From motor protein to toxin: Mutations in the zonula occludens toxin (Zot) of Vibrio cholerae phage CTXΦ suggest a loss of phage assembly function

Prophages, i.e. dormant viruses residing in bacterial cells, are not just passive passengers in the bacterial host. Several prophage-encoded genes have been shown to be contributors to bacterial virulence by mediating antimicrobial resistance or by providing toxins. Other prophage genes exhibit beneficial effects on the host by modulating e.g. motility or biofilm formation. In this study, we used an in vivo phage assembly assay and tested an extensive array of single point mutations or their combinations found in Zot, the zonula occludens toxin encoded by the Vibrio cholerae phage CTX{phi}. The assay makes use of the highly homologous Zot-like protein g1p of the filamentous Coliphage M13, a motor protein that mediates the trans-envelope assembly and secretion of filamentous phages. We also measured the in vitro ATP hydrolysis of purified proteins, and quantified virus production in V. cholerae mediated by Zot or the Zot-like protein of the two Vibrio phages CTX{phi} and VFJ{phi}. In addition, we investigated sequence variations of the Walker motifs in Vibrio species using bioinformatics method, and revealed the molecular basis of ATP binding using molecular docking and molecular dynamics simulation based on the structure predicted by AlphaFold2. Our data indicates that g1p proteins in Vibrio can easily accumulate deleterious mutations and likely lose the ability to efficiently hydrolyse ATP, while the CTX{phi} Zot was further exapted to now act as an auxiliary toxin during the infection by Vibrio cholerae.