Anaerobic HgII reduction is driven by cellular HgII-thiol interactions

Abstract

Redox reactions play a critical role in determining the availability of mercury species, Hg^II and Hg⁰, to anaerobic microbes responsible for methylating inorganic mercury into toxic monomethylmercury. Some anaerobes also contribute to Hg cycling in methylation hotspots by reducing Hg^II to its gaseous elemental form, Hg⁰. However, their contributions remain poorly quantified due to limited mechanistic insights and the absence of genetic targets. In this study, we investigated the mechanisms of anaerobic Hg^II reduction in the versatile anoxygenic photoheterotroph and fermenter Heliomicrobium modesticaldum Ice1. Given Hg^II strong electrophilic affinity for thiol groups, we hypothesized that cellular thiols are key interaction sites mediating Hg^II reduction. Exposure of H. modesticaldum to the thiol-alkylating agent N-ethylmaleimide (NEM), which irreversibly binds thiols, resulted in a concentration-dependent inhibition of Hg⁰ production during both photoheterotrophy and fermentation. Hg partitioning assays with Escherichia coli cells revealed no significant differences in Hg-cell partitioning in the presence or absence of NEM, suggesting that Hg^II reduction is dependent on intracellular thiol interactions. These findings highlight the importance of thiol-mediated pathways in Heliobacterial Hg^II reduction. Although the exact cellular components remain unidentified, we discuss potential thiol-containing coupling sites that warrant further investigation.

Keywords: bacteria; fermentation; mercury; phototrophy; redox; thiol.

Fig. 1.. Total Hg quantified from partitioning assays of E. coli cultures in medium PYE and exposed to NEM for 3 h. Hg-associated fractions are represented in order from left to right: E. coli cell pellet-associated Hg, glass-bound Hg from Balch tubes, Hg remaining in 0.2 µm-filtered spent growth media and the total Hg initially present in the exposure assay. We maintained equal ratios of cell OD 600 nm: Hg: NEM between the exposure assay and bioreactor experiments, i.e. 0.400 OD: 500 pM Hg: 400 µM NEM for these E. coli assays and 0.200: 250 pM: 200 µM for H. modesticaldum bioreactors. At the start of the exposure, approximately [Hg^II] = 500 pM or 1 ng in 10 ml of culture was added. The bottom and top of the boxes show the first and third quartiles, respectively; the bar in the middle shows the median, and the whiskers show the minimum and maximum for each treatment. The black circles overlain represent the individual sample measurements for n=3 E. coli cultures. Assumptions of normally distributed residuals (P=0.16) and homoscedasticity (P=0.99) were met for a two-way parametric ANOVA, which showed there was a significant difference between Hg-associated fractions (P=4.8×10⁻⁹) but not between NEM concentrations (P=0.77) or their interaction (P=0.248). Letters that are not shared between fractions indicate a significant difference according to the Tukey HSD test (P<0.05).

Fig. 2.. Cumulative Hg⁰ produced during bioreactor experiments with H. modesticaldum Ice1 grown photoheterotrophically (light) and fermentatively (dark) in medium PYE exposed to NEM concentrations ranging from 0 to 200 µM. The bottom and top of the boxes show the first and third quartiles, respectively; the bar in the middle shows the median, and the whiskers show the minimum and maximum for each treatment. The circles overlain represent the individual sample measurements. Assumptions of normally distributed residuals (P=0.85) and homoscedasticity (P=0.93) were met for a two-way parametric ANOVA, which showed there was a significant effect of NEM addition and metabolism on cumulative Hg⁰ production (P=2.5×10⁻⁸ and P=6.7×10⁻⁴, respectively) and a significant effect of their interaction (P=0.043). Letters that are not shared between fractions indicate a significant difference according to the Tukey HSD test (P<0.05).

Fig. 3.. Hourly rate of Hg⁰ production for individual representative bioreactor assays with H. modesticaldum Ice1 in medium PYE during (a) dark (fermentative) and (b) light (photoheterotrophic) exposure to NEM.