Quorum sensing orchestrates parallel cell death pathways in <i>Vibrio cholerae</i> via Type 6 secretion-dependent and -independent mechanisms

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In <i>Vibrio cholerae</i> colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type 6 secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control <i>t6ss</i> gene expression nor T6SS-mediated killing activity. Nonetheless, a Δ<i>hapR</i> strain displays no cell death at the colony rim. RNA-Sequencing (RNA-Seq) analyses reveal that HapR activates expression of an operon containing four genes of unknown function, <i>vca0646-0649.</i> Epistasis and overexpression studies show that two of the genes, <i>vca0646</i> and <i>vca0647</i>, are required to drive cell death in both a Δ<i>hapR</i> and a Δ<i>hapR</i> Δ<i>t6ss</i> strain. Thus, <i>vca0646</i>-<i>0649</i> are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in <i>V. cholerae</i>.