The ArcB Leucine Zipper Domain Is Required for Proper ArcB Signaling

Abstract

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.

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

“…In previous reports from our group [24], [29] and others [31], the relative level of ArcA phosphorylation was measured indirectly via a LacZ-based reporter enzyme assay. With this method the relative ArcA-P level is measured in the E. coli MC4100 derivative ASA12 via binding of ArcA-P to a modified cydAB promoter, which controls expression of the reading frame of lacZ, and was engineered to be ArcA-P selective [24].…”

Routes of phosphoryl group transfer during signal transmission and signal decay in the dimeric sensor histidine kinase ArcB

“…Plasmid pQE30ArcB 78 -778,H292Q,D576A,H717Q was created by replacing the MluI-HindIII fragment of plasmid pQE30 ArcB 78 -778,H292Q with the corresponding fragment of plasmid pQE30ArcB 78 -778,D576A,H717Q . Plasmids pBADHis-ArcB 78 -778 * (where the asterisk refers to specific amino acid replacements; see Table S1), used for L-arabinose-induced expression of His 6 -ArcB 78 -778 *, were constructed by cloning the NdeI-HindIII fragment from the respective pQE30ArcB 78 -778 * between the same restriction sites of plasmid pMX517 (8). Plasmid pMAL-ArcB 78 -778 , used for expression of ArcB 78 -778 fused to the maltose-binding protein (MBP-H1-D1-H2), was created by cloning the BamHI-HindIII fragment from pQE30ArcB 78 -778 between the same restriction sites of pMAL-c2x (New England Biolabs).…”

“…In contrast, ArcB is an unorthodox sensor kinase that contains three catalytic cytosolic domains: a transmitter domain (H1) with a conserved His 292 residue, a central receiver domain (D1) with a conserved Asp 576 residue, and a C-terminal histidine phosphotransfer domain (H2) with a conserved His 717 residue (6,7). In addition, the ArcB protein contains a functional leucine zipper (8) and a PAS domain (9), both located in the linker region, which is the segment connecting the transmembrane domain with the transmitter domain.…”

Ubiquinone and Menaquinone Electron Carriers Represent the Yin and Yang in the Redox Regulation of the ArcB Sensor Kinase

“…ArcB-enriched inverted vesicle preparation, purification of His 6 -tagged proteins, and phosphorylation assays. ArcB-enriched inverted membrane vesicles and His 6-tagged ArcB 78-778 , used in phosphorylation assays, were prepared as described previously (9,15,33). Phosphorylation assays were carried out at room temperature in the presence of 40 mM [␥- 32 P]ATP (specific activity, 2 Ci mmol Ϫ1 ; New England Nuclear), 33 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, and 10% glycerol.…”

“…Moreover, ArcB contains three catalytic domains: an N-terminal transmitter domain with a conserved His292 residue, a central receiver domain with a conserved Asp576 residue, and a C-terminal phosphotransfer domain with a conserved His717 residue (6,8). Finally, in the linker, that is, the region connecting the catalytic domains with the transmembrane domain, there are a functional leucine zipper (9) and a PAS domain (10).…”