Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background

Abstract

Cholera is a severe diarrheal disease that claims approximately 30,500 lives annually, mainly in regions with limited access to clean water. Vibrio cholerae, the causative agent, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. cholerae pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previous researchers created a transposon mutant library for V. cholerae to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed marked vibrio-specific activity. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. cholerae9s open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, luxO mutations transferred at a low frequency only with insertions near luxO. Our research uncovered density-dependent epistasis in motility genes, relevant to V. cholerae pathogenesis. Additionally, Grant Library mutants retain a plasmid conferring natural competence, enabling rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent.