Fig. 2: Identification of SAM-AMP from cells containing the activated Cmr complex.

a, HPLC analysis of E.coli extracts expressing the wild-type (WT) or mutant (ΔCy) B. fragilis Cmr system with target or non-target crRNA. The putative signal molecule was only observed for the activated system (trace i). b, Characterization of the extracted signal molecule by liquid chromatography–mass spectrometry (LC–MS) in positive mode. [M + H]⁺ and [M + 2H]²⁺ represent two different ionization forms. c, MS/MS analysis of the signal molecule with m/z 728.1963. d, The proposed structure of the signalling molecule, whose fragmentation pattern is shown by dashed arrows. The MS/MS data cannot distinguish between 2′–5′ and 3′–5′ phosphodiester bonds. The 3′–5′ phosphodiester bonds are more likely and is shown here, but a 2′–5′ bond cannot be completely ruled out. e, HPLC analysis of compounds synthesized by the purified wild-type B. fragilis Cmr complex in vitro. Cmr synthesizes the signal molecule SAM-AMP from ATP and SAM (trace i). Cmr also accepts SAH and sinefungin (SFG) as substrates (traces iii and v, respectively). Traces ii, iv and vi are control reactions. f, TLC analysis of in vitro reaction products. SAM, SAH and sinefungin plus ATP yielded radioactive products (red stars) but ATP alone did not. cA₃ generated by wild-type V. metoecus Cmr complex¹⁵ is shown for comparison. Uncropped HPLC and TLC data are presented in Supplementary Data Fig. 2.