Time-dependent changes in levels of phosphorylation of wild-type EvgS (WT) and the two mutants D1009A and H1137A.

<p>(A) Autophosphorylation reactions of WT, D1009A, and H1137A were performed in the presence of 30 mM ATP and analyzed by Phos-tag SDS-PAGE. The incubation times are shown above each lane. Each lane contained 2 μg of protein. (B) The values of the ratios of the phosphorylated forms to the total proteins were calculated by densitometry and are plotted versus the reaction times. (C) The values of the ratios for each phosphorylated form derived from the WT to the total proteins were calculated and are plotted versus the reaction times. Each plot was gained as average of three independent experiments using the same sample. Standard deviations were within almost 20%.</p

<i>In vivo</i> analysis of EvgS phosphorylation.

<p>(A) β-galactosidase assays to confirm the arabinose-induced activities of EvgS, EvgS1, and their mutants H721A, D1009A, and H1137A, expressed by a pBAD vector. When using <i>E</i>. <i>coli</i> MG1655 <i>evgS ydeP-lacZ</i> as a host cell, the activation of the EvgAS system induces transcription of <i>lacZ</i>, which controls the promoter of <i>ydeP</i> (evgA regulon). For both EvgS and EvgS1, the proteins without the Ala substitution at each phosphorylation site are described as WT. Data were represented by mean values and standard deviation bars obtained from three independent experiments. (B) <i>In vivo</i> profiling of autophosphorylation reactions of EvgS, EvgS1, and their mutants by using Phos-tag SDS-PAGE followed by Western blotting. Half of each lysate samples was boiled for 3 min to hydrolyze phosphoryl groups on His and Asp residues. Each lane contains 20 μg of the crude cellular protein. Bands for each of the two site-specific phosphorylated forms were assigned and are shown by arrows on the right-hand side of the panel.</p

Autophosphorylation reactions of other hybrid sensor kinases, BarA and ArcB.

<p>(A) Phosphorylation sites in each domain of BarA and ArcB are shown. (B) Autophosphorylation reactions of wild-type BarA (WT) and the two mutants D718A and H861A were performed in the presence of 10 mM ATP, and the products were then analyzed by Phos-tag SDS-PAGE. The incubation times are shown above each lane. Each lane contained 2 μg of protein. Bands for each of the two site-specific phosphorylated forms were assigned and are shown by arrows on the right-hand side of the panel. (C) Autophosphorylation reactions of wild-type ArcB (WT) and the two mutants D576A and H717A were performed and analyzed in the same manner as BarA in B. (D) Values of the ratio of the phosphorylated forms to the total proteins in B and C were calculated by densitometry and are plotted versus the reaction times. Each plot was gained as average of three independent experiments using the same sample. Standard deviations were within almost 20%.</p

Comparisons of the reaction modes for autophosphorylation of wild-type EvgS and of the mutant of D1009A.

<p>(A) Analysis of the kinetics of autophosphorylation of the mutant D1009A. The profile of the phosphorylation ratio (%) versus the reaction times (the same profile as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132598#pone.0132598.g004" target="_blank">Fig 4B</a>) was fitted to a first-order kinetic model; the half-life time (<i>t</i>_1/2) was 10 min (<i>k</i>' = 1 × 10⁻³ s^–1). In the early stages of the reaction (≤15 min), a plot of the natural logarithm of the phosphorylation ratio (%) against the reaction time formed a straight line. (B) Two schemes (scheme 1 and 2) for the autophosphorylation of the mutant D1009A dimer, (D1009A)₂, suggested by the kinetic analysis. Scheme 1 is a model of sequential autophosphorylation reactions. Scheme 2 is a model of a flip-flop autophosphorylation reaction followed by an exchange of subunits of the (D1009A)₂ dimer. (C) Scheme for the autophosphorylation reaction of wild-type EvgS. In the presence of sufficient ATP, a cycle of phosphorylation, intramolecular phosphate transfer, dephosphorylation is repeated.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p

Schematic representation of the EvgAS system.

<p>The system is composed of a hybrid sensor kinase (EvgS) and a cytoplasmic response regulator (EvgA). Both EvgS and EvgA function as dimers. Each subunit of EvgS contains an HK domain, a receiver domain, and an HPt domain. The HK domain consists of two subdomains: an ATP-binding subdomain and a dimerization-inducing His-containing subdomain. The ATP-binding subdomain can catalyze the transfer of the γ-phosphate of ATP to the H721 residue in the dimerization-inducing His-containing subdomain of the opposing subunit. The phosphoryl group would be then transferred in a <i>cis</i> manner to the D1009 residue of the receiver domain and then onward to the H1137 residue in the HPt domain. Phosphorylated H1137 would serve as a substrate for reversible phosphorylation of D52 in the receiver domain of EvgA.</p