The intake of mannitol, a sugar produced by plants and algae, by Vibrio cholerae increases biofilm formation and thereby persistence of the pathogenic bacterium. The process of mannitol uptake is mediated by the mannitol phosphotransferase system (PTS). Within this system, the protein MtlA is directly responsible for the intracellular transport of mannitol, and is encoded by the mtlA gene located within a group of genes (mtlA, D, and R) on the mannitol (mtl) locus. While it is known that protein MtlR represses the expression of mtlA at the transcriptional level, the mechanism of this repression is unknown. The aim of this project is to better understand the dynamics between MtlR and other protein regulators of the mtl locus, such as the CRP activator protein, to gain insight into the mechanism of MtlR regulation of mtlA expression. This was accomplished through assessment of MtlR and CRP levels across V. cholerae via Western Blots, as well as through measuring mtlA expression in the absence of CRP and/or MtlR via LacZ assays. Previous work has shown that MtlR levels fluctuate depending on available carbon source, but we found CRP levels to be relatively unaffected. We also found that while deletion of the mtlR gene increases mtlA expression, this is not observed when the crp gene is also deleted, suggesting that MtlR repression is secondary to CRP activation of mtlA expression. This work helps shed light on possible mechanisms by which MtlR might be negatively regulating the expression of mtlA and overall intracellular mannitol intake by V. cholerae.Support or Funding InformationNational Institutes of Health; The Camille and Henry Dreyfus Foundation