Developing Tools to Monitor Vibrio cholerae Type VI Secretion During Range Expansion on Solid Surfaces

Abstract

The waterborne bacterium Vibrio cholerae causes the fatal cholera diarrhea, and thrives in aquatic environments attached to chitinous surfaces with other bacteria. V. cholerae has been used to study natural competence, which promotes DNA uptake through horizontal gene transfer (HGT). A newly described Type VI Secretion System (T6SS) that V. cholerae employ to kill neighboring cells could increase the chance for a competent V. cholerae to take up and utilize the released DNA. The T6SS apparatus, which is similar to a bacteriophage spike, injects toxic effector proteins into prey cells causing lysis that can aid V. cholerae in acquiring DNA from its neighbors. Although previous studies have investigated the regulation and mechanism of T6SS in V. cholerae at a single cell level, the role of T6SS in the dynamics of mixed bacterial populations, such as those found in the environment, remains poorly understood. Fluorescence microscopy was used in this study to determine the effect of T6SS by growing V. cholerae predators and isogenic prey populations on agar surfaces as a model for competitors undergoing spatial expansion. These conditions may mimic conditions encountered on chitinous surfaces in marine settings. In this study, plasmids expressing green and red fluorescent proteins were constructed to visualize the predator and prey populations. Preliminary spatial expansion experiments were ultimately performed using different fluorescent protein alleles that were encoded on the chromosome of competing bacteria. Preliminary results suggest that the presence of a functional T6SS plays an important role in competition. The tools developed for this study are now being used to study interaction between diverse V. cholerae isolates, and as a platform for experimental evolution.