Figure 3. Biochemical activities associated with reconstituted AgrC-I, see also Figure S3.

(A) AgrC-I autokinase activity: AgrC-I discs were phosphorylated with 1 mM ATP at 37 °C for 3 minutes. AIP peptide was included as indicated. pHis levels in AgrC-I were analyzed by anti-pHis western blotting (upper). The blot was CBB stained thereafter as a loading control (lower). (B and C) Autokinase assays with AgrC-I variants or AIP analogs: nanodiscs containing AgrC-I wild-type (WT), constitutive mutant R238H (Con), DHp-inactive mutant H239Q (His) or CA-inactive mutant G394A/G396A (Kin) were phosphorylated with 20 μM [γ-³²P] ATP at 37 °C for 40 minutes. WT-AgrC-I was treated with indicated AIP peptides; truncated AIP-I is referred to as tr-I or tr-AIP-I. pHis levels were analyzed by autoradiography in (B) or quantified using scintillation counting in (C). Bargraph shows CPM values normalized to the AIP-free reaction. Error bars = SD (n = 3). (D and E) Lipid dependence of AgrC-I autokinase activity: AgrC-I discs reconstituted with various lipids (D) or DMPC and DMPG at a series of ratios (E) were autophosphorylated in the presence or absence of excess AIP-I and analyzed by anti-pHis western-blotting as in A. (F) Phospho-relay from AgrC-I to AgrA-I: AgrA-I was incubated with AgrC-I discs labeled with [γ-³²P]-ATP and indicated AIPs. Aliquots removed at various time-points were analyzed by SDS-PAGE followed by autoradiography. (G) Quantification of the autoradiograms in panel F showing decay of pHis levels in AgrC-I (normalized to t = 0) as a function of time. Error bars = range (n = 2). (H) Dephosphorylation of AgrA-I: AgrA-I (2 μM) was phosphorylated by incubation with the constitutively active chimeric protein GC214 (0.1 μM dimer – see text for details) labeled with a [³²P]-phosphoryl group. This reaction was then supplemented with 2μM AgrC-I discs and AIPs as indicated.