Fig. 3: Genes involved in PICMI115 activation.
a Quantification of PICMI115 activation after infection by Φ115pure in wild-type and single-gene mutants (e.g. Δint) was performed using qPCR. Standard curves facilitated the determination of DNA copies per 20 ng of DNA, normalized by bacterial copies (gyrB) per sample. Source data are provided as a Source Data file. Fold change was calculated by comparing DNA samples collected 30 min post-infection to the initial input before phage addition. The dashed line indicates the limit of detection for this assay. Bar charts represent the mean ± SEM from three independent experiments (individual dots). In the case of circular/concatemer PICMI and empty site, ANOVA revealed a p-value of <0.0001, and Tukey’s test indicated that Δint, Δiolg ΔalpA, were significantly different from the wild type (p < 0.05). For Phage, ANOVA yielded a p-value of <0.0069, and the Tukey test showed that none were different from the wild type (p > 0.05). b Phage progenies were produced using a derivative lacking the full PICMI115 (ΔPICMI115), a marked PICMI (PICMI115-CmR) and derivatives lacking a single gene as host. Phage DNAs were separated on an agarose gel and Southern blotted with PICMI115 or Φ115 probes. M: molecular marker (Smart ladder Eugentec). Images are representative of two independent experiments. Uncropped gel and blots are provided as a Source Data file. c For complementation assays, the PICMI115 genes (int/iolg and alpA), or as a control, gfp, were cloned under the conditional PBAD promoter. The resulting plasmids were transferred into the respective mutants. Strains were cultivated in the presence of 0.2% arabinose for PBAD induction and subsequently infected with Φ115pure for the indicated duration. The number of circular/concatemeric copies of PICMI115 was quantified by qPCR (Source Data file) and normalized to the Vibrio gyrB copy number per sample. Bar charts depict the mean ± SEM from three independent experiments (individual dots). Notably, the expression of alpA induces PICMI115 activation in the absence of phage (strain ΔalpA + PBAD-alpA in the presence of arabinose at time 0). A one-tailed t-student’s test comparing the ΔalpA complemented strain between 0 min and 30 min was not significant, while it showed significance (p < 0.05) at 60 min.
