Fig. 1
RetS downregulates GacS signalling via three distinct mechanisms. a–i Mechanism 1—RetS takes phosphoryl groups from GacS-P. Phosphorimages of SDS-PAGE gels showing phosphotransfer from a–d GacSc-P to a buffer control, b RetSc, c RetSc(HDA) and d RetSc(HAA), and from e–h GacSc(HAQ)-P to e buffer control, f RetSc, g RetSc(HDA) and h RetSc(HAA). Experiments were repeated five times and a representative image shown. i Cartoon depicting mechanism 1, where phosphotransfer occurs from the His residue in the HisKA domain of GacS to D858 in REC2 of RetS. j–l Mechanism 2—RetS has transmitter phosphatase activity against GacS-P. j Phosphorimages of SDS-PAGE gels measuring the dephosphorylation of GacSc-P alone (top) and with RetSHK (bottom). k GacS-P dephosphorylation half-times in the presence of various RetS mutant proteins. Error bars show SEM from eight replicates. *Significantly faster than GacSc-P autodephosphorylation (P < 0.05, one-way ANOVA). l Cartoon depicting mechanism 2, where RetS uses transmitter phosphatase activity against the REC domain of GacS-P. m–o Mechanism 3—RetS inhibits the GacS autophosphorylation reaction. Phosphorimages of SDS-PAGE gels comparing the autophosphorylation of: m GacSc(HAQ) on its own (top), with RetSc(HAA) (middle), and with RetSc(T428A,HDA) (bottom). n GacSHK on its own (top) and with RetSHK (bottom). Experiments were repeated eight times and a representative image shown. o Cartoon depicting mechanism 3, where the catalytic core of RetS blocks the autophosphorylation of GacS
