Abstract
Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. cholerae's pathogenesis hinges on its ability to switch between low- and high-cell-density gene regulatory states, enabling transmission between the host and the environment. Previously, a transposon mutant library for V. cholerae was created 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 wild-type genomic background. Library transfer was aided by a novel gDNA extraction method we developed using thymol, which also showed high lysis specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. cholerae's open reading frames. Whole-genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain in every strain analyzed. Notably, in no instance did the luxO mutation transfer into the wild-type background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal-relevant genetic contexts absent in the low-cell-density-locked library equivalent.Ordered transposon mutant libraries are essential tools for catalyzing research by providing access to null mutants of all non-essential genes. Such a library was previously generated for Vibrio cholerae, but whole-genome sequencing revealed that this library was made using a parent strain that is unable to exhibit cell-cell communication known as quorum sensing. Here, we utilize natural competence combined with a novel, high-throughput genomic DNA extraction method to regenerate the signaling incompetent V. cholerae ordered transposon mutant library in quorum-sensing-competent strain. Our library provides researchers with a powerful tool to understand V. cholerae biology within a genetic context that influences how it transitions from an environmentally benign organism to a disease-causing pathogen.