S10 (II) Mechanisms of microbial mercury resistance

Organisms that respire oxygen widely employ sulfur and selenium in macromolecular catalysts, informational molecules and related metabolites, making them (including us!) vulnerable to mercury compounds that bind strongly to these essential chalcogens. Nonetheless, creatures ranging from bacteria to humans routinely survive even very intense and prolonged contact with many forms of Hg that are demonstrably toxic in laboratory conditions. Drawing on recent published and unpublished observations, this presentation will cover two approaches we take to dissect the paradoxes of Hg toxicity. One approach is to define the "Hg exposome" of the model microbe E. coli using high-throughput global proteomics to identify its most vulnerable proteins as well as various biophysical techniques to identify non-protein targets of Hg. In addition to understanding the molecular basis of Hg intoxication, we aim to develop evolutionarily conserved specific and sensitive biomarkers of Hg exposure for environmental and public health applications. Our second approach is to understand the mechanism of the Hg resistance (mer) locus widely found in bacteria and archaea. This co-regulated suite of enzymes and transporters converts organic mercurials to inorganic ionic mercury and thence to volatile, monoatomic Hg(0) vapor which diffuses from their environment. Recent biochemical, biophysical and computational analyses reveal how the enzymes interact with each other and how the transcriptional regulator distinguishes Hg(II) from closely related metal ions. The mer system has evolved in prokaryotes from ubiquitous and ancient housekeeping genes, becoming tuned over millennia to deal precisely and efficiently with just the kinds of damage that Hg inflicts. Unfortunately, while higher organisms have many Hg-vulnerable proteins, none has an equivalent of the mer locus, although the bacterial detoxification enzymes work well in plants engineered for bioremediation.

Microbial resistance to mercury is based on their enzymatic reaction of mercurial compounds. Clustered genes in operons (mer operons) for the conversion of mercurials to metallic mercury by enzymatic reaction have been intensively studied. Because mercury and its compounds have widely contaminated as results of geological and industrial activities, mercury-resistant microbes are distributed widely. Recently, it is discovered that many mercury resistant mer operons are located on the transposable elements such as bacterial transposons or plasmids.

From a bacterium Bacillus megaterium MB1 that was isolated from sediment of Minamata Bay, Japan, we found a class II transposon, TnMERI1, that carries genes for broad-spectrum resistance to mercurial compounds, and whose mer operon is highly similar in nucleotide sequence to those of Tn5084 from Bacillus cereus VKM684 and Tn5085 from Exiguobacterium sp. TC38-2b isolated from Carpathia, Ukraine, and of Tn5084-like transposon from Bacillus cereus RC607 isolated from Boston Harbor, USA.

We also isolated fifty-six mercury-resistant Bacillus strains from natural environments at various sites in the world. Southern hybridization and PCR analysis showed that 21 out of the 56 isolates have closely relating or identical mer operons to that of B. megaterium MB1. These 21 isolates demonstrated a broad-spectrum mercury resistance and volatilized Hg⁰. PCR amplification with a single primer and restriction fragment length polymorphism analysis showed that these 21 isolates have TnMERI1-like class II transposons.

Other than Bacillus strains, we analyzed mercuric reductase genes (merAs) in seven heavily contaminated soil samples by using PCR primer sets specific to Firmicutes merA and specific to alpha- or beta- Proteobacteria merA. Phylogenetic analysis data of the amplified merA genes showed that some merA is dominat in the soils but still diversified. It was previously found that a strictly anaerobic Firmicutes bacterium Clostridium butyricum and an aerobic bacterium Bacillus sp. RC607 possess the identical merA genes. Therefore, the mercury resistance determinant was transferred beyond the boarders of anaerobic and aerobic environments and the bacterial genera.

From these experimental and analytical results, we propose a novel concept of in situ molecular breeding for bioremediation of mercury-contaminated sites. The concept of in situ molecular breeding technology may be more environmentally friendly, more economic and effective in practical use, because the breeding of the special microorganisms is based on natural gene dissemination phenomenon and because indigenous microorganisms in the environment are utilized as the recipient microorganisms for the gene propagation.

As a global pollutant, which has both natural and anthropogenic sources, mercury (Hg) is present in the biosphere. Thus, bacterial communities developed resistance mechanisms against its toxic effects overtime. The most reported mechanism is a redox reaction catalyzed by the mercury reductase encoded by the merA gene. The objective of this study was to investigate the presence of the merA gene in mercury resistant Gram-negative bacteria isolated from aquatic systems of different Brazilian regions. Water samples were collected, using syringes coupled to filtration devices containing 0.22 µm membranes, either in polluted or pristine environments, from coastal to inland aquatic ecosystems. Isolation of mercury resistant bacteria was performed using enrichment method and directly plating on medium containing Hg (5 µM). The isolated strains were identified using BD BBL Crystal^TM kits. Hg Minimum Inhibition Concentration (MIC) was determined using increasing Hg concentrations (from 10 to 45 M) added to the growth medium. Bacterial strains (n = 119) that grew overnight in Nutrient Agar plates containing at least 20 µM of Hg, were considered Hg resistant. Total genomic DNA was extracted for PCR reactions. Primers A1 (5 ACC ATC GGC GGC ACC TGC GT3 ) and A5 (5 ACC ATC GTC AGG TAG GGG ACC AA3 ) were used to amplify the conserved merA 1239bp fragment. The genetic variability of sequence was investigated by restriction fragment length polymorphism (RFLP) analysis. AluI, EcoRI and DdeI restriction enzymes were employed to digest 7 PCR products from different sampling regions. Results from PCR reactions detected the fragment in 33 samples (27.8%) and revealed genetic polymorphism in 73 samples (61.3%), generating electrophoretic profiles with fragments ranging from 500bp to 2500bp. PCR-RFLP assays yielded 3 restriction profiles for all enzymes tested. Our results from amplification and restriction assays revealed a high genetic diversity within merA sequence in Gram-negative bacteria from Brazilian aquatic systems. The results will be confirmed by DNA sequencing and comparison with known deposited merA sequences.

We report recent progress toward understanding the structure, function, and underlying mechanisms of proteins involved in bacterial mercury resistance using biochemical, biophysical and computational approaches. Specific systems include the metalloregulator MerR, which controls the transcription of mer genes, the organomercurial lyase MerB, which catalyzes the cleavage of mercury-carbon bonds, and the mercuric reductase MerA, which catalyzes the reduction of Hg(II) to less toxic elemental Hg(0). In the case of MerR, the structures of the apo and Hg(II)-bound forms of the protein in aqueous solution were examined using small-angle X-ray scattering (SAXS), and the conformational dynamics of the Hg(II)-bound form were characterized using molecular dynamics (MD) simulations. Simulations revealed interdomain motions on a timescale of ~10 ns involving large amplitude (~20 Å) domain opening-and-closing, coupled to ~40° variations of the interdomain torsional angle. This correlated domain motion may propagate allosteric changes from the metal-binding site to the DNA-binding site while maintaining DNA contacts required to initiate DNA underwinding. In MerB, it is known that two conserved cysteines and an aspartate residue are required for catalysis, but many details of the reaction mechanism have not been determined. Proposed reaction mechanisms of MerB were investigated using density functional theory (DFT) calculations, and the results support a mechanism in which the two cysteines coordinate with methylmercury and the aspartic acid protonates the hydrocarbon leaving group. For the mercuric reductase MerA, the structure and dynamics of the multi-domain protein were investigated using SAXS and MD simulations. The results provide insight into how the N-terminal domain (NmerA) acquires Hg(II) and delivers it to the catalytic core domain for reduction. We also discuss structural characterization and mechanistic work on the outer-membrane cytochrome OmcA, and progress toward improved understanding of the interaction of Hg(II) with low molecular weight ligands in aqueous solution.