Microbiological oxidation of antimony(III) with oxygen or nitrate by bacteria isolated from contaminated mine sediments.

Bacterial oxidation of arsenite [As(III)] is a well-studied and important biogeochemical pathway that directly influences the mobility and toxicity of arsenic in the environment. In contrast, little is known about microbiological oxidation of the chemically similar anion antimonite [Sb(III)]. In this study, two bacterial strains, designated IDSBO-1 and IDSBO-4, which grow on tartrate compounds and oxidize Sb(III) using either oxygen or nitrate, respectively, as a terminal electron acceptor, were isolated from contaminated mine sediments. Both isolates belonged to the Comamonadaceae family and were 99% similar to previously described species. We identify these novel strains as Hydrogenophaga taeniospiralis strain IDSBO-1 and Variovorax paradoxus strain IDSBO-4. Both strains possess a gene with homology to the aioA gene, which encodes an As(III)-oxidase, and both oxidize As(III) aerobically, but only IDSBO-4 oxidized Sb(III) in the presence of air, while strain IDSBO-1 could achieve this via nitrate respiration. Our results suggest that expression of aioA is not induced by Sb(III) but may be involved in Sb(III) oxidation along with an Sb(III)-specific pathway. Phylogenetic analysis of proteins encoded by the aioA genes revealed a close sequence similarity (90%) among the two isolates and other known As(III)-oxidizing bacteria, particularly Acidovorax sp. strain NO1. Both isolates were capable of chemolithoautotrophic growth using As(III) as a primary electron donor, and strain IDSBO-4 exhibited incorporation of radiolabeled [(14)C]bicarbonate while oxidizing Sb(III) from Sb(III)-tartrate, suggesting possible Sb(III)-dependent autotrophy. Enrichment cultures produced the Sb(V) oxide mineral mopungite and lesser amounts of Sb(III)-bearing senarmontite as precipitates.

Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2.

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons (mer) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2, contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria, but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK, with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 µM inorganic mercury [Hg(II)] and 0.05 µM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA. We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.

Mercury speciation and mobilization in a wastewater-contaminated groundwater plume.

We measured the concentration and speciation of mercury (Hg) in groundwater down-gradient from the site of wastewater infiltration beds operated by the Massachusetts Military Reservation, western Cape Cod, Massachusetts. Total mercury concentrations in oxic, mildly acidic, uncontaminated groundwater are 0.5-1 pM, and aquifer sediments have 0.5-1 ppb mercury. The plume of impacted groundwater created by the wastewater disposal is still evident, although inputs ceased in 1995, as indicated by anoxia extending at least 3 km down-gradient from the disposal site. Solutes indicative of a progression of anaerobic metabolisms are observed vertically and horizontally within the plume, with elevated nitrate concentrations and nitrate reduction surrounding a region with elevated iron concentrations indicating iron reduction. Mercury concentrations up to 800 pM were observed in shallow groundwater directly under the former infiltration beds, but concentrations decreased with depth and with distance down-gradient. Mercury speciation showed significant connections to the redox and metabolic state of the groundwater, with relatively little methylated Hg within the iron reducing sector of the plume, and dominance of this form within the higher nitrate/ammonium zone. Furthermore, substantial reduction of Hg(II) to Hg(0) within the core of the anoxic zone was observed when iron reduction was evident. These trends not only provide insight into the biogeochemical factors controlling the interplay of Hg species in natural waters, but also support hypotheses that anoxia and eutrophication in groundwater facilitate the mobilization of natural and anthropogenic Hg from watersheds/aquifers, which can be transported down-gradient to freshwaters and the coastal zone.

The impact of ionic mercury on antioxidant defenses in two mercury-sensitive anaerobic bacteria.

While the toxicological effects of mercury (Hg) are well studied in mammals, little is known about the mechanisms of toxicity to bacterial cells lacking an Hg resistance (mer) operon. We determined that Shewanella oneidensis MR-1 is more sensitive to ionic mercury [Hg(II)] under aerobic conditions than in fumarate reducing conditions, with minimum inhibitory concentrations of 0.25 and 2 µM respectively. This increased sensitivity in aerobic conditions is not due to increased import, as more Hg is associated with cellular material in fumarate reducing conditions than in aerobic conditions. In fumarate reducing conditions, glutathione may provide protection, as glutathione levels decrease in a dose-dependent manner, but this does not occur in aerobic conditions. Hg(II) does not change the redox state of thioredoxin in MR1 in either fumarate reducing conditions or aerobic conditions, although thioredoxin is oxidized in Geobacter sulfurreducens PCA in response to Hg(II) treatment. However, treatment with 0.5 µM Hg(II) increases lipid peroxidation in aerobic conditions but not in fumarate reducing conditions in MR-1. We conclude that the enhanced sensitivity of MR-1 to Hg(II) in aerobic conditions is not due to differences in intracellular responses, but due to damage at the cell envelope.

Identification of a possible respiratory arsenate reductase in Denitrovibrio acetiphilus, a member of the phylum Deferribacteres.

Denitrovibrio acetiphilus N2460(T) is one of the few members of the phylum Deferribacteres with a sequenced genome. N2460(T) was capable of growing with dimethyl sulfoxide, selenate, or arsenate provided as a terminal electron acceptor, and we identified 15 genes that could possibly encode respiratory reductases for these compounds. The protein encoded by one of these genes, YP_003504839, clustered with respiratory arsenate reductases on a phylogenetic tree. Transcription of the gene for YP_003504839, Dacet_2121, was highly induced when arsenate was provided as a terminal electron acceptor. Dacet_2121 exists in a possible operon that is distinct from the previously characterized respiratory arsenate reductase operon in Shewanella sp. ANA-3.

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments.

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 µM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

Reduction of Hg(II) to Hg(0) by magnetite.

Mercury (Hg) is a highly toxic element, and its contamination of groundwater presents a significant threat to terrestrial ecosystems. Understanding the geochemical processes that mediate mercury transformations in the subsurface is necessary to predict its fate and transport. In this study, we investigated the redox transformation of mercuric Hg (Hg[II]) in the presence of the Fe(II)/Fe(III) mixed valence iron oxide mineral magnetite. Kinetic and spectroscopic experiments were performed to elucidate reaction rates and mechanisms. The experimental data demonstrated that reaction of Hg(II) with magnetite resulted in the loss of Hg(II) and the formation of volatile elemental Hg (Hg[0]). Kinetic experiments showed that Hg(II) reduction occurred within minutes, with reaction rates increasing with increasing magnetite surface area (0.5 to 2 m2/L) and solution pH (4.8 to 6.7), and decreasing with increasing chloride concentration (10(-6) to 10(-2) mol/L). Mössbauer spectroscopic analysis of reacted magnetite samples revealed a decrease in Fe(II) content, corresponding to the oxidation of Fe(II) to Fe(III) in the magnetite structure. X-ray photoelectron spectroscopy detected the presence of Hg(II) on magnetite surfaces, implying that adsorption is involved in the electron transfer process. These results suggest that Hg(II) reaction with solid-phase Fe(II) is a kinetically favorable pathway for Hg(II) reduction in magnetite-hearing environmental systems.

Novel reduction of mercury (II) by mercury-sensitive dissimilatory metal reducing bacteria.

The dissimilatory metal reducing bacterium (DMRB) Shewanella oneidensis MR-1 reduces ionic mercury (Hg[II]) to elemental mercury (Hg[0]) by an activity not related to the MerA mercuric reductase. In S. oneidensis, this activity is constitutive and effective at Hg(II) concentrations too low to induce mer operon functions. Reduction of Hg(II) by MR-1 required the presence of electron donors and electron acceptors. Reduction occurred with oxygen or fumarate, but had the highest rate when ferric oxyhydroxide was used as a terminal electron acceptor. Geobacter sulfurreducens PCA and Geobacter metallireducens GS-15 reduced Hg(II) to Hg(0) with activity comparable to MR-1; however, neither the DMRB Anaeromyxobacter dehalogenans 2CP-C nor the nitrate reducer Pseudomonas stutzeri OX-1 reduced Hg(II) during growth. This discovery of constitutive mercury reduction among anaerobes has implications to the mobilization of mercury and production of methylmercury in anoxic environments.

Monitoring of microbial metal transformations in the environment.

The biotransformation of metals is an exciting, developing strategy to treat metal contamination, especially in environments that are not accessible to other remediation technologies. However, our ability to benefit from these strategies hinges on our ability to monitor these transformations in the environment. This involves monitoring metals in both solid and aqueous samples, distinguishing between different chemical states, and obtaining information on the activities of specific microbial taxa in communities that inhabit the treated site. Accomplishing these goals requires cooperation among scientists from various disciplines and would benefit from both new, innovative approaches and the tailoring of established methods to control metal mobility in the environment.

Mutations in the gal83 glycogen-binding domain activate the snf1/gal83 kinase pathway by a glycogen-independent mechanism.

The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Yap1 accumulates in the nucleus in response to carbon stress in Saccharomyces cerevisiae.

Yap1 is a transcription factor of the AP-1 family that is required for the adaptive response to oxidative stress in Saccharomyces cerevisiae. We recovered Yap1 in a two-hybrid screen for proteins that interact with the Sip2 subunit of the Snf1 protein kinase, which is required for the adaptation of cells to glucose limitation. Yap1 becomes enriched in the nucleus when cells are subjected to oxidative stress. We show that the localization of Yap1 is similarly sensitive to carbon stress. When glucose-grown cells were shifted to medium containing glycerol or no added carbon source, green fluorescent protein (GFP)-Yap1 accumulated in the nucleus. After adaptation to growth in glycerol, GFP-Yap1 was again primarily cytoplasmic. Nuclear accumulation was independent of respiration and of the Snf1, PKA, TOR, and Yak1 pathways, and the mechanism is distinct from that involved in the response to hydrogen peroxide. Addition of glutathione to the medium inhibited nuclear accumulation of GFP-Yap1 in response to carbon stress but did not affect the relocalization of Gal83 or Mig1. Other stresses such as increased temperature, acidic pH, and ionic stress did not cause nuclear enrichment of GFP-Yap1. These findings suggest a role for Yap1 in the response to carbon stress.