Mercury (Hg) associated with mixed waste generated by nuclear weapons manufacturing has contaminated vast areas of the Oak Ridge Reservation (ORR). Neurotoxic methylmercury (MeHg) has been formed from the inorganic Hg wastes discharged into headwaters of East Fork Poplar Creek (EFPC). Thus, understanding the processes and mechanisms that lead to Hg methylation along the flow path of EFPC is critical to predicting the impacts of the contamination and the design of remedial action at the ORR. In part I of our project, we investigated Hg(0) oxidation and methylation by anaerobic bacteria. We discovered that the anaerobic bacterium Desulfovibrio desulfuricans ND132 can oxidize elemental mercury [Hg(0)]. When provided with dissolved elemental mercury, D. desulfuricans ND132 converts Hg(0) to Hg(II) and neurotoxic methylmercury [MeHg]. We also demonstrated that diverse species of subsurface bacteria oxidizes dissolved elemental mercury under anoxic conditions. The obligate anaerobic bacterium Geothrix fermentans H5, and the facultative anaerobic bacteria Shewanella oneidensis MR-1 and Cupriavidus metallidurans AE104 can oxidize Hg(0) to Hg(II) under anaerobic conditions. In part II of our project, we established anaerobic enrichment cultures and obtained new bacterial strains from the DOE Oak Ridge site. We isolated three new bacterial strains from subsurface sediments collected from Oak Ridge. These isolates are Bradyrhizobium sp. strain FRC01, Clostridium sp. strain FGH, and a novel Negativicutes strain RU4. Strain RU4 is a completely new genus and species of bacteria. We also demonstrated that syntrophic interactions between fermentative bacteria and sulfate-reducing bacteria in Oak Ridge saprolite mediate iron reduction via multiple mechanisms. Finally, we tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge site, where nitrate is a major contaminant. We showed that there is an inverse relationship between Hg concentrations and rates of denitrification in enrichment cultures. In part III of our project, we examined in more detail the effects of microbial interactions on Hg transformations. We discovered that both sulfate reducing and iron reducing bacteria coexist in freshwater sediments and both microbial groups contribute to mercury methylation. We showed that mercury methylation by sulfate reducing and iron reducing bacteria are temporally and spatially separated processes. We also discovered that methanogens can methylate mercury. We showed that Methanospirillum hungatei JF-1 methylated Hg at comparable rates, but with higher yields, than those observed for sulfate-reducing bacteria and iron-reducing bacteria. Finally, we demonstrated that syntrophic interactions between different microbial groups increase mercury methylation rates. We showed that Hg methylation rates are stimulated via inter-species hydrogen and acetate transfer (i) from sulfate-reducing bacteria to methanogens and (ii) from fermenters to the sulfate-reducing bacteria. In part IV of the project, we studied Hg bioavailability and Hg isotope fractionation. We demonstrated that thiol-bound Hg is bioavailable to mercury resistant bacteria. We found that uptake of Hg from Hg-glutathione and Hg-cysteine complexes does not require functioning glutathione and cystine/cysteine transport systems. We demonstrated that a wide range of methylmercury complexes (e.g. MeHgOH, MeHg-cysteine, and MeHg-glutathione) are bioavailable to mercury resistant bacteria. The rate of MeHg demethylation varies more between different species of mercury resistant bacteria than among MeHg complexes. We showed that microbial demethylation of MeHg depends more on the species of microorganism than on the types and relative concentrations of thiols or other MeHg ligands present. Finally, we demonstrated that Hg methylation by Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132 imparts mass-dependent discrimination against 202Hg relative to 198Hg. G. sulfurreducens PCA and D. desulfuricans ND132 have similar kinetic reactant/product Hg fractionation factors. Using the Hg isotope data, we showed that there are multiple intra- and/or extracellular pools provide substrate inorganic Hg for methylation.
The concentration and distribution of selenium species in near surface geologic and aquatic environments is strongly affected by microbial processes. Under aerobic and microaerophilic conditions, a wide variety of phylogentically distinct bacteria species have been shown to reduce Se(VI) and Se(IV) to form sparingly soluble elemental selenium. In order to quantify the geochemical impact of these microorganisms, accurate models must be developed to predict when these organisms will be active, and how rapidly they will reduce selenate and selenite. In this study, we quantified the kinetics of the selenate reduction reaction by soil microorganism Enterobacter cloacae and we identified the genes that control the selenate reduction process. The kinetics of selenate reduction by E. cloacae were examined using batch experiments as a function of pH, temperature, and cell density. Electron microscopy and X-ray diffraction were employed to determine the morphology and crystallinity of the reduction products. Finally, we used transposon mutagenesis to produce mutants that have loss the ability to reduce selenate, and we characterized some of these mutants. A rate law based on the Michaelis-Menten equation was developed to describe the selenate reduction kinetics over a range of pH conditions, temperatures, and cell densities. The reduction reaction formed nanoparticulate elemental selenium granules that were both excreted into solution and attached to the cell surface. Mutants constructed from transposon mutagenesis were unable to reduce selenate to Se but were still able to reduce selenite, demonstrating that the selenate and selenite reductases are distinct enzymes. The link between the genetics and geochemistry of microbial selenium oxyanion reduction will be discussed. New insights into the molecular mechanism of microbial metal respiration
The redox cycling of selenium in aquatic environments is strongly affected by microbial activity. Citrobacter freudii is a facultative anaerobic bacterium found in marine and freshwater settings that is known to reduce selenate. In this study, we conducted selenate reduction experiments with C. freundii to investigate the mechanisms involved the selenate reduction process. The results indicate that C. freundii catalyzes the reduction of selenate to elemental selenium in the absence of oxygen. We detected the functional selenate reductase gene ynfE, which is predicted to encode for a molybdenum-binding Tat-secreted protein. A FNR binding site was located immediately upstream of the ynfE gene suggesting the expression of the selenate reductase occurs under anaerobic conditions and is regulated by oxygen-sensing transcription factors. To gain a more complete understanding of the selenate reduction process, we sequenced the genome of C. freundii. The genome analysis revealed the complete selenate reductase operon ynfEGHdmsD, showing high sequence identity to selenate reductases found in related gammaproteobacteria. Genes for molybdate uptake, molybdopterin guanine dinucleotide biosynthesis, twin arginine translocation, and FNR regulation were also identified in the genome sequence. Based on the experimental results and genome sequence, we developed a model to describe anaerobic selenate reduction in C. freundii. The results of this work provide new insights into the molecular basis of selenate reduction activity in a geochemically relevant facultative anaerobic bacterium.
Molybdoenzymes are an ancient protein family found in phylogenetically and ecologically diverse prokaryotes. Under anaerobic conditions, respiratory molybdoenzymes catalyze redox reactions that transfer electrons to a variety of substrates that act as terminal electron acceptors for energy generation. Here, we used probe sequences to conduct an extensive genomic survey and phylogenetic inference for NarG, DmsA, TorA and nine other respiratory molybdoenzyme subfamilies. Our analysis demonstrates their abundance in 60% of prokaryotic phyla. In contrast to many other autonomic genetic units in prokaryotes, the major route of evolution of their predominant subfamilies is vertical gene transfer, gene duplication and divergence. Our results show the robustness of genomic co-occurrence of respiratory molybdoenzymes genes, found in the majority of studied species, for most of the enzyme subfamilies. Genomes which encode for multiple respiratory molybdoenzymes are also enriched in genes regulating replication, recombination and mobility of genetic elements. Respiratory molybdoenzymes were found in prokaryotes associated with diverse environments occupying terrestrial, aquatic, food and host-related habitats, emphasizing their essential role in adaptation of prokaryotes to changing environments. Interestingly, host-associated prokaryotes such as human pathogens more frequently carry multiple respiratory molybdoenzyme genes compared with non-host-associated prokaryotes, highlighting the importance of metabolic flexibility in host-microbiome environments.
ABSTRACT The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA , moaB , moaE , or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme.
In this study, we conducted synchrotron radiation Fourier transform infrared (IR) spectroscopy, potentiometric titration, and metal sorption experiments to characterize metal−cyanobacteria sorption reactions. Infrared spectra were collected with samples in solution for intact cyanobacterial filaments and separated exopolymeric sheath material to examine the deprotonation reactions of cell surface functional groups. The infrared spectra of intact cells sequentially titrated from pH 3.2 to 6.5 display an increase in peak intensity and area at 1400 cm-1 corresponding to vibrational COO- frequencies from the formation of deprotonated carboxyl surface sites. Similarly, bulk acid−base titration of cyanobacterial filaments and sheath material indicates that the concentration of proton-active surface sites is higher on the cell wall compared to the overlying sheath. A three-site model provides an excellent fit to the titration curves of both intact cells and sheath material with corresponding pKa values of 4.7 ± 0.4, 6.6 ± 0.2, 9.2 ± 0.3 and 4.8 ± 0.3, 6.5 ± 0.1, 8.7 ± 0.2, respectively. Finally, Cu2+, Cd2+, and Pb2+ sorption experiments were conducted as a function of pH, and a site-specific surface complexation model was used to describe the metal sorption data. The modeling indicates that metal ions are partitioned between the exopolymer sheath and cell wall and that the carboxyl groups on the cyanobacterial cell wall are the dominant sink for metals at near neutral pH. These results demonstrate that the cyanobacterial surfaces are complex structures which contain distinct surface layers, each with unique molecular functional groups and metal binding properties.
