Metabolic turnover of cysteine-related thiol compounds at environmentally relevant concentrations by Geobacter sulfurreducens

Abstract

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.

The Combined Effect of Hg(II) Speciation, Thiol Metabolism, and Cell Physiology on Methylmercury Formation by Geobacter sulfurreducens

“…We recently demonstrated that G. sulfurreducens methylate cysteine to penicillamine in response to the addition of exogenous cysteine. 42 This process has potentially substantial implications on MeHg production since it has been demonstrated that Hg(II) methylation by G. sulf urreducens is consistently enhanced by cysteine, but not by penicillamine. 16,22 The response in Hg(II) methylation following the addition of 100 or 600 nM exogenous cysteine differed among the three assays.…”

“…A sample was collected by filtration (0.2 μm pore size PES syringe filters) at the late exponential growth phase. This filtrate was added to the assay buffer (10% v/v) to comprise the metabolite medium, which contained biogenic metabolites including enhanced concentrations of cysteine and other LMM-thiols …”

“…The concentrations of specific LMM-thiol compounds in bacteria assays were reproduced from the study by Gutensohn et al, which was partly based on samples collected from the very same experiment assays as used in this study. The LMM-thiol compounds were determined by liquid chromatography mass spectrometry following the method by Liem-Nguyen et al Sulfide was measured in the growth culture and in specific methylation assays by the methylene blue method using UV–vis absorption spectroscopy at 670 nm (UV-1201 spectrophotometer, Shimadzu). , …”

“…Mercury methylation assays were performed in three different assay buffers in closed glass serum vials: (I) a standard assay buffer as described by Schaefer and Morel, 18 (II) nutrient assay buffer, and (III) metabolite assay buffer (detailed assay buffer compositions are given in Table S1 ). 42 The nutrient concentration increased in the order from standard < metabolite < nutrient assay with 1 mM acetate ( e – donor) and fumarate ( e – acceptor), 1.0–1.9 mM acetate and 1.0–3.9 mM fumarate, and 1.9 mM acetate and 3.9 mM fumarate for each assay system, respectively ( Table S1 ). Assay buffers were prepared in acid-clean glass serum bottles, flushed with N 2 , crimp sealed with Teflon stoppers, and autoclaved.…”

“…This filtrate was added to the assay buffer (10% v/v) to comprise the metabolite medium, which contained biogenic metabolites including enhanced concentrations of cysteine and other LMM-thiols. 42 Mercury and exogenous cysteine were added to the assay buffers to a final concentration of 30 nM Hg(II) (as HgNO 3 ; Sigma-Aldrich certified standard) and 0−600 nM cysteine (anoxic solution, L-cysteine (≥97%), Sigma-Aldrich). The used Hg(II) concentration is typical for Hg(II) methylation experiments with G. sulf urreducens, 16,19,[21][22][23]39,40 and the added cysteine concentrations were chosen based on reported concentrations of this thiol in environmental samples.…”