Browsing by Author "Boyd, E. Fidelma"
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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 Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky and E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.Item Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology Journals, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky, E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.