Abstract
Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. The receiver domain therefore plays a crucial role in controlling the phosphorelay to the response regulator. Furthermore, our in vitro assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro.
Highlights
Bacterial and plant cells have a two-component signal-transduction regulatory system for certain cellular responses [1, 2]
The autophosphorylation reaction of the H1137A mutant proceeded in almost the same manner as the wild-type protein. These results suggest that the phosphoryl group on the Asp residue (D1009) in the receiver domain is hydrolyzed in preference to the His residue (H721) in the histidine kinase (HK) domain or the His residue (H1137) in the histidine-containing phosphotransmitter (HPt) domain
We separated various site- phosphorylated species of hybrid sensor kinases by using Phos-tag SDS-PAGE and we examined the rates of the phosphoryl-transfer reactions of the enzymes qualitatively and quantitatively
Summary
Bacterial and plant cells have a two-component signal-transduction regulatory system for certain cellular responses [1, 2] This system consists of a histidine sensor kinase and a response regulator. Some histidine sensor kinases, known as hybrid sensor kinases, have a more complex type of phosphorelay consisting of two additional domains: a receiver domain containing a conserved Asp residue and a histidine-containing phosphotransmitter (HPt) domain. In such cases, signals are transmitted through a His-AspHis-Asp phosphorelay: a phosphoryl group moves successively from the HK domain to the first receiver domain, the HPt domain, and the receiver domain of the response regulator
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