Extended Data Fig. 6: Control experiments showing details of toxicity during WonB overproduction.
From: West African–South American pandemic Vibrio cholerae encodes multiple distinct phage defence systems
(a) Toxicity assay evaluating the growth of 10-fold serial dilutions of cultures of V. cholerae strain A1552 derivatives with a chromosomally integrated transposon carrying the indicated genes under the control of the arabinose-inducible PBAD- promoter, in the absence (LB) and presence of induction (+ 0.2% ara), as compared to a negative control strain. (b) Toxicity assay evaluating the growth of 10-fold serial dilutions of V. cholerae strain A1552 and the indicated derivatives encoding site-directed variants of WonA, compared to the indicated deletion control strains, in the absence (LB) and presence (+ 0.2% ara) of WonB overproduction. Note that the control strains are also shown separately in Fig. 3a. (c) Western blots showing the protein levels of WonA and WonB in the strains used for the toxicity assay shown in (b). Samples were taken from exponentially growing cultures, 15 minutes after induction of PBAD-wonB. (d) Time-course microscopy snapshots showing the effect of WonB overproduction on cell morphology and cellular DNA content, as monitored by a HU-mNeonGreen fusion, compared to a negative control strain and a strain overproducing the inactive WonB[K71A] variant. Scale bar = 5 µm. The panels depict the full time-series for the examples presented in Fig. 3d. (e, f) Agarose gels evaluating the integrity of genomic DNA extractions (e) and comparison of wonA and wonB transcript levels as determined by qRT-PCR (f), prepared from cultures over time upon WonB overproduction following induction in exponentially growing cells at time 0, as compared to a negative control strain and a strain overproducing the inactive WonB[K71A] variant. All data are representative of the results of three independent experiments. Bar charts show the mean + s.d.
