Developing a predictive understanding of Hg transformation in ecosystems requires knowledge of exchange and feedback processes at Critical Interfaces. Task 3 involves understanding microbial community functions and geochemical influences on mercury biotransformations.
The overarching goals of Task 3 are twofold: (1) Determine the breadth and depth of mercury-methylating species, and (2) determine the native biochemical function(s) of HgcA and HgcB and their participation in other cellular biochemical pathways.
We have made considerable progress in understanding the processes governing microbially mediated mercury transformations and the physicochemical factors that influence these processes across a range of scales. A summary of this progress is presented in the following two sections.
Homology model of the corrinoid-binding domain of HgcA from Desulfovibrio desulfuricans ND132. The corrinoid cofactor is illustrated at the top of the figure with the cobalt shown in pink. The proximity of the strictly conserved Cys (sulfur in yellow) to cobalt in the corrinoid suggests a unique 'base-off' 'Cys-on' configuration and thus a possible functional role for this interaction in the methylation reaction. The 'cap-helix' is shown as a green ribbon (N90 – K97). Amino acid side chains are represented as stick models. Additional elements are oxygen, red; nitrogen, blue; and carbon, gray.
Based on the discovery of the mercury-methylation genes hgcAB and their ability to be used as biomarkers for mercury methylation, we have collaborated with Task 4 on two milestones (3j and 3k). At the molecular level, we determined the cysteine residues that are essential to methylation to understand how Hg(II) is transferred and bound to the methyl group to form methylmercury (see research highlight, Key Amino Acid Residues for Mercury Methylation Confirmed). Through targeted codon substitutions, we found that many residues in and out of the active site are necessary for mercury methylation. At the biochemical level, we are determining HgcAB native function (i.e., what these proteins do in the absence of mercury) and are mapping the path of the methyl group for methylmercury (i.e., identifying the methyl donor).
As an independent task that will benefit the overall project, we have completed development of degenerate primers for the detection, identification, and quantification of hgcAB from all environments. Three other reports exist but are incomplete. Two reports determine and quantify only hgcA, which we have shown cannot methylate without hgcB, suggesting that false positives may occur. The third report uses primers for hgcAB and suggests that Firmicutes are the dominant mercury methylators. However, the Bae et al. 2014 study did not quantify hgcA. We tested all three methods using their protocols and materials and found that the first two reports yielded amplification of two to five pure cultures out of the 28 tested that spanned all four clades, while the latter amplified only Firmicutes. These findings suggest that Liu et al. 2014 and Schaefer et al. 2014 captured only a small fraction of the mercury-methylating community. The Bae et al. study led to incorrect conclusions that Firmicutes are the major methylators in the Florida Everglades because, according to our results, they preferentially amplified those organisms. Our latest results show that we have both a universal set of degenerate primers that amplify hgcAB from 26 of the 28 tested organisms and clade-specific quantitative polymerase chain reaction (qPCR) primers for the Deltaproteobacteria, Firmicutes, and the Archaea. These primers will enable the overall capture and identification of organisms containing hgcAB while yielding quantitative hgcA values that are clade specific, thus providing data that tie community metabolism to mercury methylation.
Investigations into the microbial ecology and physiology of mercury methylation have yielded an increased understanding of methylmercury production at the community level and the geochemical influences on its generation. We have investigated the role of Deltaproteobacteria in mercury methylation on the Oak Ridge Reservation (ORR) and found that they are likely the major methylators. While working with Task 2 to understand cell surface interactions, Task 3 researchers have been determining methylation kinetics and the role of DOM in mercury methylation during sulfate reduction. These findings will be tested in the proposed work using model (synthetic) microbial communities with results that can be extrapolated to and tested in the field.
Global relative frequency of HgcA based on metagenomic projects. Overlay is the estimated continental emission of mercury (in tons), based on the United Nations Environment Programme Global Mercury Assessment 2013 report. Diamonds represent pelagic ocean water samples, and circles represent all other samples.
We have taken this knowledge a step further and surveyed >3,500 publicly available microbial metagenomes (with >1.6 billion genes) across various databases. This survey yielded a global picture of mercury methylation potential in many environments (see research highlight, Global Prevalence and Distribution of hgcA, a Gene Encoding Microbial Mercury Methylation). We found that while hgcAB was absent from mammalian microbiomes and the open oceans, it was prevalent in coastal dead zones, contaminated sites, engineered sites, and Arctic permafrost. We also discovered associations of methylating and nonmethylating organisms that co-occur in nature. These findings are important in understanding the metabolic associations of key types of organisms and their impact on mercury methylation. For example, we have determined that some fermenters (e.g., Clostridium spp.) frequently associate with selected sulfate-reducers and methanogens. These symbiotic relationships are known to increase nutrient sharing and thus create more efficient lifestyles. Similar relationships may transfer to more efficient mercury methylation potentials than have been observed in monocultures in the laboratory. This knowledge will guide the types of organisms used in synthetic communities.