Extended Data Fig. 4: DncV is produced in V. cholerae and does not interact with SMX.
a) DncV is present in V. cholerae, and SMX treatment does not increase its levels. Immunoblot analysis of DncVAIA-6xHis (∼50 kDa) using anti-His antibodies. The experiment was done in Δcap3 background to avoid tag cleavage at the C-terminal24. Shown are three replicates of non-treated cells, and cells treated with 200 µg/ml SMX for 2 h (upper panel) or 5 h (lower panel) post SMX-addition. Overnight culture in both panels is shown as a reference. Total protein loaded per lane was controlled by Coomassie stain (Supplementary Fig. 1). b) Colony forming units of SMX treated cultures used for cGAMP quantification. Bar height represents mean over n independent biological replicates. c) CBASS overexpression exacerbates CBASS-antifolate interaction. Growth (AUC10h) of wild-type V. cholerae and CBASS-inducible mutant where the endogenous promoter region is replaced by araBAD promoter. L-arabinose and SMX concentrations are shown above and on the right side of the plot, respectively. d) Thermal shift assay confirms binding of 5-MTHF to DncV, but no direct SMX-DncV interaction. Shown are derivate melting curves (-dF/dT) over the applied temperature gradient of purified DncV treated with 5MTHF, SMX or buffer control. The maximum of the curves indicates the melting temperature (Tm) of DncV in each treatment, shifted to higher temperature only in the case of 5MTHF. e) Prevalence of CBASS and SMX resistance genes in all sequenced V. cholerae strains (Methods). n equals the number of independent biological replicates (b, c & d).
