Functional Characterization in Vitro of All Two-component Signal Transduction Systems from Escherichia coli

Abstract

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Selfphosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

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

“…5). This is consistent with a recently published study of in vitro transphosphorylation tested for all possible combinations of E. coli histidine-sensor kinases and response regulators, which reported that phosphotransfer could occur between ArcB and RssB (Yamamoto et al 2005). However, RssB could also be phosphorylated by the chemotaxissensor kinase CheA, which seems of questionable physiological relevance in view of the highly dynamic CheA phosphorylation and dephosphorylation operating on a millisecond-to-second time scale (Sourjik 2005).…”

“…5D,E). This raises the question of whether one response regulator can allosterically affect phosphotransfer from a sensor kinase dimer to another response regulator, even though its own phosphorylation is less efficient (or even absent, as suggested by the interference of RssB with in vitro phosphotransfer in several other twocomponent systems, as reported by Yamamoto et al 2005). Such biochemical details and physiological implications of protein interactions and phosphotransfer reactions in two-component networks, in particular if they consist of phosphorelay systems, will have to be clarified in future studies.…”

“…However, RssB could also be phosphorylated by the chemotaxissensor kinase CheA, which seems of questionable physiological relevance in view of the highly dynamic CheA phosphorylation and dephosphorylation operating on a millisecond-to-second time scale (Sourjik 2005). Moreover, RssB seemed to interfere with transphosphorylation within many "cognate" two-component pathways, without being itself phosphorylated by the corresponding sensor kinases (Yamamoto et al 2005). Our data shown here not only provide evidence that phosphorylation of RssB by ArcB is physiologically relevant and results in active RssB able to bind S , but also demonstate intricate differences in ArcA and RssB phosphorylation by ArcB that can contribute to fine-tuning of the physiological roles of phosphorylated ArcA and RssB.…”

“…Which of all possible signal-transducing and regulatory circuits actually operate, may strongly depend on the actual conditions. This also means that large-scale studies-e.g., of putative interactions between the proteins of an organism (Butland et al 2005) or between all members of a certain protein family such as the two-component system proteins (Yamamoto et al 2005) under a single standard condition or in vitro-reveal an overall regulatory potential, but not the relevant regulatory circuitry under certain physiological conditions.…”

Determinants of specificity in two-component signal transduction

“…This process of pathway insulation following duplication has resulted in extant organisms harboring large sets of two-component pathways that can transduce signals without significant cross-talk [4,11,40]. Consistently, an examination of the specificity residues in two-component proteins from an individual organism typically reveals significant differences in almost all possible pairwise comparisons of kinases or regulators [37].…”