Thermoacidophilic archaea are found in heavy metal-rich environments, and, in some cases, these microorganisms are causative agents of metal mobilization through cellular processes related to their bioenergetics. Given the nature of their habitats, these microorganisms must deal with the potentially toxic effect of heavy metals. Here, we show that two thermoacidophilic Metallosphaera species with nearly identical (99.99%) genomes differed significantly in their sensitivity and reactivity to uranium (U). Metallosphaera prunae, isolated from a smoldering heap on a uranium mine in Thüringen, Germany, could be viewed as a "spontaneous mutant" of Metallosphaera sedula, an isolate from Pisciarelli Solfatara near Naples. Metallosphaera prunae tolerated triuranium octaoxide (U 3 O 8 ) and soluble uranium [U(VI)] to a much greater extent than M. sedula. Within 15 min following exposure to "U(VI) shock," M. sedula, and not M. prunae, exhibited transcriptomic features associated with severe stress response. Furthermore, within 15 min post-U(VI) shock, M. prunae, and not M. sedula, showed evidence of substantial degradation of cellular RNA, suggesting that transcriptional and translational processes were aborted as a dynamic mechanism for resisting U toxicity; by 60 min post-U(VI) shock, RNA integrity in M. prunae recovered, and known modes for heavy metal resistance were activated. In addition, M. sedula rapidly oxidized solid U 3 O 8 to soluble U(VI) for bioenergetic purposes, a chemolithoautotrophic feature not previously reported. M. prunae, however, did not solubilize solid U 3 O 8 to any significant extent, thereby not exacerbating U(VI) toxicity. These results point to uranium extremophily as an adaptive, rather than intrinsic, feature for Metallosphaera species, driven by environmental factors. uranium oxidation | uranium resistance | chemolithotrophy | terminal oxidases

Extreme thermoacidophiles (Topt > 65 °C, pHopt < 3.5) inhabit unique environments fraught with challenges, including extremely high temperatures, low pH, as well as high levels of soluble metal species. In fact, certain members of this group thrive by metabolizing heavy metals, creating a dynamic equilibrium between biooxidation to meet bioenergetic needs and mechanisms for tolerating and resisting the toxic effects of solubilized metals. Extremely thermoacidophilic archaea dominate bioleaching operations at elevated temperatures and have been considered for processing certain mineral types (e.g., chalcopyrite), some of which are recalcitrant to their mesophilic counterparts. A key issue to consider, in addition to temperature and pH, is the extent to which solid phase heavy metals are solubilized and the concomitant impact of these mobilized metals on the microorganism's growth physiology. Here, extreme thermoacidophiles are examined from the perspectives of biodiversity, heavy metal biooxidation, metal resistance mechanisms, microbe-solid interactions, and application of these archaea in biomining operations.

The extremely thermoacidophilic archaeon Metallosphaera sedula mobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by "shocking" M. sedula with representative metals ( ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu 2؉ (6-fold) but also in response to UO 2 2؉ (4-fold) and Zn 2؉ (9-fold). Cu 2؉ challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu 2؉ resistance or issues in sulfur metabolism. The results indicate that M. sedula employs a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions. IMPORTANCEThe mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics. E xtremely thermoacidophilic archaea from the genera Sulfolobus, Acidianus, and Metallosphaera can inhabit natural and anthropogenic environments laden with metals (1) and, as a consequence, require strategies to avert the deleterious impact of these metals on cellular function (2). For some species within the Sulfolobales, metal oxidation mediated by membrane-bound, oxidase clusters provides a cellular bioenergetic benefit (3-5). However, at the same time, utilization of this energy source contributes to the toxicity of the biotope by mobilizing metal cations that can subsequently inhibit biological function (6, 7). As a consequence, the interconnections between metal biooxidation, toxicity, and resistance are important to consider to have a full appreciation of how these unique microorganisms survive in extreme environments (8).For extremely thermoacidophilic archaea, the most studied metal thus far has been copper, because of its importance in biohydrometallurgy (9). Resistance to copper in extreme thermoacidophiles is mediated at least by mechanisms involving active transport and metal sequestr...

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Summary When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during ‘uranium-shock’ implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Certain species from the extremely thermoacidophilic genus Metallosphaera directly oxidize Fe(II) to Fe(III), which in turn catalyzes abiotic solubilization of copper from chalcopyrite to facilitate recovery of this valuable metal. In this process, the redox status of copper does not change as it is mobilized. Metallosphaera species can also catalyze the release of metals from ores with a change in the metal’s redox state. For example, Metallosphaera sedula catalyzes the mobilization of uranium from the solid oxide U3O8, concomitant with the generation of soluble U(VI). Here, the mobilization of metals from solid oxides (V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3) was examined for M. sedula and M. prunae at 70°C and pH 2.0. Of these oxides, only V and Mo were solubilized, a process accelerated in the presence of FeCl3. However, it was not clear whether the solubilization and oxidation of these metals could be attributed entirely to an Fe-mediated indirect mechanism. Transcriptomic analysis for growth on molybdenum and vanadium oxides revealed transcriptional patterns not previously observed for growth on other energetic substrates (i.e., iron, chalcopyrite, organic compounds, reduced sulfur compounds, and molecular hydrogen). Of particular interest was the upregulation of Msed_1191, which encodes a Rieske cytochrome b6 fusion protein (Rcbf, referred to here as V/MoxA) that was not transcriptomically responsive during iron biooxidation. These results suggest that direct oxidation of V and Mo occurs, in addition to Fe-mediated oxidation, such that both direct and indirect mechanisms are involved in the mobilization of redox-active metals by Metallosphaera species. IMPORTANCE In order to effectively leverage extremely thermoacidophilic archaea for the microbially based solubilization of solid-phase metal substrates (e.g., sulfides and oxides), understanding the mechanisms by which these archaea solubilize metals is important. Physiological analysis of Metallosphaera species growth in the presence of molybdenum and vanadium oxides revealed an indirect mode of metal mobilization, catalyzed by iron cycling. However, since the mobilized metals exist in more than one oxidation state, they could potentially serve directly as energetic substrates. Transcriptomic response to molybdenum and vanadium oxides provided evidence for new biomolecules participating in direct metal biooxidation. The findings expand the knowledge on the physiological versatility of these extremely thermoacidophilic archaea.