Phosphotransfer circuitry of the putative multi‐signal transducer, ArcB, of Escherichia coli: in vitro studies with mutants

Abstract

Recently we demonstrated the occurrence of a novel device of signal transducers in Escherichia coli. This class of bacterial sensory kinases, typified by ArcB and BarA, possesses two phospho-donor (His) sites, together with a phospho-accepting (Asp) site. These multi-phosphorylation sites were suggested to make a phosphotransfer circuit. To clarify this complex circuitry, we carried out a series of in vitro assays involving a set of ArcB mutant proteins which have an amino acid substitution at each putative phosphorylation site (His-292, Asp-576 and His-717). By these in vitro phosphorylation and/or phosphotransfer assays, the followings were assessed: (i) ArcB autophosphorylation; (ii) ArcB-mediated phosphorylation of the cognate response regulator, ArcA; (iii) ArcB-mediated phosphorylation of its truncated form (ArcBc) encompassing only the C-terminal phosphorylation site (His-717); (iv) phosphotransfer from ArcBc to ArcA; and (v) phosphotransfer from ArcBc to ArcB. On the basis of these in vitro results, a complex circuitry was revealed for the signal transducer ArcB. This evidence obtained in vitro supports the view that ArcB can serve as a powerful device for not only propagating multi-signals, but also making up signalling networks, in ways more sophisticated than previously thought.

Tuning of the porin expression under anaerobic growth conditions by His‐to‐Asp cross‐phosphorelay through both the EnvZ‐osmosensor and ArcB‐anaerosensor in Escherichia coli

“…The results showed that the altered phenotype of the DarcB cells is fully complemented by the plasmidborn arcB gene. We employed another single copynumber plasmid (pIA022), which carries a mutant arcB gene encoding a defective ArcB protein with the amino acid substitution at the phosphorylated histidine site (His-717 to Leu) in its HPt domain (Tsuzuki et al 1995). When this particular plasmid was used for the complementation analysis, the mutant arcB gene failed to do so (Fig.…”

“…To examine the presumed cross-phosphorelay between ArcB and OmpR in vitro, the E. coli cytoplasmic membrane containing ArcB was puri®ed, as was previously described (Tsuzuki et al 1995). The cytoplasmic membrane containing the mutant ArcB protein with the amino acid substitution (His-717 to Leu) was also isolated.…”

“…Urea-treated cytoplasmic membranes containing ArcB were isolated, as previously described (Tokishita et al 1990;Tsuzuki et al 1995). The OmpR protein was puri®ed, as previously described ).…”

“…However, recent studies have shown that the reality is more complex, because ArcB is a hybrid sensor having multiple (at least three) phosphorelay domains, a His-kinase, a receiver and an HPt domain (see Fig. 1) (Ishige et al 1994;Tsuzuki et al 1995;Kato et al 1997). A multistep His-to-Asp phosphorelay mechanism was proposed for the ArcB-to-ArcA phosphotransfer pathway, in which the newly uncovered HPt domain plays a crucial role (Tsuzuki et al 1995;Georgellis et al 1997).…”

“…1) (Ishige et al 1994;Tsuzuki et al 1995;Kato et al 1997). A multistep His-to-Asp phosphorelay mechanism was proposed for the ArcB-to-ArcA phosphotransfer pathway, in which the newly uncovered HPt domain plays a crucial role (Tsuzuki et al 1995;Georgellis et al 1997). Nevertheless, the importance of ArcB's complex structure has not yet been fully understood.…”