Detailed Assessment of the Kinetics of Hg-Cell Association, Hg Methylation, and Methylmercury Degradation in Several Desulfovibrio Species

ABSTRACTThe kinetics of inorganic Hg [Hg(II)i] association, methylation, and methylmercury (MeHg) demethylation were examined for a group ofDesulfovibriospecies with and without MeHg production capability. We employed a detailed method for assessing MeHg production in cultures, including careful control of medium chemistry, cell density, and growth phase, plus mass balance of Hg(II)iand MeHg during the assays. We tested the hypothesis that differences in Hg(II)isorption and/or uptake rates drive observed differences in methylation rates amongDesulfovibriospecies. Hg(II)iassociated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ∼3 h. All MeHg produced was rapidly exported. We also tested the idea that allDesulfovibriospecies are capable of Hg(II)imethylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in theDesulfovibriois not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylatingDesulfovibriospecies even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive cross-species differences in Hg methylation rates. As part of this study, we identified four new Hg methylators (Desulfovibrio aespoeensis,D. alkalitolerans,D. psychrotolerans, andD. sulfodismutans) and four nonmethylating species (Desulfovibrio alcoholivorans,D. tunisiensis,D. carbinoliphilus, andD. piger) in our ongoing effort to generate a library of strains for Hg methylation genomics.

Biogeochemical factors affecting mercury methylation rate in two contaminated floodplain soils

An automated biogeochemical microcosm system allowing controlled variation of redox potential (EH) in soil suspensions was used to assess the effect of various factors on the mobility of mercury (Hg) as well as on the methylation of Hg in two contaminated floodplain soils with different Hg concentrations (approximately 5 mg Hg kg−1 and >30 mg Hg kg–1). The experiment was conducted under stepwise variation from reducing (approximately −350 mV at pH 5) to oxidizing conditions (approximately 600 mV at pH 5). Results of phospholipid fatty acids (PLFA) analysis indicate the occurrence of sulfate reducing bacteria (SRB) such as Desulfobacter species (10Me16:0, cy17:0, 10Me18:0, cy19:0) or Desulfovibrio species (18:2ω6,9), which are considered to promote Hg methylation. The products of the methylation process are lipophilic, highly toxic methyl mercury species such as the monomethyl mercury ion [MeHg+], which is named as MeHg here. The ln(MeHg/Hgt) ratio is assumed to reflect the net production of monomethyl mercury normalized to total dissolved Hg (Hgt) concentration. This ratio increases with rising dissolved organic carbon (DOC) to Hgt ratio (ln(DOC/Hgt) ratio) (R2 = 0.39, p<0.0001, n= 63) whereas the relation between ln(MeHg/Hgt ratio and lnDOC is weaker (R2 = 0.09; p<0.05; n = 63). In conclusion, the DOC/Hgt ratio might be a more important factor for the Hg net methylation than DOC alone in the current study. Redox variations seem to affect the biogeochemical behavior of dissolved inorganic Hg species and MeHg indirectly through related changes in DOC, sulfur cycle, and microbial community structure whereas EH and pH values, as well as concentration of dissolved Fe3+/Fe2+ and Cl– seem to play subordinate roles in Hg mobilization and methylation under our experimental conditions.

Biogeochemical factors affecting mercury methylation rate in two contaminated floodplain soils

An automated biogeochemical microcosm system allowing controlled variation of redox potential (EH) in soil suspensions was used to assess the effect of various factors on the mobility of mercury (Hg) as well as on the methylation of Hg in two contaminated floodplain soils with different Hg concentrations (approximately 5 mg kg−1 Hg and >30 mg kg−1 Hg). The experiment was conducted under stepwise variation from reducing (approximately −350 mV at pH 5) to oxidizing conditions (approximately 600 mV at pH 5). Results of phospholipid fatty acids (PLFA) analysis indicate the occurrence of sulfate reducing bacteria (SRB) such as Desulfobacter species (10me16:0, cy17:0, 10me18:0, cy19:0) or Desulfovibrio species (18:2ω6,9), which are considered to promote Hg methylation. The products of the methylation process are lipophilic, highly toxic methyl mercury species such as the monomethyl mercury ion [MeHg+], which is named as MeHg here. The ln(MeHg/Hgt) ratio is assumed to reflect the net production of monomethyl mercury normalized to total dissolved Hg (Hgt) concentration. This ratio increases with rising dissolved organic carbon (DOC) to Hgt ratio (lnDOC/lnHgt ratio) (R2 = 0.39, p < 0.0001, n = 63) whereas the relation between ln(MeHg/Hgt) ratio and lnDOC is weaker (R2 = 0.09; p < 0.05; n = 63). In conclusion, the DOC/Hgt ratio might be a more important factor for the Hg net methylation than DOC alone in the current study. Redox variations seem to affect the biogeochemical behavior of dissolved inorganic Hg species and MeHg indirectly through related changes in DOC, sulfur cycle, and microbial community structure whereas E,H and pH values, as well as concentration of dissolved Fe,3+/Fe2+ and Cl− seem to play subordinate roles in Hg mobilization and methylation under our experimental conditions.

An HcpR homologue from Desulfovibrio desulfuricans and its possible role in nitrate reduction and nitrosative stress

The Escherichia coli CRP (cAMP receptor protein), is a global regulator of transcription that modulates gene expression by activation or repression at a range of promoters in E. coli. A major function is to regulate the selection of nutrients required for growth. The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC27774 is capable of utilizing sulfate, nitrite and nitrate as terminal electron acceptors. In the presence of both sulfate and nitrate, sulfate is reduced preferentially despite nitrate being the thermodynamically more favourable electron acceptor. Three inverted repeat sequences upstream of the D. desulfuricans ATCC27774 nap (nitrate reduction in the periplasm) operon have high levels of similarity to the consensus sequence for the E. coli CRP DNA-binding site. In other Desulfovibrio species a putative CRP homologue, HcpR [regulator of hcp (hybrid cluster protein) transcription], has a predicted regulon comprising genes involved in sulfate reduction and nitrosative stress. The presence of CRP consensus sites within the D. desulfuricans ATCC27774 nap promoter prompted a search for CRP homologues in the genomes of sulfate-reducing bacteria. This revealed the presence of a potential CRP homologue that we predict binds to CRP consensus sites such as those of the nap operon. Furthermore, we predict that much of the core HcpR regulon predicted in other Desulfovibrio species is conserved in D. desulfuricans.