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

Schematic representation of domain composition in ArcB and coiled coil

<p><b>prediction. </b><b>A</b>) The ArcB sensor kinase is attached to the plasma membrane by TM1 corresponding to residues 23 to 41 and TM2 corresponding to residues 58 to 77. The linker region contains a coiled coil motif with a putative leucine-zipper (gray filled block) and two redox-active cysteine residues 180 and 241. 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 histidine phosphotransfer domain (Hpt/H2) contains the conserved His717. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038187#pone.0038187-Georgellis2" target="_blank">[7]</a> but with some modifications. <b>B</b>) Prediction of coiled coil motifs along the ArcB amino acid sequence. Above is plotted the probability of coiled coil structure occurrence along the ArcB sequence, as predicted by the COILS/PCOILS tool. Below is presented the sequence of the ArcB coiled-coil motif with its heptad repeats, and the leucine zipper as predicted by the program 2ZIP. <b>C</b>) Model of the ArcB coiled coil section covering residues 73–121. The structure prediction was generated based on the crystal structure of a model dimeric coiled coil (PDB code 3he5). The positions of leucine residues 73, 80, 87, 94, 108 and 115 are indicated in yellow and the position of Leu 102, which was used as an experimental control, is shown in blue.</p

Effect of mutations in the leucine zipper of ArcB on its phosphatase activity and their dominant phenotypes.

<p><b>A</b>) Strain ECL5003 [<i>arcB</i>^wt, λΦ(<i>cydA’-lacZ</i>)] carrying low copy plasmids with the <i>arcB</i> mutant variants was grown aerobically (solid bars) or anaerobically (empty bars) to mid-exponential growth phase (OD₆₀₀ ∼ 0.5), and the ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated. <b>B</b>) Effect of Leu replacements on the phosphatase activity of ArcB. Strain ECL5004, transformed with low-copy plasmids carrying different <i>arcB</i> forms, was grown aerobically to mid-exponential growth phase (OD₆₀₀ ∼ 0.5), on minimal medium with pyruvate as sole carbon source, and ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated.</p

Effect of DTT and ubiquinone-0 on the rate of ArcB net-phosphorylation.

<p>Membrane vesicles (1 µg) containing high amounts of wild type ArcB^1–778 (circles) or the mutant ArcB variants (ArcB^L80V (squares) and ArcB^L87V (diamonds)) were incubated at room temperature with 40 µM [γ-³²P]ATP in the presence of 5 mM DTT (open symbols) or 250 µM Q0 (closed symbols) in a 20 µl reaction mixture. At the indicated time intervals a 5 µl sample was withdrawn for SDS-PAGE analysis. Left panel: autoradiograms of the gels. Right panel: net increase of ArcB-P with time, as quantitated with a PhosphorImager.</p

<i>Escherichia coli</i> strains and plasmids used in this study are listed.

<p><i>Escherichia coli</i> strains and plasmids used in this study are listed.</p

Effect of mutations in the leucine zipper of ArcB on the expressions of λΦ(<i>cydA’-lacZ</i>) and λΦ(<i>lldP’- lacZ</i>) operon fusions.

<p><b>A</b>) Strains ECL5012 [λΦ(<i>lldP’- lacZ</i>)] and ECL5004 [λΦ(<i>cydA’-lacZ</i>)] carrying low copy plasmids that harbor the <i>arcB</i> mutant variants were grown aerobically (solid bars) or anaerobically (empty bars) in Luria-Bertani broth containing 0.1 M MOPS (morpholinepropanesulfonic acid; pH 7.4) and 20 mM D-xylose. In the case of the λΦ(<i>lldP’- lacZ</i>)-bearing strains the growth medium was supplemented with 20 mM L-lactate as an inducer. At mid-exponential growth phase (OD₆₀₀ ∼ 0.5) the cells were harvested and the ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated. (<b>B</b>) Equal number of bacteria of the above aerobic cultures were analyzed by Western blot analysis, using ArcB polyclonal antibodies as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038187#pone.0038187-Kwon3" target="_blank">[34]</a>.</p