The ArcB Sensor Kinase of Escherichia coli : Genetic Exploration of the Transmembrane Region

Abstract

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 (⌬ 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 ⌬ 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.The Arc two-component signal transduction system of Escherichia coli regulates the expression of more than 30 operons, depending on the redox conditions of growth (20,22,30,31). The system consists of ArcB, the membrane-bound sensor kinase, and ArcA, the cognate response regulator. ArcB (17,23,27,48) (see Fig. 1A) belongs to the tripartite sensor kinase subfamily (1,16,25,35,37,44,49), is attached to the cytoplasmic membrane by two transmembrane segments (TM1 and TM2) near the N-terminal end (24), and catalyzes a phosphorelay via His292, Asp576, and His717 of ArcB to Asp54 of ArcA (14). The autophosphorylation step is stimulated by effectors, such as D-lactate, pyruvate, and acetate. These metabolites accumulate when exogenous electron acceptors limit respiration during growth (13, 18). Dephosphorylation of ArcA-P occurs by a reverse phosphorelay from the Asp54 of ArcA to the His717 and Asp576 of ArcB. The phosphoryl group is then released as P i (12).Most sensor kinases receive their signal from the periplasmic domain, resulting in conformation changes that trigger autophosphorylation. As a sensor kinase, ArcB is unusual in having a short putative periplasmic bridge (24,29). Relatively little is known about the nature of the signal for ArcB and what role the membrane-associated region plays in signal reception, except that autophosphorylation seems to be activated by excessive reducing equivalents (7,11,21,22,24,38). Two studies involving growth of cells at high pH and treatment of cells by protonophores during growth, however, led to the suggestion that ArcB kinase is activated by a decrease in proton motive force (PMF) across the cytoplasmic membrane (2, 5). Here, we confirm the transmembrane topology of ArcB by genetic analysis and probe the function of the membrane region by replacing the chromosomal arcB ϩ by a single copy of a mutant allele ( Fig. 2; Tables 1 and 2).Transmembrane topology of ArcB. To test the suggested topology based on hydrophobicity analysis, we constructed four phoA protein fusions (32) of arcB (Fig. 1B). The PhoA fusions at residues 22 and 102 of ArcB exhibited very low levels of alkaline phosphatase activity. In contrast, the PhoA fusions at residues 41 and 57 of ArcB showed very high levels of the enzyme activity (Fig. 1B). On the basis of this genetic analysis and a more recent algorithm for determining membr...

Identification of a quinone-sensitive redox switch in the ArcB sensor kinase

“…The proteins were separated by nonreducing SDS͞PAGE, and ArcB was visualized by Western blot analysis as described in ref. 6.…”

“…In this context, it is of relevance to mention that the membranepermeating reductants generate an intracellular reducing environment that affects protein folding and, in particular, disulfide bond formation (18,19). Considering that ArcB possesses a cytoplasm-located signal reception site (6), it is activated only by Four parallel cultures of strain ECL5203 (6) and its isogenic ECL5204 (⌬arcB) (6) were grown aerobically in Luria-Bertani broth containing 0.1 M MOPS (pH 7.4) and 20 mM D-xylose. At OD600 of 0.3, one aliquot was withdrawn for measuring the ␤-galactosidase activity (depicted as Ϫ15 min).…”

“…As a sensor, ArcB is deviant because in contrast to typical sensor kinases that have a substantial periplasmic domain for environmental sensing, ArcB has a very short periplasmic sequence of only 16 amino acid residues delimited by two canonical transmembrane segments. Interestingly, the ArcB transmembrane domain (amino acids does not directly participate in signal sensing but rather serves as an anchor that keeps the protein close to the source of the signal (6). As a kinase, ArcB is atypical because it contains three catalytic domains: an N-terminal transmitter domain with a conserved His-292 residue, a central receiver domain with a conserved Asp-576 residue, and a C-terminal phosphotransfer domain with a conserved His-717 residue (5,7).…”

A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of σS (RpoS) in E. coli

“…The following mutant alleles were introduced by P1 transduction (Miller 1972) into either MC4100, FS1 (MC4100 carrying the rpoS742ϻlacZ operon fusion; see below) or FS110 (MC4100 carrying the arcA159ϻlacZ operon fusion, see below): arcAϻkan, arcBϻkan, arcAϻtet, arcBϻtet (Kwon et al 2000;Georgellis et al 2001b;Liu and de Wulf 2004), rpoSϻkan (Bohannon et al 1991), and rssBϻTn10 (Muffler et al 1996). To provide RssB in trans, the plasmids pBadRssB or pBadRssB D58P (Becker et al 2000) were introduced into rssBϻTn10 strains.…”

The ArcB Sensor Kinase of Escherichia coli Autophosphorylates by an Intramolecular Reaction

“…The lldPЈ-lacZ and cydAЈ-lacZ target operons were chosen as an ArcA-P-repressible reporter and as an ArcA-P-activatable reporter, respectively. To construct plasmid pMX041, we first created plasmid pMX712 by cloning the BamHI-HindIII fragment from plasmid pIBW (20), which carries the arcB promoter, the arcB ribosomal binding site, an introduced NdeI site that includes the initiation codon of arcB, and the arcB open reading frame (ORF) and stop codon into pBlueScript II KSϩ. Subsequently, the MluI-HindIII fragment of pMX025 was used to replace the MluI-HindIII wild-type ArcB fragment of pMX712, generating plasmid pMX040.…”