ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1

Abstract

The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.

FIG. 1.

Domain organization of Shewanella ArcS compared to that of E. coli ArcB. The numbers in boxes mark the positions of the domains in the amino acid sequence. The position of conserved amino acid residues putatively involved in phosphotransfer is marked within the corresponding domain. The numbers in shaded areas between the domains of ArcS and ArcB display levels of identity/similarity between the domains (n/h, no homologies). Black vertical bars show the positions of transmembrane domains. CaChe, CaChe-sensing domain; PAS, energy-sensing domain; HisKA, histidine kinase dimerization domain; HATPase_c, histidine kinase ATPase domain; REC, receiver domain.

FIG. 2.

Growth and biofilm formation of ΔarcS, ΔhptA, and ΔarcA mutans. The doubling times of the corresponding strains under aerobic (top) and anaerobic conditions with fumarate (middle) and DMSO (bottom) as electron acceptors, respectively, are indicated. At the bottom, the biofilm formation of the mutants relative to that of the wild type is indicated. The error bars represent the standard deviations. n/a, not applicable.

FIG. 3.

Transcriptomic analysis of ΔarcS, ΔhptA, and ΔarcA mutants. (A) Hierarchical clustering of genes significantly regulated in both ΔarcA and ΔarcS mutant strains under aerobic conditions as analyzed by microarrays. Expression differences between mutants (left, ΔarcA; right, ΔarcS) and the wild type are represented by colors (yellow, induced; blue, repressed). (B) Changes in transcriptional levels of csgB, rpoS, aggA, and dmsB in ΔarcS, ΔhptA, and ΔarcA mutants grown under aerobic and anaerobic conditions compared to the wild type. Transcript levels were analyzed by q-RT-PCR. ORF, open reading frame.

FIG. 4.

In vivo and in vitro interactions of ArcS, HptA, and ArcA. (A) Analysis of in vivo protein-protein interactions in a bacterial two-hybrid system. Interactions of the indicated proteins fused to the T18 and T25 fragments, respectively, of the B. pertussis adenylate cyclase result in a red appearance of the colonies on MacConkey agar. +, positive control (T18-zip/T25-zip); −, negative control (T18/T25 empty vectors). (B) Autoradiographic analysis of phosphotransfer between ArcS(646-1188), GST-HptA, and ArcA. Phosphorylated ArcA (10 μM) was incubated for the given amount of time (top) with equimolar amounts of the indicated components and then separated by SDS-PAGE.

FIG. 5.

Restoration of transcriptional levels in arc mutants by E. coli ArcB. arcB from E. coli was cloned into the inducible vector pBAD33, and the resulting vector was electroporated into ΔarcS, ΔarcS ΔhptA, and ΔarcA mutants. The transcriptional levels of csgB (black bars) and SO_2427 (white bars) were determined by q-RT-PCR of RNA obtained from the corresponding cultures grown aerobically under inducing and noninducing (“silent”) conditions. The bars display the expression levels relative to that of the wild type. The error bars represent standard deviations.

FIG. 6.

Gene organization alignments of the S. oneidensis hptA locus and the E. coli arcB locus. Genes are displayed as arrows indicating the direction of transcription. The shaded areas display regions of significant similarity. The coordinates in the genome are given to the left and right of the corresponding region. The genetic organization strongly indicates that the major part of arcB was lost in Shewanella, leaving the phosphotransfer domain hptA.

FIG. 7.

Phylogenetic analysis of the Shewanella ArcS histidine kinase region (A) and HptA (B). The appropriate protein sequences were aligned with those of the BarA sensor kinase, the ArcB sensor kinase, and the putative RcsC sensor kinase of S. oneidensis MR-1 (outgroup). Bold lines represent the positions of S. oneidensis RcsC, ArcS, and BarA and E. coli ArcB (A) and S. oneidensis MR-1 BarA and HptA and E. coli ArcB (B) (in clockwise order). The numbers display the corresponding bootstrap values. The Shewanella Arc and BarA proteins are marked in red and blue, respectively. ArcB proteins are marked in gray. Alt., Alteromonadales; Ae., Aeromonadales.