Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with cooccurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G؉C-and low-G؉C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb r ) isolates was amplified with PCR primers specific for P IB -type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P IB -type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb r P IB -type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO 2 2؉ ) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P IB -type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.The remediation of hazardous mixed-waste sites, particularly those cocontaminated with heavy metals and radionuclides, is one of the most costly environmental challenges currently faced by the United States and other countries. Interactions between microorganisms, radionuclides, and metals that promote their precipitation and immobilization in situ are promising strategies for treatment and cleanup of the contaminated subsurface (1, 15). At mixed-waste sites where the concentrations of metal contaminants can reach toxic levels, the metal resistance of indigenous microbial populations could be critical for the success of in situ biostimulation efforts. For example, while a number of microbes can carry out reductive precipitation of radionuclides (e.g., Desulfovibrio sp., Geobacter sp., and Shewanella sp.) (28,44,63), the sensitivity of these organisms to heavy metals could possibly limit their in situ activities. Thus, the metal sensitivity of some radionuclide-reducing microbes suggests that the acquisition of metal resistance traits (e.g., P IB -type ATPases that regulate the transport of heavy metals) might be conducive to facilitating and/or enhancing microbial metabolism during subsequent biostimulation activities in metal-and radionuclide-contaminated subsurface environments.The P-type ATPases represent a chromosomally en...

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9°N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 M Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45°C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65°C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.Heavy metals are highly enriched in hydrothermal vent fluids of mid-oceanic ridge systems (35), reaching concentrations that are considered to be toxic to living organisms (26). The steep physical-chemical gradients that occur when reduced, hot, element-and sulfur-rich vent fluids are diluted with oxygenated, cold seawater create a gradient in metal toxicity in the vent environment (20). As oxygen mixes with the anoxic, sulfur-rich fluid, metal speciation can shift from metal sulfides that show poor bioavailability and low toxicity to more soluble and oxidized forms with increased bioavailability and toxicity. With such a change in metal speciation, high tolerance to metals is expected among microbes inhabiting niches of the vent ecosystem where mixing between hydrothermal fluids and oxygenated seawater occurs, such as diffuse flow vents and associated plumes. This hypothesis is supported by experimental data showing that thermophilic archaea and bacteria from highly reduced vent microhabitats were metal su...

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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 ␤ subclass of the Proteobacteria (␤-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of ␤-and ␥-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.The role of lateral gene transfer (LGT) in the evolution of microorganisms becomes more and more apparent with every new microbial genome that is sequenced and annotated (54, 58), which has led many workers to question our basic concepts of microbial speciation (18, 23). The prevalence of laterally transferred genes clearly suggests that this mode of evolution is advantageous to microbial life, possibly by providing the means for genetic innovation in the absence of frequent sexual recombination (37). This variation is likely to increase metabolic diversity, and consequently competence, in environments subject to frequent change (13, 61). The possibility that LGT is an important force in shaping the structure and function of microbial communities in their natural habitats is suggested by (i) the fact that abundant and diverse plasmids (12, 73), viruses (8, 89), insertion sequences (70), transposons (59, 78), integrons (49), and other elements that contribute to genomic plasticity are commonly isolated from environmental samples and strains; (ii) the fact that natural competence is common among microbes (11) and occurs in soils (17, 50) and natural water (88); (iii) the fact that LGT has been demonstrated in model ecosystems (i.e., microcosms) (52, 62, 75, 80) and in intact environmental samples (19); and (iv) the fact that seeding soils with bacteria carrying conjugal catabolic plasmids results in transfer of the plasmids to indigenous soil microbes and the stimulation of plasmid-specified activit...

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.