Hg methylation is driven by sulfides
The SRB P. hydrargyri strain BerOc1, able to use sulfate or fumarate as electron acceptors, was used in this study to determine the link between MeHg production and sulfide. The production of MeHg was assessed at three different Hg(II) concentrations (0.05, 2 and 5 µM of Hg(II)) under non-sulfidogenic growth (Pyruvate (Pyr)/Fumurate) with cysteine or sulfide added exogenously (0.1 mM) and under sulfidogenic growth (Pyr/Sulfate). The chosen cysteine and sulfide concentrations correspond to the minimal sulfur concentration needed for optimal growth without Hg(II) (Online Resource 2a). The Hg(II) concentrations were chosen from a previous study that showed a maximum of mercury methylation rate at 5 µM of Hg(II) (Isaure et al. 2020). Sulfide concentrations measured at the end of the growth in the presence of cysteine (0.010 ± 0.003 - 0.020 ± 0.005 mM, Table 1) are consistent with previous sulfide concentrations reported for degradation of cysteine by bacteria, including the well-known Hg methylators Geobacter sulfurreducens PCA and Pseudodesulfovibrio mercurii ND132 (Graham et al. 2012a, b; Thomas and Gaillard 2017; Thomas et al. 2018). Moreover, for a given growth condition, addition of Hg(II) did not impact neither bacterial growth nor sulfide concentration (Table 1, Online Resource 2d-f).
Interestingly, the Hg(II) methylation potential under non-sulfidogenic growth with 0.1 mM sulfide was higher than in the other tested conditions, regardless of the Hg(II) concentration (Figure 1a, Online Resource Table). While a progressive decrease of Hg methylation potential over an increase of Hg(II) concentrations was detected under sulfidogenic growth, a drastic decline over 0.05 µM was noticed under non-sulfidogenic growth with cysteine (from 15% to < 1%) (Figure 1a). However, under non-sulfidogenic growth with sulfide only a moderate decline was observed above 2 µM Hg(II). Furthermore, the maximal rate of production of MeHg per cell (τmax calculation) is between 50 and 100 fold higher (Table 1) in presence of sulfide. The saturation of MeHg production (from KMichaelis calculation) of BerOc1 grown with sulfide is reached at higher Hg(II) concentrations than when grown with sulfate (10 times lower than sulfide) or cysteine (1000 times lower than sulfide). These results indicated that the Hg methylation mechanism is saturable in the various cell growth conditions tested as previously described under a different growth condition than tested here (Isaure et al. 2020). Abiotic controls confirmed that measured Hg methylation was a fully biotic process and that the observed differences between growth conditions were not related to differences in abiotic Hg methylation (Online Resource 3). Furthermore, our study reported for the first time that the concentration at which this saturation is reached and the capacity of a single cell to produce MeHg depends on growth conditions.
Importantly, Hg methylation potentials were higher with 0.1 mM of sulfide, compared to cysteine condition (expected to favor Hg methylation (Schaefer and Morel 2009)) regardless of the Hg(II) concentration added (Figure 1a). However, in presence of cysteine around 10 µM of sulfides were measured (Table 1). We thus hypothesized that the variation observed in Hg methylation depends on sulfide concentration (added or produced) rather than directly on the added cysteine. Therefore, we investigated Hg(II) methylation at varying concentration of sulfides (from 0.0005 to 5 mM). The range of sulfide concentrations were chosen following the measured sulfide production under cysteine and sulfate reduction growth (Table 1). Two Hg(II) concentrations, 0.05 and 2 µM Hg(II) (where the differences between Hg(II) potential are the most prominent (Figure 1a)), were used to test the effect of sulfide concentration on Hg(II) methylation potentials. Addition of low exogenous sulfide concentrations (< 0.05 mM) limited BerOc1 growth only at 2 µM of Hg (II) while sulfide above 2 mM was toxic for the growth, regardless of the Hg(II) concentration added (Table 1, Online Resource 2b-c). At 0.05 µM of Hg(II), Hg methylation potentials increased with the increase of sulfide concentrations up to 0.5 mM, and then decreased at higher sulfide concentrations (Figure 1b). The same pattern was observed with 2 µM of Hg(II) with a maximal Hg methylation reached at 0.1 mM of sulfides (Figure 1c). Overall, our data showed that sulfide at concentration ranged from 0.1 to 0.5 mM favors Hg methylation in P. hydrargyri BerOc1 and that lower sulfide concentration (< 0.1 mM) and higher sulfide concentration (> 0.5 mM) from that range are disadvantageous to the process. Several previous studies concluded that the presence of sulfide has an inhibitory effect on Hg methylation (Benoit et al. 1999a, a). The inhibitory effect on Hg methylation at high sulfide concentration was for instance observed in Desulfobulbus propionicus (1pr3) by testing sulfide concentration from 0.05 to 2 mM (Benoit et al. 2001a). However, because only one concentration of sulfide below 0.1 mM was tested in this study, the beneficial effect of sulfide on Hg methylation was not noticed on the plot. Additionally, an optimum of sulfide concentration (between 0.01 and 0.1 mM) favoring Hg methylation has been observed in methanogens (Gilmour et al. 2018). The modeling linking sulfate concentration to MeHg production in natural ecosystems also identified a similar pattern, with an optimum of Hg methylation ranging from 0.2 to 0.5 mM of sulfate (Gilmour and Henry 1991). Since the optimal concentration of sulfide favoring Hg methylation in our study is close to the optimal concentration of sulfate favoring Hg methylation in Gilmour and Henry study (Gilmour and Henry 1991), we can suggest that sulfide is the main parameter here favoring Hg methylation, since sulfate can be reduced to sulfide.
The mechanism by which S containing molecules favors Hg methylation is far from being understood. Thomas et al (Thomas et al. 2020), identified Hg-S complexes in non-growing Geobacter sulfurreducens PCA (a Hg(II) methylating bacterium) incubated with cysteine. In this study, the addition of cysteine also increased Hg(II) methylation and the authors suggested that the degradation of cysteine produces sulfide that forms Hg-S complexes and those complexes would favor Hg(II) methylation through a facilitated Hg uptake, as demonstrated in E. coli (Thomas et al. 2019). Unfortunately, sulfide production by G. sulfurreducens PCA was not provided. Further spectroscopic analysis and characterization of Hg-S complexes under a range of sulfide concentrations would shed light on the role of sulfide at 0.1-0.5 mM in the production of Hg-S forms and the increase of Hg(II) methylation.
Overall, how sulfide, sulfate (Gilmour and Henry 1991) and cysteine (Schaefer and Morel 2009) affect Hg methylation depends mainly on their concentrations. In marine environments, sulfate can be found at concentrations up to 20 mM (Jørgensen et al. 2019) and cysteine at fM to nM concentrations (Joe-Wong et al. 2012; Liem-Nguyen et al. 2017; Bouchet et al. 2018; Adediran et al. 2019). At those concentrations, sulfate and cysteine are not expected to play a major role in MeHg production (Gilmour and Henry 1991; Schaefer and Morel 2009). However, other S-containing molecules, especially those involved in the sulfur biogeochemical cycle (sulfide, sulfite, thiosulfate, and polysulfide), reach concentrations between 0.1 and 1 mM in marine and freshwater sediments (Findlay 2016; Findlay and Kamyshny 2017; Gentès et al. 2017) and can all be transformed into sulfide by reduction or degradation. Here, we reported an increase of Hg methylation at sulfide concentration encountered in the environment. We hypothesize that in natural environments, sulfate will enhance Hg methylation because it favors SRB metabolism but sulfide (regardless of its origin, exogenous or endogenous) will govern the extent of Hg methylation potentials.
Sulfides influence methylmercury partitioning
In a previous study, a link between Hg species partitioning and Hg methylation was observed with various thiols molecules (Schaefer and Morel 2009). Thus, to further understand differences on Hg methylation observed with various S containing molecules and sulfide concentration in our study, we investigated the Hg species (Hg(II) and MeHg) partitioning between the extracellular and the cell-associated (cell-sorbed and intracellular) fractions in BerOc1 grown under the three growth conditions (Pyr/Fumarate with cysteine or sulfide and, Pyr/Sulfate) and with sulfide gradient under Pyr/Fumarate condition. Distribution of Hg species was measured at the beginning of the growth (Ti, Hg(II), Online Resource 4) and at the end of exponential phase (Tf, Hg(II) and MeHg produced) at increasing Hg(II) concentrations (Figure 2, Figure 3). For a given growth condition, increasing Hg(II) concentrations did not affect neither Hg(II) (Figure 2a) nor MeHg (Figure 3a) partitioning. Hg(II) became more associated with the cells over time (Online Resource 4a, Figure 3a) and, was mostly associated with cells at the end of the exponential growth phase (80-100%) regardless of the growth condition (Figure 3a). On the contrary, MeHg distribution between extracellular and the cell fractions depends on the growth condition (Figure 3a): while most of the MeHg was detected in the extracellular fraction under sulfidogenic growth (96 ± 2%), the proportion in the extracellular fraction reached only 30 ± 4% under non-sulfidogenic condition with cysteine. An intermediate distribution (50 ± 5%) of MeHg was observed in non-sulfidogenic growth with sulfide. Thus, the proportion of extracellular MeHg increased with the increasing sulfide concentrations measured in the cultures (Figure 3a, Table 1). The dominance of produced MeHg in the extracellular medium has been already observed and was interpreted as an export of MeHg by the bacteria or desorption from the cells (Graham et al. 2012b; Liu et al. 2016; Pedrero et al. 2012). We thus infer that the export/desorption of MeHg from the bacterial cells can be favored by sulfide.
In order to test this hypothesis, we measured the distribution of Hg species in the extracellular and the cell-associated fractions in BerOc1 cells grown with increasing exogenous sulfide concentrations. At both 0.05 and 2 µM of Hg(II), Hg(II) became more associated to the cell fraction over time for all of the sulfide concentrations tested (Online Resource 4b-c and Figure 2b-c), as observed for BerOc1 under Pyr/Fumarate with cysteine and Pyr/Sulfate conditions (Online Resource 4a and Figure 2a). Thus, Hg(II) partitioning appear not to be influenced by sulfide concentration. Remarkably, the extracellular MeHg increased with the increase of sulfide concentrations regardless of the Hg(II) concentration added (Figure 3b, Figure 3c). A strong positive correlation was observed (Spearman correlation rho = 0.89, p value < 0.001). Previous studies showed an increase of the extracellular MeHg in presence of thiols (Ndu et al. 2016). More specifically, cysteine strongly enhanced the extracellular MeHg produced by G. sulfurreducens PCA and more slightly by Pseudodesulfovibrio mercurii ND132. Accordingly, a facilitated MeHg cell export and desorption from the cells was proposed via the cysteine (Lin et al. 2015). However, no data of sulfide contents was provided (Lin et al. 2015) and, in the light of the recent papers (Thomas et al. 2019), it may be possible that the facilitated export/desorption was the consequence of sulfide production due to cysteine degradation. Our results show that sulfide, either exogenous or endogenous (produced by the cell via cysteine degradation or sulfate reduction), could be a strong parameter involved in MeHg export and/or desorption from the cell.
A recent study on abiotic interaction of MeHg and sulfide predicted the formation of bismethylmercury sulfide as the dominant MeHg species in sulfidic solutions (Kanzler et al. 2018). In mammalian cells, the bismethylmercury sulfide formation from MeHg has been proposed as a mechanism of cell detoxification (Yoshida et al. 2011). In this study, sulfide (exogenous or produced via cysteine and homocysteine degradation) form bismethylmercury sulfide in the presence of MeHg, avoiding MeHg binding to cellular proteins (Yoshida et al. 2011). In our study, the progressive decrease of cell associated MeHg observed when sulfide concentration increased could be explained by the formation of bismethylmercury sulfide that avoid the binding of MeHg to cellular proteins. However, it is difficult to speculate on the combined role of Hg methylation and MeHg export for Hg cell detoxification since no link between both Hg methylation potentials and MeHg export could be found. MeHg export probably protects BerOc1 cells against MeHg toxicity. In the other hand, the decrease of Hg methylation potential observed at high sulfide concentrations may be the consequence of a lower availability of Hg because of Hg-S complexes, limiting Hg toxicity.
The increase of Hg methylation in presence of sulfide is not associated to hgcA overexpression
hgcAB are the only genetic determinisms identified as necessary for Hg methylation(Parks et al. 2013). To investigate if the increase of Hg methylation observed with 0.1 mM of sulfide was related to the overexpression of the enzymatic mechanism of Hg methylation, the expression of hgcA gene (involved in the methyl transfer to Hg) was measured in BerOc1. We selected cells growing under non-sulfidogenic growth with 0.1 mM sulfide and 0.1 mM cysteine, where Hg methylation was lower (Figure 1a). Low hgcA overexpression (from 1.8 to 2.2) was observed for cysteine for all the Hg(II) concentrations tested (Figure 4a). In contrast, no overexpression could be detected for 0.1 mM sulfide, where the methylation potentials were higher (Figure 1a and Figure 4b). Thus, no direct link could be established between hgcA expression and Hg methylation potentials. The high Hg methylation potential observed is likely linked to the cellular environment, such as sulfides reported here or substrates used for bacterial growth as previously reported (Goñi-Urriza et al. 2015). In addition, our results revealed that hgcA gene is expressed at basal level even at increasing Hg(II) concentrations. Hg(II) seems not control hgcA expression, unlike other metals (Zn, Cu, As, Fe) which regulate the expression of genes encoding enzymes controlling their transformation (Andrei et al. 2020; Andrews et al. 2003; Pederick et al. 2015; Silver 1996; Silver and Phung 2005). Nevertheless, the available data regarding the expression of hgcAB gene are so far insufficient to ascertain that hgcAB genes are not regulated. A transcriptional regulator, arsR, located upstream hgcAB genes and cotranscribed with hgcA clearly suggests a regulated process (Goñi-Urriza et al. 2020). Its implication in the regulation of hgcAB genes expression and the factors that trigger the ArsR response are still unknown and should be addressed in future studies.