Methylmercury is a potent neurotoxin produced in natural environments from inorganic mercury by anaerobic bacteria. However, until now the genes and proteins involved have remained unidentified. Here, we report a two-gene cluster, hgcA and hgcB, required for mercury methylation by Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. In either bacterium, deletion of hgcA, hgcB, or both genes abolishes mercury methylation. The genes encode a putative corrinoid protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, consistent with roles as a methyl carrier and an electron donor required for corrinoid cofactor reduction, respectively. Among bacteria and archaea with sequenced genomes, gene orthologs are present in confirmed methylators but absent in nonmethylators, suggesting a common mercury methylation pathway in all methylating bacteria and archaea sequenced to date.

A global metagenome assessment reveals a low risk of methylmercury production in humans and a high potential in Arctic permafrost.

Subsurface amendments of slow-release substrates (e.g., emulsified vegetable oil [EVO]) are thought to be a pragmatic alternative to using short-lived, labile substrates for sustained uranium bioimmobilization within contaminated groundwater systems. Spatial and temporal dynamics of subsurface microbial communities during EVO amendment are unknown and likely differ significantly from those of populations stimulated by soluble substrates, such as ethanol and acetate. In this study, a one-time EVO injection resulted in decreased groundwater U concentrations that remained below initial levels for approximately 4 months. Pyrosequencing and quantitative PCR of 16S rRNA from monitoring well samples revealed a rapid decline in groundwater bacterial community richness and diversity after EVO injection, concurrent with increased 16S rRNA copy levels, indicating the selection of a narrow group of taxa rather than a broad community stimulation. Members of the Firmicutes family Veillonellaceae dominated after injection and most likely catalyzed the initial oil decomposition. Sulfate-reducing bacteria from the genus Desulforegula, known for long-chain fatty acid oxidation to acetate, also dominated after EVO amendment. Acetate and H 2 production during EVO degradation appeared to stimulate NO 3 ؊ , Fe(III), U(VI), and SO 4 2؊ reduction by members of the Comamonadaceae, Geobacteriaceae, and Desulfobacterales. Methanogenic archaea flourished late to comprise over 25% of the total microbial community. Bacterial diversity rebounded after 9 months, although community compositions remained distinct from the preamendment conditions. These results demonstrated that a one-time EVO amendment served as an effective electron donor source for in situ U(VI) bioreduction and that subsurface EVO degradation and metal reduction were likely mediated by successive identifiable guilds of organisms.

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB ؉ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB. IMPORTANCEThe neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivationindependent strategy. T he trace metal mercury (Hg) is a widespread global pollutant with a Ͼ4-fold increase in atmospheric emissions since antiquity (1). While all forms of Hg are toxic, the neurotoxin monomethyl Hg (MeHg) poses the most health risks due to its toxicity and biomagnification in food webs (2). MeHg production in nature is predominantly microbial (3) and is limited to strictly anaerobic microorganisms (4, 5). Methylation has commonly been tied to microbial sulfate reduction (6-8), but only a subset of sulfate-reducing microbes are capable of Hg methylation (4, 9). Hg methylation has also...