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

Abstract

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.

FIG. 1

Schematic representation of ArcB and ArcA. (Top) The N-terminal transmembrane domain of ArcB was determined by alkaline phosphatase fusions (16). A putative leucine zipper (6) and a PAS domain (26) were predicted on the basis of amino acid sequence homology. The primary transmitter domain (H1) contains the conserved His292 and the catalytic determinants N, G1, and G2. The G1 and G2 sequences typify nucleotide-binding motifs. The receiver domain (D1) contains the conserved Asp576, and the secondary transmitter domain (H2) contains the conserved His717 (13, 27). (Bottom) ArcA consists of an N-terminal receiver domain (D2) containing the conserved Asp54 and a C-terminal helix-turn-helix (HTH) domain (12).

FIG. 2

Allele replacement strategy. (A) Construction of the ΔarcB::Tet^r strains. The 5′- and 3′-flanking DNA fragments of arcB (fragments 1 and 2) were prepared by PCR, using chromosomal DNA from strain MC4100 as the template and, respectively, the primer pairs DAB-5N–DAB-5C and DAB-3N–DAB-3C (16). The PCR products were cloned into pUC18. A Tet^r cassette, isolated from pNK81 (30), was then inserted between the two arcB-flanking fragments to generate pDB3. This plasmid was transformed into strain JC7623 (22) to create a ΔarcB::Tet^r strain (ECL5000) by homologous recombination. The ΔarcB::Tet^r allele was then P1 transduced into strains ECL5002 and ECL5003, resulting in ECL5004 and ECL5012, respectively. (B) Introduction of modified arcB sequences into the ΔarcB::Tet^r strain. The 5′- and 3′-flanking DNA fragments of arcB (fragments 3 and 4) were prepared by PCR, using chromosomal DNA from strain MC4100 as the template and, respectively, the primer pairs IAB-5N–IAB-5C and IAB-3N–IAB-3C (16). The PCR products were cloned into pBluescript II KS (+). A Kan^r cassette, isolated from pUC4-KIXX (2), was then inserted between the two arcB-flanking fragments to generate pIB3. Fragment 3 includes the arcB promoter, the ribosome-binding site, and an introduced NdeI site that includes the initiation codon of arcB followed by a HindIII site. A modified arcB sequence (arcB*) was cloned into the pIB3 between the NdeI site and HindIII site, generating pIB*. This plasmid was transformed into strain ECL5000 to replace the ΔarcB::Tet^r allele with arcB* Kan^r by homologous recombination. Recombinants were selected by their Tet^s Kan^r Amp^s phenotypes and confirmed by PCR. The arcB* Kan^r construct was then P1 transduced into strains ECL5004 and ECL5012.

FIG. 3

Effects of mutations in the conserved amino acid residues of ArcB on the expressions of λΦ(cydA′-lacZ) or a λΦ(lldP′-lacZ). The cydAB operon encodes cytochrome d oxidase (5), and the lldPRD operon encodes proteins involved in l-lactate utilization (4). The Φ(cydA′-lacZ)-bearing strains were grown in Luria-Bertani broth containing 0.1 M MOPS (morpholinepropanesulfonic acid; pH 7.4) and 20 mM d-xylose. The Φ(lldP′-lacZ)-bearing strains were grown in the above medium supplemented with 20 mM l-lactate as an inducer (4). β-Galactosidase activity was assayed and expressed in Miller units (20). The data are averages from four experiments (variations were <10% from the mean). Solid bars, aerobically grown cells; hatched bars, anaerobically grown cells.

FIG. 4

Western blot analysis. A 1-ml sample of cultures grown aerobically in Luria-Bertani broth was harvested at an optical density at 600 nm of 0.5. The pelleted cells were washed with 1 ml of 10 mM Tris-HCl (pH 8.0) and solubilized by incubation at 95°C for 5 min in 100 μl of 2× sodium dodecyl sulfate sample buffer. Samples of 10 μl were subjected to electrophoresis in a sodium dodecyl sulfate–12% polyacrylamide gel, and the resolved proteins were electrotransferred to a Hybond-ECL filter (Amersham). Immunoblot analyses were subsequently performed, using ArcB polyclonal antibodies as previously described (16). Lane 1, ΔarcB; lane 2, arcB⁺; lane 3, arcB^H292Q; lane 4, arcB^D576A; lane 5, arcB^H717Q.

FIG. 5

Model for signal transduction by the Arc system. Heavy solid arrows indicate the forward phosphotransfer reactions leading to formation of ArcA-P. Hatched arrows indicate the reverse phosphotransfer reactions leading to signal decay (7). Arrows with crosses indicate phosphotransfer reactions not substantiated by this study.