Gregory, Gwendolyn J.2021-02-192021-02-192020https://udspace.udel.edu/handle/19716/28772Bacteria have evolved mechanisms that allow them to adapt to changes in osmolarity and some species have adapted to live optimally in high salinity environments such as the marine ecosystem. Many bacteria that live in high salt environments do so by the biosynthesis or uptake of compatible solutes, small organic molecules, that maintain the turgor pressure of the cell by balancing the internal and external osmolarity. The mechanisms by which bacteria regulate osmoadaptations in response to osmotic stress is poorly understood. Vibrio parahaemolyticus is a marine halophile that grows optimally at 0.5M NaCl, and also encounters changes in osmolarity, both hypo- and hyper-salinities. The bacterium copes with hyper-salinity by accumulating a range of compatible solutes by uptake from the environment via multiple compatible solute transporters: four betaine-carnitine-choline transporter (BCCT) family transporters and two ATP-Binding Cassette (ABC)-family transporters, ProU1 and ProU2. In addition, V. parahaemolyticus contains the compatible solute biosynthesis operons ectoine (ectABC-asp_ect) and glycine betaine (betIBA). This is triple the number of systems compared to those present in Escherichia coli and V. cholerae. We hypothesized that V. parahaemolyticus can grow optimally in high salinity conditions due to the presence of multiple compatible solute systems. The work in this dissertation seeks to elucidate how these evolutionary adaptations are coordinated and regulated at the transcriptional level and how these adaptations allow halophiles such as V. parahaemolyticus to thrive in high salinity environments. Chapter 2 focuses on the role of the quorum sensing regulatory system in controlling ectoine biosynthesis. Using genetic and biochemical analyses, we determined that low cell density regulator AphA was a direct positive regulator of ectoine biosynthesis, whereas the high cell density regulator OpaR was a direct negative regulator of the operon. This study also identified an additional regulator CosR that repressed the ectoine biosynthesis operon. In addition, CosR was positively regulated by the quorum sensing master regulators AphA and OpaR. This regulation mechanism formed a feed-forward loop to tightly control expression of ectoine. ☐ In chapter 3, we demonstrated that the CosR repressor is a global regulator of the osmotic stress response. We showed that CosR was a repressor of multiple compatible solute transporters and both biosynthesis operons. DNA binding assays demonstrated that CosR binds directly to each of the regulatory regions of these osmotic stress response genes. Plasmid-based reporter assays in E. coli demonstrated that CosR directly represses bccT3, both proU operons, and the glycine betaine operon. CosR distribution is widespread within Vibrionaceae, and in Gamma-proteobacteria in general, indicating that CosR regulation of the osmotic stress response is pervasive among bacteria. ☐ In chapter 4, we demonstrated for the first time that N-N dimethylglycine (DMG), dimethylsulfoniopropionate (DMSP), trimethylamine-N-oxide (TMAO), and γ-amino-N-butyric acid (GABA), amongst others, are effective compatible solutes for V. parahaemolyticus. DMG was a highly effective osmoprotectant and we show that it is also utilized as an osmoprotectant by V. harveyi, V. fluvialis, V. cholerae and V. vulnificus. We determined that, with the exception of BccT4, all of the BccTs in V. parahaemolyticus could uptake DMG. Of the four BCCT-family transporters present in V. parahaemolyticus, BccT1 had the broadest substrate uptake ability in terms of number and diversity of compounds. To determine how substrate coordination and transport by BccT1 has evolved, we examined amino acid residues known to be important for coordination of glycine betaine. Utilizing mutagenesis and functional complementation approaches, our results showed the binding pocket for glycine betaine is more flexible than for ectoine and DMG. ☐ In chapter 5, we examined the role of DMSP in Vibrio osmoprotection. DMSP is an organosulfur compound produced by phytoplankton in huge quantities in the marine environment and used as an osmoprotectant. Whether marine bacteria also use DMSP as an osmoprotectant is largely unknown. Our work demonstrated that DMSP is a highly effective compatible solute used by Vibrio species. Our work showed that DMSP is transported into bacterial cells using a single BCCT transporter BccT2 with high efficiency. ☐ In chapter 6, we set out to identify novel ectoine biosynthesis regulators. To accomplish this, we performed a DNA affinity chromatography-pulldown with the regulatory region of the ectABCasp_ect operon as a bait. Pulldowns were performed under inducing conditions and non-inducing conditions to capture both positive and negative regulators of the ectABCasp_ect operon. In total, we identified 37 candidate proteins that bound to the regulatory region. Four proteins were examined further, regulators NhaR, TorR, LeuO, and OmpR. Our work showed that NhaR is a repressor while LeuO is an activator of the ectABCasp_ect operon.Compatible solutesOsmotic stressQuorum sensingTransportersThesis1237815484https://doi.org/10.58088/df71-wj962020-10-13en