Title: An ordered mutant library and its use in defining flagellar biosynthesis and biofilm formation in Vibrio cholerae
Description: V. cholerae in the environment exists both as individual motile cells and as members of surface attached biofilm communities. As a planktonic cell, V. cholerae forms a single polar sheathed flagella, but upon contacting a suitable surface the bacterium represses flagellar biosynthesis and instead produces a carbohydrate-rich matrix known as vibrio polysaccharide (VPS). The bacterium uses quorum sensing, c-diGMP signaling, and direct physical sensing by its flagella to coordinately regulate its transition between these two distinct developmental stages. The biosynthetic and regulatory systems needed for these processes are complex, and a systematic approach is required to fully define gene function at each stage in V. cholerae development. In this dissertation, I describe the creation of a sequence-defined mutant library in the V. cholerae O1 El Tor strain C6706. Automated sequencing analysis of 23,312 mutants allowed me to build a 3156-member subset library containing a representative insertion in every disrupted open reading frame. I used this subset library in a screen for motility to define almost all genes required for flagellar assembly and function in V. cholerae .

I chose to further examine three uncharacterized genes, flgOPT , identified in this screen. Transcriptional profiling demonstrates that they are members of the flagellar regulatory hierarchy and subcellular localization places the three proteins in the periplasmic space at the cell pole. Protein stability assays suggest that they function together as a complex, and the fact that the flagellar L-ring is required for both FlgT localization and FlgO stability suggests that the FlgOPT complex interacts directly with the flagellar basal body. Finally, I used a directed screen to further define the regulatory mechanisms that coordinate flagellar biosynthesis with repression of biofilm formation. I screened for positive regulators of VPS production and found that the Pst phosphate transporter, diguanylate cyclase VC2285, and uncharacterized gene vpsX (VC1349) are all necessary for proper biofilm formation. VpsX was further characterized using transcriptional profiling and colony morphology assays to show that its periplasmic domain functions in parallel to the VpsR and VpsT regulatory networks to control vps transcription and biofilm formation through an unknown mechanism.