Fig. 3: ThsB1, ThsB2 and Mhp form an active complex in vivo.
From: Bacterial TIR-based immune systems sense phage capsids to initiate defense
a, Immunoblot analysis of proteins extracted from staphylococci expressing hexahystidyl- (H) or Flag- (F) tagged versions of ThsB1 or ThsB2, uninfected (−) or infected with wild type or Δgp43 ΦNM1 phage, either before (input) or after affinity chromatography using a cobalt resin. Proteins were separated by SDS–PAGE and electrotransferred to a polyvinylidene difluoride membrane. Tagged proteins were detected with antihexahystidyl (a-His) or anti-Flag (a-Flag) antibodies and chemiluminescence staining. b, Coomassie Blue-stained SDS–PAGE of proteins isolated after cobalt resin affinity chromatography in the experiment described in a. His6-ThsB1, 41.8 kDa; ThsB2-His6, 23.4 kDa; Mhp, 36.8 kDa. Protein molecular weight (kDa) markers are shown. c, HPLC analysis of the products resulting from the incubation of the proteins purified from staphylococci expressing His6-ThsB1 and ThsB2-Flag, uninduced or induced with wild type or Δgp43 ΦNM1 phage, with NAD+, using a cobalt resin. Retention times (RT) of reactants and products are marked with dashed lines. Absorbance (Abs) at 250 nm is reported. d, The same as in c but using proteins extracted from staphylococci expressing Flag-ThsB1 and ThsB2-His6. e, Tenfold serial dilutions of Φ80α-vir on lawns of S. aureus RN4220 harbouring plasmids carrying either an incomplete (thsB1/B2) or full (thsA/B1/B2) Thoeris operon carrying wild type or mutant versions of thsB1 or thsB2. f, Coomassie Blue-stained SDS–PAGE of proteins isolated from staphylococci expressing ThsB1 with ThsB2, ThsB2-His6 or ThsB2F6A-His6, uninfected or infected with Φ80α-vir, after cobalt resin affinity chromatography. Protein molecular weight (kDa) markers are shown. g, HPLC analysis of the products resulting from the incubation of the proteins purified from staphylococci expressing ThsB1 with ThsB2-His6 or ThsB2F6A-His6, uninfected or infected with Φ80α-vir, with NAD+, using a cobalt resin. RT of reactants and products is marked with dashed lines. Absorbance (Abs) at 250 nm is reported.
