Fig. 2. Distinct viral nucleases target CBASS and Pycsar immune signals.
a, Schematic and representative example of activity-guided biochemical fractionation and mass spectrometry (MS) to identify Acb1 candidate genes from phage T4. Fractions were collected from an S75 size-exclusion column and tested for 3′3′-cGAMP activity. In, crude lysate input. Data are representative of two independent experiments. b, Comparison of 3′3′-cGAMP cleavage by T4 lysate and recombinant Acb1. Data are representative of three independent experiments. c, Summary of HPLC analysis testing Acb1 substrate specificity (20-min incubation). Acb1 cleaves dinucleotide and trinucleotide CBASS signals containing one or more AMP. Data are presented as mean ± s.d. from n = 3 independent experiments. d, Bioinformatic analysis identifies candidate Apyc1 genes from genomic regions exclusive to cCMP-cleaving phages. TLC data are representative of two independent experiments. e, Comparison of cCMP cleavage by SBSphiJ lysate and recombinant Apyc1. Data are representative of three independent experiments. f, Summary of HPLC analysis testing Apyc1 substrate specificity (20-min incubation). Apyc1 cleaves all cNMP signals with equal efficiency. Data are presented as mean ± s.d. from n = 3 independent experiments. g, h, Schematics showing genes neighbouring T4 Acb1 (g) and SBSphiJ Apyc1 (h); dNMP, deoxyribosenucleoside monophosphate. i, Phylogenetic tree showing T4 Acb1 and 271 related protein sequences from phages, including 112 sequences derived from prophages. Colour strips indicate the order of the bacterial host. Red circles indicate proteins tested for cleavage of 3′3′-cGAMP and cAAA. j, Phylogenetic tree displaying SBSphiJ Apyc1 and 106 related protein sequences from phages. Colour strips indicate the genus of the bacterial host. Red circles indicate proteins tested for cleavage of cAMP and cCMP.