Browsing by Author "Gregory, Gwendolyn J."
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Item NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression(Applied and Environmental Microbiology, 2023-06-06) Boas Lichty, Katherine E. Boas; Gregory, Gwendolyn J.; Boyd, E. FidelmaBacteria accumulate compatible solutes to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, among others, 3 regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed in exponential and stationary phase cells. PectA-gfp expression was significantly repressed in the ΔleuO mutant and significantly induced in the ΔnhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δhns mutant, PectA-gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a ΔleuO/Δhns mutant, PectA-gfp showed reduced expression, but significantly more than ΔleuO, suggesting H-NS and LeuO interact to regulate ectoine expression. However, ΔnhaR/Δhns had no additional effect compared to ΔnhaR, suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a PleuO-gfp reporter analysis was examined that showed significantly increased expression in the ΔleuO, Δhns, and ΔleuO/Δhns mutants compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance outside of regulating ectoine biosynthesis gene expression. IMPORTANCE Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and showed that, similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.Item Regulation and evolutionary adaptations of the osmotic stress response in Vibrio parahaemolyticus(University of Delaware, 2020) Gregory, Gwendolyn J.Bacteria 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.