Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

New findings on evolution of metal homeostasis genes: evidence from comparative genome analysis of bacteria and archaea.

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P(IB)-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P(IB)-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P(IB)-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.

Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface.

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.

Metal resistance among aerobic chemoheterotrophic bacteria from the deep terrestrial subsurface.

The metal resistance of 350 subsurface bacterial strains from two U.S. Department of Energy facilities, the Savannah River Site (SRS), South Carolina, and the Hanford site, Washington, was determined to assess the effect of metal toxicity on microorganisms in the deep terrestrial subsurface. Resistance was measured by growth inhibition around discs containing optimized amounts of Hg(II), Pb(II), and Cr(VI). A broad range of resistance levels was observed, with some strains of Arthrobacter spp. demonstrating exceptional tolerance. A higher level of resistance to Hg(II) and Pb(II) (P < 0.05) and a higher occurrence of multiple resistances suggested that metals more effectively influenced microbial evolution in subsurface sediments of the SRS than in those of the Hanford site. Common resistance to heavy metals suggests that toxic metals are unlikely to inhibit bioremediation in deep subsurface environments that are contaminated with mixed wastes.

Metal and radionuclide bioremediation: issues, considerations and potentials.

Recent demonstrations of the removal and immobilization of inorganic contaminants by microbial transformations, sorption and mineralization show the potential of both natural and engineered microbes as bioremedial tools. Demonstrations of microbe-mediated mineral formation in biofilms implicate this mode of microbial life in geological evolution and remediation of inorganic contaminants.

Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase.

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.

Mercury biogeochemistry in the Idrija river, Slovenia, from above the mine into the Gulf of Trieste.

The Idrija Mine is the second largest Hg mine in the world which operated for 500 years. Mercury (Hg)-laden tailings still line the banks, and the system is a threat to the Idrija River and water bodies downstream including the Soca/Isonzo River and the Gulf of Trieste in the northern Adriatic Sea. A multidisciplinary study was conducted in June 1998 on water samples collected throughout the Idrija and Soca River systems and waters and sediments in the Gulf. Total Hg in the Idrija River increased >20-fold downstream of the mine from <3 to >60 ng liter(-1) with methyl mercury (MeHg) accounting for approximately 0.5%. Concentrations increased again downstream and into the estuary with MeHg accounting for nearly 1.5% of the total. While bacteria upstream of the mine did not contain mercury detoxification genes (mer), such genes were detected in bacteria collected downstream. Benthic macroinvertebrate diversity decreased downstream of the mine. Gulf waters near the river mouth contained up to 65 ng liter(-1) total Hg with approximately 0.05 ng liter(-1) MeHg. Gulf sediments near the river mouth contained 40 microgram g(-1) total Hg with MeHg concentrations of about 3 ng g(-1). Hg in sediment pore waters varied between 1 and 8 ng liter(-1), with MeHg accounting for up to 85%. Hg methylation and MeHg demethylation were active in Gulf sediments with highest activities near the surface. MeHg was degraded by an oxidative pathway with >97% C released from MeHg as CO(2). Hg methylation depth profiles resembled profiles of dissolved MeHg. Hg-laden waters still strongly impact the riverine, estuarine, and marine systems. Macroinvertebrates and bacteria in the Idrija River responded to Hg stress, and high Hg levels persist into the Gulf. Increases in total Hg and MeHg in the estuary demonstrate the remobilization of Hg, presumably as HgS dissolution and recycling. Gulf sediments actively produce MeHg, which enters bottom waters and presumably the marine food chain.

Enhancement of solubilization and biodegradation of polyaromatic hydrocarbons by the bioemulsifier alasan.

Alasan, a high-molecular-weight bioemulsifier complex of an anionic polysaccharide and proteins that is produced by Acinetobacter radioresistens KA53 (S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E. Z. Ron, and E. Rosenberg, Appl. Environ. Microbiol. 61:3240-3244, 1995), enhanced the aqueous solubility and biodegradation rates of polyaromatic hydrocarbons (PAHs). In the presence of 500 microg of alasan ml-1, the apparent aqueous solubilities of phenanthrene, fluoranthene, and pyrene were increased 6.6-, 25.7-, and 19.8-fold, respectively. Physicochemical characterization of the solubilization activity suggested that alasan solubilizes PAHs by a physical interaction, most likely of a hydrophobic nature, and that this interaction is slowly reversible. Moreover, the increase in apparent aqueous solubility of PAHs does not depend on the conformation of alasan and is not affected by the formation of multimolecular aggregates of alasan above its saturation concentration. The presence of alasan more than doubled the rate of [14C]fluoranthene mineralization and significantly increased the rate of [14C]phenanthrene mineralization by Sphingomonas paucimobilis EPA505. The results suggest that alasan-enhanced solubility of hydrophobic compounds has potential applications in bioremediation.

Utilization of phenoxyacetic acid, by strains using either the ortho or meta cleavage of catechol during phenol degradation, after conjugal transfer of tfdA, the gene encoding a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase.

The degradation of recalcitrant pollutants in contaminated soils and waters could be facilitated by broadening the degradative capabilities of indigenous microbes by the conjugal transfer of catabolic genes. The feasibility of establishing bacterial populations that degrade phenoxyacetic acid by conjugal transfer of tfdA, the gene encoding 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase, to phenol-degrading strains of Pseudomonas and Ralstonia was examined. The mobilizable plasmid pKJS32 served as a vector for delivery of tfdA and the regulatory gene, tfdS. Transconjugant strains that degraded phenol by an ortho cleavage of catechol grew well on phenoxyacetic acid while those employing a meta cleavage could only grow on phenoxyacetic acid in the presence of benzoic acid or after a prolonged lag period and the appearance of mutants that had gained catechol 1,2-dioxygenase activities. Thus, an ortho cleavage of catechol was essential for degradation of phenoxyacetic acid, suggesting that a product of the ortho-cleavage pathway, probably cis, cis-muconic acid, is an inducer of tfdA gene expression. Establishment of phenoxyacetic-acid-degrading soil populations by conjugal transfer of tfdA would depend on the presence of phenol-degrading recipients employing an ortho cleavage of catechol.

Effects of dissolved organic carbon and salinity on bioavailability of mercury.

Hypotheses that dissolved organic carbon (DOC) and electrochemical charge affect the rate of methylmercury [CH3Hg(I)] synthesis by modulating the availability of ionic mercury [Hg(II)] to bacteria were tested by using a mer-lux bioindicator (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993). A decline in Hg(II)-dependent light production was observed in the presence of increasing concentrations of DOC, and this decline was more pronounced at pH 7 than at pH 5, suggesting that DOC is a factor controlling the bioavailability of Hg(II). A thermodynamic model (MINTEQA2) was used to select assay conditions that clearly distinguished among various Hg(II) species. By using this approach, it was shown that negatively charged forms of mercuric chloride (HgCl3-/HgCl(4)2-) induced less light production than the electrochemically neutral form (HgCl2), and no difference was observed between the two neutral forms, HgCl2 and Hg(OH)2. These results suggest that the negative charge of Hg(II) species reduces their availability to bacteria and may be one reason why accumulation of CH3Hg(I) is more often reported to occur in freshwater than in estuarine and marine biota.

Cell-density-dependent sensitivity of a mer-lux bioassay.

The sensitivity of a previously described assay (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993) for the detection of bioavailable inorganic mercury (Hg2+) by the activation of a mer-lux fusion was increased from nanomolar to picomolar concentrations by reducing biomass in the assays from 10(7) to 10(5) cells ml-1. The increase in sensitivity was due to a reduction in the number of cellular binding sites that may compete with the regulatory protein, MerR, for binding of the inducer, Hg2+. These results show that (i) the sensitivity of the mer-lux assay is sufficient for the detection of Hg2+ in most contaminated natural waters and (ii) mer-specified reactions, Hg2+ reduction and methylmercury degradation, can be induced in natural waters and may participate in the geochemical cycling of mercury.

Detection of the merA gene and its expression in the environment

Bacterial transformation of mercury in the environment has received much attention owing to the toxicity of both the ionic form and organomercurial compounds. Bacterial resistance to mercury and the role of bacteria in mercury cycling have been widely studied. The genes specifying the required functions for resistance to mercury are organized on the mer operon. Gene probing methodologies have been used for several years to detect specific gene sequences in the environment that are homologous to cloned mer genes. While mer genes have been detected in a wide variety of environments, less is known about the expression of these genes under environmental conditions. We combined new methodologies for recovering specific gene mRNA transcripts and mercury detection with a previously described method for determining biological potential for mercury volatilization to examine the effect of mercury concentrations and nutrient availability on rates of mercury volatilization and merA transcription. Levels of merA-specific transcripts and Hg(II) volatilization were influenced more by microbial activity (as manipulated by nutrient additions) than by the concentration of total mercury. The detection of merA-specific transcripts in some samples that did not reduce Hg(II) suggests that rates of mercury volatilization in the environment may not always be proportional to merA transcription.

Methylmercury oxidative degradation potentials in contaminated and pristine sediments of the carson river, nevada.

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [(sup14)C]MeHg was detected at all sites as indicated by the formation of (sup14)CO(inf2) and (sup14)CH(inf4). Oxidative demethylation was indicated by the formation of (sup14)CO(inf2) and was present at significant levels in all samples. Oxidized/reduced demethylation product ratios (i.e., (sup14)CO(inf2)/(sup14)CH(inf4) ratios) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of (sup14)CO(inf2) was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having a high proportion of oxidized, coarse-grain sediments, sulfate reduction and methanogenic activity levels were very low and (sup14)CO(inf2) accounted for 98% of the product formed from [(sup14)C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurrence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed oxidized/reduced demethylation product ratios, but overall demethylation rates of inhibited and uninhibited samples were comparable. Addition of sulfate to sediment slurries stimulated production of (sup14)CO(inf2) from [(sup14)C]MeHg, while 2-bromoethanesulfonic acid blocked production of (sup14)CH(inf4). These results reveal the importance of sulfate-reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments.

merA gene expression in aquatic environments measured by mRNA production and Hg(II) volatilization.

The relationship of merA gene expression (specifying the enzyme mercuric reductase) to mercury volatilization in aquatic microbial communities was investigated with samples collected at a mercury-contaminated freshwater pond, Reality Lake, in Oak Ridge, Tenn. Levels of merA mRNA transcripts and the rate of inorganic mercury [Hg(II)] volatilization were related to the concentration of mercury in the water and to heterotrophic activity in field samples and laboratory incubations of pond water in which microbial heterotrophic activity and Hg(II) concentration were manipulated. Levels of merA-specific mRNA and Hg(II) volatilization were influenced more by microbial metabolic activity than by the concentration of mercury. merA-specific transcripts were detected in some samples which did not reduce Hg(II), suggesting that rates of mercury volatilization in environmental samples may not always be proportional to merA expression.

Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon.

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.

Bioluminescent sensors for detection of bioavailable Hg(II) in the environment.

Biosensors for the detection of pollutants in the environment can complement analytical methods by distinguishing bioavailable from inert, unavailable forms of contaminants. By using fusions of the well-understood Tn21 mercury resistance operon (mer) with promoterless luxCDABE from Vibrio fischeri, we have constructed and tested three biosensors for Hg(II). Bioluminescence specified by pRB28, carrying merRo/pT, by pOS14, mediating active transport of Hg(II), and by pOS15, containing an intact mer operon, was measured in rich and minimal media. The highest sensitivities were achieved in minimal medium and were 1, 0.5, and 25 nM Hg(II) for pRB28, pOS14, and pOS15, respectively. The utility of the biosensors in natural waters was demonstrated with freshwater, rain, and estuarine samples supplemented with Hg(II). mer-lux carried by pRB28 and pOS14 responded to Hg(II) in mercury-contaminated water samples collected from a freshwater pond. Semiquantitative analyses based on light emission in samples collected from the inlet (analytically determined total mercury, approximately 20 nM) and outlet (total mercury, approximately 7 nM) of the pond showed bioavailable mercury at approximately 20 and 1 to 2 nM, respectively. Thus, the biosensors described here semiquantitatively detect bioavailable inorganic mercury (at a nanomolar to micromolar concentration range) in contaminated waters.

Conjugal gene transfer to aquatic bacteria detected by the generation of a new phenotype.

An experimental approach based on the assembly of genes of a catabolic pathway was used to detect transconjugants in aquatic communities. Resistance to phenylmercury acetate was established in transconjugants when wide-host-range conjugal plasmids containing merB, the gene encoding organomercurial lyase, were transferred to strains from aquatic communities that had been acclimated to inorganic mercury and thus enriched for populations containing merA, the gene encoding mercuric reductase (T. Barkay, Appl. Environ. Microbiol. 53:2725-2732, 1987). Conjugation was confirmed by using the plasmids' encoded antibiotic resistance patterns and by hybridization with a eukaryotic gene. Three merB-conjugal plasmids, belonging to incompatibility groups W (pGTE16), P1 (pGTE26), and N (pGTE25), were prepared. Transfers by filter matings of pGTE16 and pGTE26 from Pseudomonas aeruginosa PA01 to indigenous strains were at efficiencies of 4.5 x 10 and 4.8 x 10 transconjugant per potential recipient, respectively. These efficiencies were from 1 to 2 orders of magnitude below those observed for intraspecies matings with genetically marked recipients. The third plasmid, pGTE25, was not stably maintained in P. aeruginosa donors, and its transfer from Escherichia coli donors was below the level of detection. Characterized transconjugant strains were shown to be Pseudomonas spp. Potential applications of the described experimental approach in the creation of bacterial populations with new catabolic capabilities in hazardous waste sites and in the detection of transfer of recombinant DNA from engineered microorganisms to indigenous bacteria are discussed.

The relationships of Hg(II) volatilization from a freshwater pond to the abundance ofmer genes in the gene pool of the indigenous microbial community.

The role of biological activities in the reduction and volatilization of Hg(II) from a polluted pond was investigated. Elemental mercury was evolved from pond water immediately following spiking with(203)Hg(NO3)2, whereas an acclimation period of 36 hours was required in control samples collected from a nearby, unpolluted river before onset of volatilization. Genes encoding the bacterial mercuric reductase enzyme (mer genes) were abundant in DNA fractions extracted from biomass of the pond microbial community, but not in samples extracted from control communities. Thus, evolution of Hg(0) was probably due to activities mediated by the bacterial mercuric reductase. Of four characterizedmer operons, the system encoded by transposon 501 (mer(Tn501)) dominated and likely contributed to the majority of the observed Hg(II) volatilization. Thus,mer-mediated reduction and volatilization could be used to reduce Hg(II) concentrations in polluted waters, in turn decreasing rates of methylmercury formation by limiting substrate availability.