FIG 3.

MnoS shows autophosphorylation and phosphotransferase activity in vitro. (A) Conserved domains present in the MnoS protein from the N to C terminus. GAF domain corresponds to a domain present in cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA proteins. The histidine kinase domain is represented as HisKA_3, whereas the HATPase domain is responsible for ATPase activity of the protein. (B) Autoradiogram for the autophosphorylation activities of MnoS and MnoR. The phosphorylation activity was carried out for the specified time (in minutes) in the presence or absence of [γ-³²P]ATP. The data show that MnoS undergoes autophosphorylation upon incubation with [γ-³²P]ATP, whereas MnoR is unable to perform autophosphorylation reaction under similar conditions. (C) Autoradiogram for the phosphotransferase activity of MnoS (presented in all of the lanes) toward either MnoR or MnoR^D60A proteins. The position of both MnoS and MnoR proteins in the autoradiogram is marked. The reaction was carried out for the specified time (in seconds). Only the wild-type MnoR undergoes phosphorylation upon incubation with phosphorylated MnoS, which is accompanied by the loss of signal from MnoS.