Microbial Biosynthesis of Thiol Compounds: Implications for Speciation, Cellular Uptake, and Methylation of Hg(II)

Abstract

Cellular uptake of inorganic divalent mercury (Hg(II)) is a key step in microbial formation of neurotoxic methylmercury (MeHg), but the mechanisms remain largely unidentified. We show that the iron reducing bacterium Geobacter sulfurreducens produces and exports appreciable amounts of low molecular mass thiol (LMM-RSH) compounds reaching concentrations of about 100 nM in the assay medium. These compounds largely control the chemical speciation and bioavailability of Hg(II) by the formation of Hg(LMM-RS) 2 complexes (primarily with cysteine) in assays without added thiols. By characterizing these effects, we show that the thermodynamic stability of Hg(II)-complexes is a principal controlling factor for Hg(II) methylation by this bacterium such that less stable complexes with mixed ligation involving LMM-RSH, OH − , and Cl − are methylated at higher rates than the more stable Hg(LMM-RS) 2 complexes. The Hg(II) methylation rate across different Hg(LMM-RS) 2 compounds is also influenced by the chemical structure of the complexes. In contrast to the current perception of microbial uptake of Hg, our results adhere to generalized theories for metal biouptake based on metal complexation with cell surface ligands and refine the mechanistic understanding of Hg(II) availability for microbial methylation.

Determination of picomolar levels of methylmercury complexes with low molecular mass thiols by liquid chromatography tandem mass spectrometry and online preconcentration

“…Adediran et al recently reported biosynthesis and excretion of various LMM thiols in G. sulfurreducens bacteria cultures reaching up to about 120 nM total thiol concentrations in the extracellular medium [11]. To demonstrate the potential of the developed method for the determination of MeHg-thiol complexes in the presence of bio-synthesized LMM thiols, 400 nM of MeHg was added to filtered extracellular assay Table S3).…”

“…Low molecular mass (LMM) thiols are important in biological processes due to the complexation ability of the thiol functional group with metal ions and subsequent bio-transformations between the thiols and disulfides through redox reactions mediated by enzymatic activities [7,8]. LMM thiols are directly synthesized by living organisms and/or indirectly formed through the reaction of sulfide with unsaturated natural organic matter in the environment [9][10][11]. Such thiol compounds are typically found at nM to μM concentrations in the environments, but they have also been found in mM concentrations [12][13][14].…”

“…Significant increases in the Hg II methylation rate have been reported for bacteria culture assays amended with specific LMM thiols [20,21] and subsequent cellular export of MeHg has been observed to be facilitated by addition of cysteine [22]. In a recent study, a characterization of the extracellular milieu of Geobacter sulfurreducens PCA, an iron reducing bacterium capable of Hg II methylation, revealed metabolically active biosynthesis of various LMM thiol compounds [11]. Despite the scientific evidences of the important roles of LMM thiols for MeHg cycling, the measurement of specific MeHgthiol complexes has been hampered by constraints in analytical measurement techniques, and the behavior of specific MeHg-thiol in the environment as well as in biological systems remains poorly investigated.…”

“…This causes a relatively large risk for peak overlaps and misidentification in samples with many thiol compounds, in particular if the sample also contains a substantial concentration of Hg II forming Hg II -thiol complexes [12,35]. In bacteria culture and environmental systems, many different LMM thiol compound are often present [5,11] and the development of a highly selective and more robust method is therefore essential to characterize MeHg-thiol complexes at concentrations relevant for culture systems with Hg II methylating bacteria and for the environment.…”

“…In this study, we present a novel methodology for the determination of a comparatively large number of MeHg complexes (13 complexes) with LMM thiol ligands which has been frequently found in bacteria culture assays and in terrestrial and aquatic ecosystems [5,11,12,21,35]. The method is based on LC coupled with ESI triple quadrupole tandem mass spectrometry (ESI-MS/MS) with selective reaction monitoring (SRM) detection mode.…”