PHOSPHOTRANSFER CIRCUITRY OF THE PUTATIVE MULTISIGNAL TRANSDUCER, ARCB, OF ESCHERICHIA-COLI - IN-VITRO STUDIES WITH MUTANTS

Recently we demonstrated the occurrence of a novel device of signal tr ansducers 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-phosphorylat ion sites were suggested to make a phosphotransfer circuit. To clarify this complex circuitry, we carried out a series of in vitro assays in volving a set of ArcB mutant proteins which have an amino acid substit ution 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; (ii i) ArcB-mediated phosphorylation of its truncated form (ArcB(c)) encom passing only the C-terminal phosphorylation site (His-717); (iv) phosp hotransfer from ArcB(c) to ArcA; and (v) phosphotransfer from ArcB(c) to ArcB. On the basis of these in vitro results, a complex circuitry w as revealed for the signal transducer ArcB. This evidence obtained in vitro supports the view that ArcB can serve as a powerful device for n ot only propagating multi-signals, but also making up signalling netwo rks, in ways more sophisticated than previously thought.