Browsing by Author "Rozovsky, Sharon"
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Item Exploring Sulfur Sites in Proteins via Triple-Resonance 1H-Detected 77Se NMR(Journal of the American Chemical Society, 2023-11-15) Koscielniak, Janusz; Li, Jess; Sail, Deepak; Swenson, Rolf; Anklin, Clemens; Rozovsky, Sharon; Byrd, R. AndrewNMR spectroscopy has been applied to virtually all sites within proteins and biomolecules; however, the observation of sulfur sites remains very challenging. Recent studies have examined 77Se as a replacement for sulfur and applied 77Se NMR in both the solution and solid states. As a spin-1/2 nuclide, 77Se is attractive as a probe of sulfur sites, and it has a very large chemical shift range (due to a large chemical shift anisotropy), which makes it potentially very sensitive to structural and/or binding interactions as well as dynamics. Despite being a spin-1/2 nuclide, there have been rather limited studies of 77Se, and the ability to use 1H-indirect detection has been sparse. Some examples exist, but in the absence of a directly bonded, nonexchangeable 1H, these have been largely limited to smaller molecules. We develop and illustrate approaches using double-labeling of 13C and 77Se in proteins that enable more sensitive triple-resonance schemes via multistep coherence transfers and 1H-detection. These methods require specialized hardware and decoupling schemes, which we developed and will be discussed.Item Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria(mBio, 2021-07-27) Keffer, Jessica L.; McAllister, Sean M.; Garber, Arkadiy I.; Hallahan, Beverly J.; Sutherland, Molly C.; Rozovsky, Sharon; Chan, Clara S.Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.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.