments.