Proteomic and Isotopic Response of Desulfovibrio vulgaris to DsrC Perturbation

Dissimilatory sulfate reduction is a microbial energy metabolism that can produce sulfur isotopic fractionations over a large range in magnitude. Calibrating sulfur isotopic fractionation in laboratory experiments allows for better interpretations of sulfur isotopes in modern sediments and ancient sedimentary rocks. The proteins involved in sulfate reduction are expressed in response to environmental conditions, and are collectively responsible for the net isotopic fractionation between sulfate and sulfide. We examined the role of DsrC, a key component of the sulfate reduction pathway, by comparing wildtype Desulfovibrio vulgaris DSM 644T to strain IPFG07, a mutant deficient in DsrC production. Both strains were cultivated in parallel chemostat reactors at identical turnover times and cell specific sulfate reduction rates. Under these conditions, sulfur isotopic fractionations between sulfate and sulfide of 17.3 ± 0.5 or 12.6 ± 0.5 were recorded for the wildtype or mutant, respectively. The enzymatic machinery that produced these different fractionations was revealed by quantitative proteomics. Results are consistent with a cellular-level response that throttled the supply of electrons and sulfur supply through the sulfate reduction pathway more in the mutant relative to the wildtype, independent of rate. We conclude that the smaller fractionation observed in the mutant strain is a consequence of sulfate reduction that proceeded at a rate that consumed a greater proportion of the strains overall capacity for sulfate reduction. These observations have consequences for models of sulfate reducer metabolism and how it yields different isotopic fractionations, notably, the role of DsrC in central energy metabolism.publishersversionpublishe

Energy flux couples sulfur isotope fractionation to proteomic and metabolite profiles in Desulfovibrio vulgaris

Funding Information: We thank S. Moore and D. Fike for bulk sulfur isotope analyses (WashU); M. Seuss for assistance with lipid\u2010H isotope analyses (Bradley lab, WashU); X. Feng (Dartmouth) and M. Osburn (Northwestern) for water H\u2010isotope analyses; and A. Sessions and J. Adkins (CalTech) for access to HPLC\u2010ICP\u2010MS. Metabolite analyses were performed by the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center (St. Louis, MO, USA). Funding was provided: by NASA Exobiology Award 13\u2010EXO13\u20100082 (ASB, WDL, JW), NSF\u2010EAR Award 1928309 (WDL), Washington University in St. Louis Department of Earth & Planetary Sciences Fossett Fellowship (WDL), the Walter and Constance Burke Fund at Dartmouth College (WDL), and the Fulbright\u2014Bunge & Born\u2014Williams Foundation Scholarship Program (FJB), Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (Portugal) through R&D unit MOSTMICRO\u2010ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (IACP), NSF GRFP [2017250547] (SP). Funding Information: We thank S. Moore and D. Fike for bulk sulfur isotope analyses (WashU); M. Seuss for assistance with lipid-H isotope analyses (Bradley lab, WashU); X. Feng (Dartmouth) and M. Osburn (Northwestern) for water H-isotope analyses; and A. Sessions and J. Adkins (CalTech) for access to HPLC-ICP-MS. Metabolite analyses were performed by the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center (St. Louis, MO, USA). Funding was provided: by NASA Exobiology Award 13-EXO13-0082 (ASB, WDL, JW), NSF-EAR Award 1928309 (WDL), Washington University in St. Louis Department of Earth & Planetary Sciences Fossett Fellowship (WDL), the Walter and Constance Burke Fund at Dartmouth College (WDL), and the Fulbright\u2014Bunge & Born\u2014Williams Foundation Scholarship Program (FJB), Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (Portugal) through R&D unit MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (IACP), NSF GRFP [2017250547] (SP). Publisher Copyright: © 2024 The Authors. Geobiology published by John Wiley & Sons Ltd.Microbial sulfate reduction is central to the global carbon cycle and the redox evolution of Earth's surface. Tracking the activity of sulfate reducing microorganisms over space and time relies on a nuanced understanding of stable sulfur isotope fractionation in the context of the biochemical machinery of the metabolism. Here, we link the magnitude of stable sulfur isotopic fractionation to proteomic and metabolite profiles under different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expression, abundances and isotopic composition of intracellular S metabolites, and lipid structures and lipid/water H isotope fractionation values. These coupled approaches reveal which protein abundances shift directly as a function of energy flux, those that vary minimally, and those that may vary independent of energy flux and likely do not contribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope observations with quantitative proteomics, we provide novel constraints for metabolic isotope models. Together, these results lay the foundation for more predictive metabolic fractionation models, alongside interpretations of environmental sulfur and sulfate reducer lipid-H isotope data.publishersversionpublishe

Energy flux couples sulfur isotope fractionation to proteomicand metabolite profiles in Desulfovibrio vulgaris

Fil: Leavitt, William D. Dartmouth College. Department of Earth Sciences, Hanover, New Hampshire; United States of America.Fil: Leavitt, William D. Washington University in St. Louis. Department of Earth and Planetary Sciences, Missouri; United States of America.Fil: Waldbauer, Jacob. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Venceslau, Sofia S. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Sim, Min Sub. Seoul National University. School of Earth and Environmental Sciences; South Korea.Fil: Zhang, Lichun. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Flavia Jaquelina Boidi. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Flavia Jaquelina Boidi. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina.Fil: Flavia Jaquelina Boidi. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Plummer, Sydney. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Díaz, Julia M. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Pereira, Inês A. C. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Bradley, Alexander S. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Bradley, Alexander S. Washington University in St. Louis. Division of Biology and Biomedical Sciences, Missouri; United States of America.Abstract: Microbial sulfate reduction is central to the global carbon cycle and the redox evolu-tion of Earth's surface. Tracking the activity of sulfate reducing microorganisms overspace and time relies on a nuanced understanding of stable sulfur isotope fractiona-tion in the context of the biochemical machinery of the metabolism. Here, we link themagnitude of stable sulfur isotopic fractionation to proteomic and metabolite profilesunder different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expres-sion, abundances and isotopic composition of intracellular S metabolites, and lipidstructures and lipid/water H isotope fractionation values. These coupled approachesreveal which protein abundances shift directly as a function of energy flux, those thatvary minimally, and those that may vary independent of energy flux and likely do notcontribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope obser-vations with quantitative proteomics, we provide novel constraints for metabolic iso-tope models. Together, these results lay the foundation for more predictive metabolicfractionation models, alongside interpretations of environmental sulfur and sulfatereducer lipid-H isotope data.info:eu-repo/semantics/publishedVersionFil: Leavitt, William D. Dartmouth College. Department of Earth Sciences, Hanover, New Hampshire; United States of America.Fil: Leavitt, William D. Washington University in St. Louis. Department of Earth and Planetary Sciences, Missouri; United States of America.Fil: Waldbauer, Jacob. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Venceslau, Sofia S. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Sim, Min Sub. Seoul National University. School of Earth and Environmental Sciences; South Korea.Fil: Zhang, Lichun. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Flavia Jaquelina Boidi. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Flavia Jaquelina Boidi. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina.Fil: Flavia Jaquelina Boidi. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Plummer, Sydney. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Díaz, Julia M. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Pereira, Inês A. C. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Bradley, Alexander S. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Bradley, Alexander S. Washington University in St. Louis. Division of Biology and Biomedical Sciences, Missouri; United States of America

Structural and spectroscopic characterization of a HdrA-like subunit from Hyphomicrobium denitrificans

Funding Information: We thank Laurenz Heidrich for help with statistical analyses. This work was supported by grant Da 351/8‐1 (to CD) from the Deutsche Forschungsgemeinschaft and Fundação para a Ciência e Tecnologia (Portugal) (grant PTDC/BIA‐BQM/29118 and R&D units MOSTMICRO‐ITQB (UIDB/04612/2020 and UIDP/04612/2020), and European Union's Horizon 2020 research and innovation program (grant agreement No 810856). Open access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Many bacteria and archaea employ a novel pathway of sulfur oxidation involving an enzyme complex that is related to the heterodisulfide reductase (Hdr or HdrABC) of methanogens. As a first step in the biochemical characterization of Hdr-like proteins from sulfur oxidizers (sHdr), we structurally analyzed the recombinant sHdrA protein from the Alphaproteobacterium Hyphomicrobium denitrificans at 1.4 Å resolution. The sHdrA core structure is similar to that of methanogenic HdrA (mHdrA) which binds the electron-bifurcating flavin adenine dinucleotide (FAD), the heart of the HdrABC-[NiFe]-hydrogenase catalyzed reaction. Each sHdrA homodimer carries two FADs and two [4Fe–4S] clusters being linked by electron conductivity. Redox titrations monitored by electron paramagnetic resonance and visible spectroscopy revealed a redox potential between −203 and −188 mV for the [4Fe–4S] center. The potentials for the FADH•/FADH− and FAD/FADH• pairs reside between −174 and −156 mV and between −81 and −19 mV, respectively. The resulting stable semiquinone FADH• species already detectable in the visible and electron paramagnetic resonance spectra of the as-isolated state of sHdrA is incompatible with basic principles of flavin-based electron bifurcation such that the sHdr complex does not apply this new mode of energy coupling. The inverted one-electron FAD redox potentials of sHdr and mHdr are clearly reflected in the different FAD-polypeptide interactions. According to this finding and the assumption that the sHdr complex forms an asymmetric HdrAA′B1C1B2C2 hexamer, we tentatively propose a mechanism that links protein-bound sulfane oxidation to sulfite on HdrB1 with NAD+ reduction via lipoamide disulfide reduction on HdrB2. The FAD of HdrA thereby serves as an electron storage unit. Database: Structural data are available in PDB database under the accession number 6TJR.publishe

Proteomic and Isotopic Response of Desulfovibrio vulgaris to DsrC Perturbation

Dissimilatory sulfate reduction is a microbial energy metabolism that can produce sulfur isotopic fractionations over a large range in magnitude. Calibrating sulfur isotopic fractionation in laboratory experiments allows for better interpretations of sulfur isotopes in modern sediments and ancient sedimentary rocks. The proteins involved in sulfate reduction are expressed in response to environmental conditions, and are collectively responsible for the net isotopic fractionation between sulfate and sulfide. We examined the role of DsrC, a key component of the sulfate reduction pathway, by comparing wildtype Desulfovibrio vulgaris DSM 644T to strain IPFG07, a mutant deficient in DsrC production. Both strains were cultivated in parallel chemostat reactors at identical turnover times and cell specific sulfate reduction rates. Under these conditions, sulfur isotopic fractionations between sulfate and sulfide of 17.3 ± 0.5‰ or 12.6 ± 0.5‰ were recorded for the wildtype or mutant, respectively. The enzymatic machinery that produced these different fractionations was revealed by quantitative proteomics. Results are consistent with a cellular-level response that throttled the supply of electrons and sulfur supply through the sulfate reduction pathway more in the mutant relative to the wildtype, independent of rate. We conclude that the smaller fractionation observed in the mutant strain is a consequence of sulfate reduction that proceeded at a rate that consumed a greater proportion of the strains overall capacity for sulfate reduction. These observations have consequences for models of sulfate reducer metabolism and how it yields different isotopic fractionations, notably, the role of DsrC in central energy metabolism

A novel NADH dehydrogenase family widespread in bacteria

info:eu-repo/semantics/nonPublishe

The DsrD functional marker protein is an allosteric activator of the DsrAB dissimilatory sulfite reductase

Funding Information: ACKNOWLEDGMENTS. We thank Christiane Dahl from the University of Bonn for critically reading the manuscript. This work was funded by the Fundac¸ão para a Ciência e Tecnologia (Portugal) through Fellowships PD/BD/135488/2018 (A.C.C.B.), PD/BD/128204/2016 (D.F.), and SFRH/BPD/79823/2011 (S.S.V.); Grants PTDC/BIA-MIC/6512/2014 and PTDC/BIA-BQM/29118/2017; and Research unit Molecular and Structural Microbiology (MOSTMICRO-ITQB) (Grants UIDB/04612/ 2020 and UIDP/04612/2020). The European Union’s Horizon 2020 Research and Innovation Program (Grant Agreement No. 810856) is also acknowledged. Publisher Copyright: © 2022 National Academy of Sciences. All rights reserved.Dissimilatory sulfur metabolism was recently shown to be much more widespread among bacteria and archaea than previously believed. One of the key pathways involved is the dsr pathway that is responsible for sulfite reduction in sulfate-, sulfur-, thiosulfate-, and sulfite-reducing organisms, sulfur disproportionators and organosulfonate degraders, or for the production of sulfite in many photo- and chemotrophic sulfur-oxidizing prokaryotes. The key enzyme is DsrAB, the dissimilatory sulfite reductase, but a range of other Dsr proteins is involved, with different gene sets being present in organisms with a reductive or oxidative metabolism. The dsrD gene codes for a small protein of unknown function and has been widely used as a functional marker for reductive or disproportionating sulfur metabolism, although in some cases this has been disputed. Here, we present in vivo and in vitro studies showing that DsrD is a physiological partner of DsrAB and acts as an activator of its sulfite reduction activity. DsrD is expressed in respiratory but not in fermentative conditions and a ΔdsrD deletion strain could be obtained, indicating that its function is not essential. This strain grew less efficiently during sulfate and sulfite reduction. Organisms with the earliest forms of dsrAB lack the dsrD gene, revealing that its activating role arose later in evolution relative to dsrAB.publishersversionpublishe

MALDI-TOF spectrum of DsrC.

<p>30 µM DsrC was incubated with 30 µM persulfurated DsrEFH for 1 hour at 30°C. The transfer of up to three sulfur atoms from DsrEFH to DsrC is documented by mass increase in steps of 32 Da.</p

MALDI-TOF spectra of persulfurated DsrC proteins.

<p>30 µM of unmodified DsrC <b>(A)</b> and DsrC mutant proteins carrying a Cys-Ser mutation in DsrC-Cys100 (<b>B</b>) or DsrC-Cys111 (<b>C</b>) were incubated with 2 µM IscS and 2 mM cysteine or with 2 mM sulfide. Note that for the unmodified DsrC results are shown for the double charged molecule.</p