Coupling Fluorescent Probes to Characterize S-Containing Compounds in a Sulfate Reducing Bacteria Involved in Hg Methylation

Abstract

The microbial methylation of inorganic mercury Hg(II) is governed by S-containing compounds such as thiols (RSH) and sulfides (S2-). Various S-containing molecules in an environmental or culture medium can be difficult to assess because of the complexity of the medium, poor stability, and low concentration ranges of sulfide and thiol compounds. Here, we applied two fluorescence spectroscopy-based methods using α, β-unsaturated ethanoylcoumarin fluorophore (DHC) for the quantification of sulfides, and monobromo (trimethylammonio) bimane (qBBr) to quantify total thiol concentrations (in extracellular and bacterial cell fractions). The potential interferences of both organic and inorganic compounds from the matrix were evaluated. In the presence of Hg species, both methods allowed the quantification of free sulfides or thiols (not forming complexes with Hg). The two methods were highly sensitive, with detection limits of 100 nM and 20 nM for thiols and sulfides, respectively. They also exhibited high selectivity for the detection of thiols or sulfides against other tested matrix compounds. Finally, both methods were applied to characterize S-containing compounds in a culture of Pseudodesulfovibrio hydrargyri strain BerOc1, a methylating sulfate-reducing bacterium (SRB) exposed to 0.1mM of cysteine. During bacterial growth, we used (i) DHC probe to quantify sulfide concentration in the bulk fraction, (ii) qBBr for total extracellular thiols and total thiols adsorbed on the cells, and (iii) liquid chromatography-tandem mass spectrometry to track cysteine degradation and characterize other thiols. The time series until the end of BerOc1 growth showed biodegradation of cysteine, and biosynthesis of sulfides and other thiol compounds.

Keywords: Fluorescence spectroscopy-based methodsS-containing moleculesthiolssulfidesmercury-methylating bacteria