Murphy, Shannon G.2021-09-092021-09-092021-05Murphy_cornellgrad_0058F_12512http://dissertations.umi.com/cornellgrad:12512https://hdl.handle.net/1813/109780153 pagesSupplemental file(s) description: Chapter 3. Table S3, Chapter 3. Table S2, Chapter 3. Table S1, Chapter 3. Movie S1, Chapter 2. Table S2, Chapter 2. Table S1.Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands. The VSP-II island is 26-kb of mystery; most of its contents are not expressed under standard laboratory conditions, suggesting that its induction requires an unknown signal from the host or environment. In this work, we identify zinc starvation as one such cue. Bacteria must acquire trace metal cofactors, like zinc, from their surrounding environment for protein folding and catalysis. When zinc is scarce, bacteria employ a set of genes known as the “zinc starvation response.” This regulon is transcriptionally repressed by Zur when zinc is abundant. Using a variety of molecular approaches — including transposon mutagenesis, RNA-seq, and lacZ transcriptional reporters — we show that VSP-II encodes several novel members of the zinc starvation response. These novel Zur targets include a cell wall hydrolase (ShyB) and a transcriptional activator (VerA). ShyB is one of V. cholerae’s numerous endopeptidases, which cleave peptide crosslinks within the cell wall sacculus to allow for elongation. ShyB is sufficient to sustain cell elongation during zinc starvation; however, it appears to overlap in function with other V. cholerae endopeptidases. Unlike these other Zn2+-dependent endopeptidases, ShyB activity is uniquely resistance to metal chelators in vitro, suggesting it is well-adapted to metal-limited environments. The other Zur-regulated genes identified on VSP-II include a three gene operon encoding an AraC-family transcriptional activator. VerA induces expression of nearby chemotaxis receptor on VSP-II (AerB). AerB is a putative energy taxis receptor, which is predicted to sense cell metabolic status and relay a signal to chemotaxis proteins that effect flagellar rotation. We show that AerB causes V. cholerae cells to congregate in nutrient poor medium away from the air-liquid interface in an oxygen-dependent manner, suggesting it might enable V. cholerae to migrate towards more anoxic microenvironments. Collectively, we identify four Zur-regulated genes and one secondary target on VSP-II that are novel additions to the El Tor zinc starvation response. These data suggest that VSP-II may serve a role in a zinc-starved environment, such as within the human host or during colonization of chitinous surfaces in aquatic reservoirs.enAttribution-NonCommercial-ShareAlike 4.0 Internationalcell wallchemotaxisEl TorVibrio choleraeVibrio Seventh Pandemiczinc starvationdissertation or thesishttps://doi.org/10.7298/7meg-0q09