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Metabolic turnover of cysteine-related thiol compounds at environmentally relevant concentrations by Geobacter sulfurreducens
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Department of Environmental Sciences, Rutgers University, NJ, New Brunswick, United States.
Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße, Münster, Germany.
Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, Umeå, Sweden.
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2023 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 13, article id 1085214Article in journal (Refereed) Published
Abstract [en]

Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic bacteria. In this work, we examined the production and turnover of nanomolar concentrations of LMM thiols with a chemical structure related to cysteine by the model iron-reducing bacterium Geobacter sulfurreducens. Our results show that G. sulfurreducens tightly controls the production, excretion and intracellular concentration of thiols depending on cellular growth state and external conditions. The production and cellular export of endogenous cysteine was coupled to the extracellular supply of Fe(II), suggesting that cysteine excretion may play a role in cellular trafficking to iron proteins. Addition of excess exogenous cysteine resulted in a rapid and extensive conversion of cysteine to penicillamine by the cells. Experiments with added isotopically labeled cysteine confirmed that penicillamine was formed by a dimethylation of the C-3 atom of cysteine and not via indirect metabolic responses to cysteine exposure. This is the first report of de novo metabolic synthesis of this compound. Penicillamine formation increased with external exposure to cysteine but the compound did not accumulate intracellularly, which may suggest that it is part of G. sulfurreducens’ metabolic strategy to maintain cysteine homeostasis. Our findings highlight and expand on processes mediating homeostasis of cysteine-like LMM thiols in strict anaerobic bacteria. The formation of penicillamine is particularly noteworthy and this compound warrants more attention in microbial metabolism studies.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2023. Vol. 13, article id 1085214
Keywords [en]
anaerobe bacteria, cysteine homeostasis, Geobacter sulfurreducens, low-molecular-mass thiols, penicillamine formation
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-204469DOI: 10.3389/fmicb.2022.1085214ISI: 000920099300001Scopus ID: 2-s2.0-85146997112OAI: oai:DiVA.org:umu-204469DiVA, id: diva2:1737774
Funder
Swedish Research Council, 2017–04537The Kempe Foundations, SMK-1753The Kempe Foundations, SMK-1243Umeå UniversityAvailable from: 2023-02-17 Created: 2023-02-17 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Unraveling the importance of thiol compounds on mercury speciation, uptake and transformation by the iron-reducer Geobacter sulfurreducens
Open this publication in new window or tab >>Unraveling the importance of thiol compounds on mercury speciation, uptake and transformation by the iron-reducer Geobacter sulfurreducens
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Betydelsen av tiolföreningar för Hg(II)-speciering, upptag och metylering av den järnreducerande mikroorganismen Geobacter sulfurreducens
Abstract [en]

The biogenic methylation of inorganic, divalent mercury (Hg(II)) by methylating microorganisms leads to formation and bioaccumulation of monomethyl mercury (MeHg) in the environment and can cause severe damage to ecosystems and human health. Diverse microorganisms carry the gene sequence hgcAB and are able to methylate Hg(II) intracellularly. The interplay of biological, chemical and physical parameters is driving mercury (Hg) transformation by microorganisms. The chemical speciation of Hg(II) with thiol compounds, both with dissolved low molecular mass (LMM) thiols and thiols present on microbial membrane surfaces, is one key factor for Hg availability and transformation. In this work the role of thiol compounds with respect to Hg speciation, uptake and transformation was studied by the iron-reducing model organisms Geobacter sulfurreducens. The turnover of dissolved thiols and the role of outer and inner membrane thiols was studied with novel experimental strategies.

In Paper I and II the formation of thiol compounds was studied under varying nutrient conditions. It was shown that the formation of LMM-thiol compounds was impacted by divalent iron, Fe(II). Furthermore, we showed the turnover of the small LMM-thiol cysteine to the branched LMM-thiol penicillamine, which was further amplified by the addition of exogenous cysteine or nutrients. This turnover of small to branched LMM-thiols impacted the Hg(II) speciation in methylation assays and the relative contribution between cysteine and penicillamine was important for Hg(II) availability, uptake and methylation. In addition, the partition of Hg(II) between the cell-adsorbed and dissolved phase was shifted towards the latter at higher LMM-thiol concentrations. Nutrient concentrations impacted cell physiology due to a shift to an active metabolism and a faster metabolization of LMM-thiols. We concluded that the interplay between thiol metabolism, Hg(II) speciation and cell physiology are key parameters for Hg(II) methylation by G. sulfurreducens. In Paper III The outer and inner membrane was characterized independently by two X-ray absorption spectroscopy techniques. The determination of the Hg speciation by both X-ray absorption spectroscopy techniques showed coherent results for both the outer and inner membrane of G. sulfurreducens. The concentration of thiol membrane groups was higher on the inner compared to the outer membrane. The differences between the outer and inner membrane suggested that thiol concentration and Hg coordination environment likely impact the Hg(II) internalization. The role of membrane thiols for Hg(II) uptake and transformation was further investigated in Paper IV by selectively blocking these functional groups. Partitioning and uptake of Hg was not affected by blocking the outer and inner membrane thiols of whole cell and spheroplast samples, respectively. However, the Hg(II) methylation was decreased by blocking thiols at the outer membrane, but no effect was observed by blocking thiols at the inner membrane. Blocking of membrane surface thiols changed the physiology in whole cells but not in spheroplasts. This result suggested weaknesses of the applied blocking approach. In addition, Hg(II) reduction was studied on the outer and inner membrane and showed the formation of liquid and gaseous elemental Hg, Hg(0), in Paper III and IV, respectively.

Overall, this work showed the central role of dissolved and cell-associated thiol compounds for Hg(II) uptake and the transformation reactions. Herby, concentration, compositions and distribution of thiols are crucial and impact the Hg(II) speciation, partitioning, uptake and availability for Hg(II) methylation and reduction. In addition, cell physiology is impacting the methylation potential and the turnover of LMM-thiol compounds. The role of membrane surface thiols for Hg(II) uptake was not fully identified, however such thiols were for the first time characterized selectively for the outer and inner membrane by X-ray absorption spectroscopy.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2023. p. 66
Keywords
mercury, low molecular mass thiols, speciation, uptake, methylation, reduction, Hg(0), Hg(II), MeHg, Geobacter sulfurreducens, ICP-MS, LC-MS/MS, X-ray absorption spectroscopy, cell physiology
National Category
Analytical Chemistry Inorganic Chemistry Geochemistry Environmental Sciences Microbiology
Identifiers
urn:nbn:se:umu:diva-207899 (URN)978-91-8070-089-4 (ISBN)978-91-8070-090-0 (ISBN)
Public defence
2023-06-02, KBE303, Stora hörsalen, byggnad KBC, Linnaeus väg 10, Umeå, 09:00 (English)
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Available from: 2023-05-12 Created: 2023-05-04 Last updated: 2023-05-04Bibliographically approved

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Gutensohn, MareikeBjörn, Erik

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