ABSTRACT
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.
INTRODUCTION
The mobilization of base, precious, and strategic metals from mineral ores by acidophilic microorganisms, often referred to as bioleaching, contributes significantly to mining operations (1). The most well-characterized acidophilic iron biooxidation mechanism is for the bacterial mesophile, Acidithiobacillus ferrooxidans, that involves an electron transfer pathway and associated molecular complexes (2, 3). Recently, significant progress has been made in understanding iron oxidation at the molecular level for extremely acidophilic archaea, including the mesophile Ferroplasma acidiphilum (4, 5). The bioleaching of metal sulfides at elevated temperatures (>65°C), however, is dominated by archaea of the genera Acidianus, Metallosphaera, and Sulfolobus, all members of the order Sulfolobales (6, 7). While no detailed pathway has yet been confirmed for iron oxidation by certain species in the Sulfolobales, experimental evidence for Sulfolobus metallicus, Sulfolobus tokodaii, Metallosphaera sedula, and Metallosphaera yellowstonensis supports a proposed model underlying this phenomenon (8–10). Interestingly, the ability to oxidize ferrous iron [Fe(II)] to ferric iron [Fe(III)], which is essential for many types of bioleaching, is not a common trait among the currently characterized members of the Sulfolobales, even though many species subsist in inorganic chemical-laden environments (1).
The present understanding of microbial metal mobilization from sulfides and oxides assumes an indirect mechanism (1). This involves an abiotic, oxidizing attack on the solid material by Fe(III), after which the resulting Fe(II) is oxidized by the microbe to Fe(III), thereby catalyzing the ensuing mobilization of the metal from the solid matrix. For minerals, such as chalcopyrite (CuFeS2), the released copper [Cu(II)] is already in its highest oxidation state and is thus mobilized but not further oxidized. However, during bioleaching, certain metal sulfides (e.g., MoS2) and metal oxides (e.g., U3O8, MoO2, and V2O3) release metals that can be further oxidized in solution. Thus, soluble metal species with more than one oxidation state and redox potentials more negative than the O2/H2O redox couple could serve as energetic substrates for acidophilic bacteria and archaea (11). A. ferrooxidans has been reported to directly oxidize Cu+ to Cu2+ (12, 13), U4+ to U6+ (14, 15), and Mo5+ to Mo6+ (16). Furthermore, oxidation of U3O8 by M. sedula and Metallosphaera prunae (17) and arsenite oxidation by Sulfolobus metallicus (18) and Acidianus brierleyi (19) have been reported. However, the complete elimination of Fe(III) as a mediator in this process is usually not possible, especially in whole-cell assays where trace amounts of iron are present. This makes differentiation between direct biological oxidation and Fe(III)-mediated abiotic oxidation of the metal difficult (11).
Here, a variety of metal oxides were screened for their susceptibility to mobilization by M. sedula or M. prunae in a microbially mediated process. Of the metal oxide substrates examined, V and Mo species could both be mobilized and oxidized, a process for which Fe(II)/Fe(III) cycling was implicated as part of an indirect mechanism. However, transcriptomic analysis revealed no induction of the ferrous iron oxidation complex (fox) cluster in these cases but instead pointed to previously unidentified open reading frames (ORFs) encoding proteins that could mediate direct oxidation of solubilized metal species. This raised the possibility that solubilization of redox-active metal species from these ores includes previously unidentified metal-oxidizing enzyme complexes in Metallosphaera species and other Sulfolobales that complement the Fe(II)/Fe(III) cycle in bioleaching applications.
RESULTS AND DISCUSSION
Solubilization and oxidation of metals from their corresponding oxides by Metallosphaera species.The capacity for M. sedula to solubilize various metal oxides was assessed by exposing M. sedula at ∼2.0 × 108 cells/ml to 0.1% (wt/vol) of the oxide under chemolithoautotrophic growth conditions. M. sedula was chosen since it was previously found to be a more effective metal mobilizer than another Metallosphaera species also examined here, M. prunae (17, 20). The metal oxides screened included V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3. Metal solubilization was compared to an abiotic control after 1 day and 7 days of incubation. The screening revealed a significant increase in solubilization compared to the abiotic control for V2O3 and MoO2 when incubated with M. sedula; no other metals were solubilized to significant levels. V2O3 and MoO2 solubilization was then tracked over a 3-day period for M. sedula, M. prunae, and an abiotic control under chemolithoautotrophic conditions (see Fig. 1). Although V2O3 solubilization happened abiotically, the presence of M. sedula or M. prunae significantly accelerated the rate (see Fig. 1A, inset), and ultimately produced a large amount of precipitate and insoluble material. This was possibly due to instability of higher oxidation state complexes in solution and the formation of cell aggregates; note that the images shown in Fig. 1 were taken after the samples were filtered (0.22 μm) to remove insoluble material. Figure 2 shows V oxidation over 2 h in the presence of viable cultures of M. sedula and M. prunae, as well as cell-free and whole-cell extracts of these two archaea. No significant color change was noted between the abiotic control and the M. prunae samples over 2 h. However, for both viable M. sedula and whole-cell extracts of M. sedula, a significant color change was noted, which is indicative of V oxidation to V5+. The fact that no coloration was noted for the cell extracts suggests that cell membranes are potentially a key factor in the oxidation process. For MoO2, solubilization by the abiotic control was minimal compared to in the presence of either M. sedula or M. prunae (see Fig. 1B). Since the initial rate (day 0 versus 1) of V solubilization was greater than Mo, this suggests that the oxide, MoO2, is either more recalcitrant than V2O3 or is oxidized by a different mechanism. Consistent with V2O3, M. sedula-treated MoO2 resulted in greater solubilization compared to M. prunae.
Vanadium and molybdenum solubilization catalyzed by M. sedula and M. prunae. (A) Vanadium; (B) molybdenum; (C) vanadium after day 1; (D) molybdenum after day 2. Symbols: blue diamond, M. sedula (Mse); red box, M. prunae (Mpr); green triangle, no-cell-added control (ctrl). The panel A inset shows the color change associated with vanadium solubilization within the first few hours. All samples were processed through a 0.22-μm filter prior to imaging.
Short-time V oxidation by Metallosphaera species. Coloration changes for incubation of V2O3 in the presence or absence of M. sedula and M. prunae viable cultures, cell extracts, and whole-cell extracts. No color change was noted for M. prunae and abiotic control samples over 2 h, but a yellow color was noted for viable cultures of M. sedula and whole-cell extracts of M. sedula, indicative of a V5+ oxidation state.
To further examine the microbial contribution to V and Mo oxidation, O2 consumption rates in the presence or absence of the metals were determined. Representative traces of O2 consumption for each metal are shown in Fig. 3, along with rates [pmol/(min · cell)] determined from three or more biological replicates. Abiotic consumption of O2 for V and Mo (no cells added) was minimal for each metal (see blue lines in Fig. 3A and B). When viable cell suspensions of either M. sedula or M. prunae were added (point 1), the O2 consumption increased significantly, with similar oxidation rates observed for both species on each metal (Fig. 3). Oxygen consumption rates for V exceeded those for Mo, reflecting the observations made in Fig. 1.
O2 consumption rates for Metallosphaera sedula (Mse) and Metallosphaera prunae (Mpr) growing in the presence of vanadium (A) and molybdenum (B) oxides. The blue trace represents O2 consumption in the absence of cells. The red trace shows M. sedula O2 consumption with ferric chloride. The green trace shows M. prunae O2 consumption with ferric chloride. The purple trace shows M. sedula O2 consumption for without FeCl3 (later added). The cells added (point 1) and FeCl3 added (point 2) points are indicated. Rates represent averages of three or more biological repeats (not shown here). The O2 consumption rates (uninoculated control) for vanadium and molybdenum oxides were subtracted prior to determining the rates on a per-cell basis. The % dissolved oxygen is measured from the point at which oxygen is in equilibrium with air at the time the reaction vessel was sealed.
The effect of iron on the observed metal oxidation rates was examined by first deleting FeCl3 from the medium recipe (DSMZ medium 88) and then spiking in FeCl3 at the nominal medium concentration to the culture after 40 min. Preparing iron-free media is problematic since residual trace amounts of iron usually remain in medium salts and buffers. Nevertheless, O2 consumption rates for both M. sedula and M. prunae were severely impacted when FeCl3 was eliminated from the medium recipe, moreso for V than for Mo (see points 1 to 2 in Fig. 3A and B). O2 consumption rates were essentially recovered for M. sedula and M. prunae upon the addition of FeCl3 (Fig. 3). However, for M. prunae, the O2 consumption rate was not recovered in all experiments (Fig. 3). Previous work showed that sudden additions of uranium to cultures triggered a dormant phase in M. prunae (but not in M. sedula) (20) involving VapC toxin-antitoxin loci (17, 20); a similar phenomenon could be a factor here. These results indicate that chemolithoautotrophic metabolism could be supported by Fe(II) oxidation in these Metallosphaera species and that the use of V and Mo as electron mediators to recycle Fe(III) to Fe(II) through their oxidation bolstered respiratory processes.
Indirect versus direct metal oxidation by Metallosphaera species.While the presence of Fe impacted V and Mo solubilization and stimulated Metallosphaera metabolism (increased O2 consumption rates), it was less clear whether any direct biological oxidation of either metal occurred. Transcriptomic analysis was used to gain further insights into the observed phenomena. The strategy was to “shock” the cells with a particular metal oxide (21) and then monitor the transcriptomic response to identify possible genes/proteins involved. It is possible that V and Mo could elicit a response related to metal toxicity or metal resistance mechanisms that could be generic to M. sedula or metal specific (21). Another possibility was that each metal oxide influenced bioenergetics in a direct way that was not related to iron metabolism.
Cells were grown to late exponential phase on yeast extract (YE), at which time the equivalent of 0.05 wt% metal oxide was added (“shocked”): V2O3, MoO2, or U3O8 (see the experimental loop in Fig. 4C). Within the experimental loop, more than 250 ORFs responded 2-fold or more to metal addition, representing ∼11% of the M. sedula genome. As shown in Fig. 4D and in Table 1, U elicited a minimal response (3 ORFs, 3↑/0↓, where the numbers and arrows indicate 3-fold upregulation and 0-fold downregulation), with only three genes being upregulated. V2O3 (251 ORFs, 112↑/139↓) triggered a larger response than MoO2 (61 total, 43↑/18↓). A significant portion of the V (215 total, 87↑/128↓) and Mo (26 total, 18↑/8↓) oxide responses was unique to the metal oxide. The overlap between V and Mo included a total of 35 ORFs, 25↑/10↓ (see Table 1; see also Table S1 in the supplemental material). The limited response observed for uranium was attributed to concentrations not exceeding ∼0.2 mM, which had been shown previously to elicit a minimal response (21). Furthermore, YE in the media likely prevented U concentrations from exceeding 0.2 mM due to precipitation (17); thus, a separate experiment with no YE was conducted (chemolithoautrophic growth) where uranium concentrations were ∼0.5 mM. At this higher uranium concentration, differential transcription of 39 ORFs, 19↑/20↓ were observed (see Table S2).
Transcriptional response of M. sedula to “oxide shock.” (A and B) Cultures were grown to the late exponential phase, at which time 0.05 wt% metal oxide (MoO2, V2O3, and U3O8) was added. (C) Cells were harvested, and the RNA obtained was used for analysis by a mixed-effect model experimental design. (D) Total ORFs responding ≥2-fold.
Genes up- and downregulated ≥5-fold for Mo (blue) and/or V (green) versus control
The metal oxide response included ORFs previously identified during metal “shock” with metal cations (Co2+, Cu2+, Ni2+, UO22+, or Zn2+) in their highest oxidation states (21). By comparing the transcriptomic data here with the previous results where metal oxidation was not occurring, some additional insights were obtained. These are discussed below.
Metal “shock” transcriptomes.The metal oxide “shock” transcriptomes suggested that M. sedula metabolism had shifted from what had been observed during growth on previously established energy sources [i.e., Fe(II), organic compounds in YE, and H2 (9, 22, 23)]. Among the Sulfolobales that are known metal oxidizers, the two key gene clusters associated with iron oxidation are the fox and soxLN-cbsAB clusters (see Fig. 5) (8–10, 22). Given that FeCl3 affected O2 consumption rates on MoO2 and V2O3, the response of these ORFs was expected but not observed (see Fig. 5). soxLN-cbsAB was strongly upregulated during chalcopyrite bioleaching by M. sedula (23), but no such response was observed here in the presence of any of the metal oxides. Aside from a transcriptional regulator (Msed_0466) and a hypothetical protein (Msed_0481) in the fox cluster, ORFs in this locus were either not responsive or downregulated (Fig. 5, Table 1). Also, several hypothetical proteins that are homologs of bacterial pyrroloquinoline quinone biosynthesis proteins (24) were upregulated on ferrous iron and chalcopyrite (9, 23), but only one of these ORFs (Msed_0515) responded to V and Mo (Table S1). The transcriptomic results indicate that either iron oxidation machinery is constitutively transcribed or that other biomolecules contributed to the biooxidation of soluble molybdenum and vanadium species.
Transcriptional response of M. sedula membrane complexes implicated in metal and reduced inorganic sulfur compound (RISC) biooxidation to vanadium, molybdenum, and uranium “shock.” The color scale relates to least-squares mean (LSM) of ORFs encoding membrane complexes, based on a mixed-effect model of the loop-structured experiment shown in Fig. 3C. LSM values are reported by condition/added metal species: no metal added, uranium octaoxide, molybdenum (IV) oxide, or vanadium (III) oxide.
Though a blue copper protein homolog, rusticyanin, has been implicated in electron transfer for the iron oxidation model of the mesophilic bioleacher, A. ferrooxidans, no response of M. sedula’s four blue copper proteins (two sulfocyanins and two rusticyanins) to iron or reduced inorganic sulfur compounds (RISCs) has been reported (9). Here, downregulation of a sulfocyanin (Msed_0826) and a rusticyanin (rus2, Msed_1206) was observed for MoO2, while upregulation of rus1 (Msed_0966) occurred for V2O3. Analysis of M. sedula’s transcriptome during chalcopyrite bioleaching showed rus1 was upregulated (day 9 versus day 0) (23); transcription of the ORF was associated with additional factors other than soluble iron levels. Thus, the upregulation of rus1 for vanadium was common to chalcopyrite bioleaching and vanadium solubilization, but this is not connected to soluble iron levels or iron cycling.
ORFs associated with the growth of M. sedula on reduced inorganic sulfur compounds were differentially transcribed in the presence of molybdenum and vanadium oxides (Table 1). A tetrathionate hydrolase (tetH; Msed_0804) and a polysulfide/sulfur/dimethyl sulfoxide reductase-like complex (Msed_0810 to Msed_0818) are connected to electron transport for RISCs and are induced by tetrathionate and elemental sulfur (9). With the exception of Msed_0810, Msed_0811, and Msed_0813, the locus was downregulated in response to molybdenum and vanadium oxide. Furthermore, tetH (Msed_0804) was downregulated 10-fold for vanadium oxide. Interestingly, ORFs associated with the soxABCDD′L terminal oxidase complex (Msed_0285 to Msed_0291) were induced for vanadium oxide, suggesting a putative role in vanadium oxidation.
Consistent with a shift in energy metabolism, ORFs within the loci encoding M. sedula’s two NiFe hydrogenases (Msed_0913 to Msed_0950) (23) and soxM supercomplex (22) experienced differential transcription when exposed to metal oxides. In particular, the large subunit of the membrane-bound hydrogenase (Msed_0945) and membrane anchor (Msed_0947) were downregulated for vanadium oxide (Table S1). Though no response of either NiFe hydrogenase was observed for uranium oxide, upregulation of hydrogenase assembly machinery occurred. soxM (Msed_0324), a bb3 type cytochrome (25) known to be involved heterotrophic growth of M. sedula on yeast extract (22), was downregulated in response to vanadium oxide. The observation is consistent with M. sedula shifting energy metabolism in response to vanadium oxide.
Possibility of direct metal oxidation in Metallosphaera species.Based on the results presented here, a key question to be addressed is whether Metallosphaera species directly oxidize redox-active metal oxides. There may be some indication that this is the case. Msed_1191, encoding a Rieske cytochrome b6 fusion protein (Rcbf), was upregulated 5-fold for molybdenum and vanadium oxides, referred to here as V/MoxA (Table 1, Fig. 5 and 6). Previously, rcbf was reported to be induced 4-fold for YE+FeSO4 versus YE (26), and a role of Rcbf was considered for oxidation of Fe(II) by M. yellowstonensis, although no transcript for the gene was detected under any tested condition (10). A close homolog is present only in a few Metallosphaera spp., while a more distant homolog is present in other Sulfolobales and more distant organisms, primarily due to the Rieske, C-terminal domain. In addition, Msed_1190 is annotated as an arsR (trmB-like) transcriptional regulator; these are known to derepress in the presence of metal ions (the “ars” refers to arsenic) (27).
Proposed role of Msed_1191 (V/MoxA) in vanadium and molybdenum oxidation. Based on transcriptional response to metal “shock,” a putative mechanism for the involvement of a previously annotated hypothetical protein in the Metallosphaera genome is depicted.
In addition, biological, and not only abiotic, processes were responsible for the visible color change of the oxide-containing supernatants, color changes commensurate with oxidation to higher ionic states. Based on the discussion above, a model is proposed whereby Rus1 (Msed_0966) initially accepts electrons from solubilized vanadium or molybdenum species and shuttles electrons downhill to the soxABCDD′L terminal oxidase complex for ATP generation or uphill through Rcbf (V/MoxA) to the quinone pool for NADH production. The model is analogous to what has been proposed for iron oxidation in Metallosphaera species (10). The fact that cell membrane proteins are possibly implicated in this process, at least for V oxidation (Fig. 2), further supports the proposed model.
Summary.Both M. sedula and M. prunae contribute to the solubilization of both vanadium and molybdenum oxides by Fe2+/Fe3+ cycling. Oxygen consumption assays showed a direct reduction in the rate of oxygen consumption for iron-limited conditions, which was recovered by the addition of ferric chloride. However, the recovery was not complete for M. prunae and could indicate a VapC toxin-mediated dormant phase, previously observed during uranium “shock” for M. prunae (17, 20). Solubilized molybdenum and vanadium anions could exist in more than one oxidation state, with a higher oxidation state possible, and thus serve as energetic substrates capable of being biooxidized. The transcriptomic profiles for molybdenum, vanadium, and uranium varied markedly from previous work on other energetic substrates (9, 22, 23), suggesting alternative biomolecules being involved in metal oxidation. For vanadium, the increased expression of rus1 and the soxABCDD′L terminal oxidase complex genes suggest that Rus1 initially accepts electrons from solubilized vanadium species and shuttles electrons downhill to the soxABCDD′L terminal oxidase complex for ATP generation. Alternatively, transcriptomic analysis for vanadium and molybdenum oxides implicate Rcbf (V/MoxA) being involved in the uphill transfer of electrons for production of NADH. The results here open up the possibility that both direct and indirect metal oxidation is catalyzed by Metallosphaera species, possibly for bioenergetic benefit or to detoxify their microenvironment.
MATERIALS AND METHODS
Growth of Metallosphaera species.Metallosphaera sedula (DSM 5348T) and Metallosphaera prunae (DSM 10039T) were cultured aerobically in DSMZ medium 88 (pH 2.0), supplemented with 0.2% (wt/vol) YE, and incubated at 70°C in an oil bath agitated at 100 rpm. Chemolithoautotrophic media contained no YE. The metal oxides tested (V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3) were obtained from Sigma-Aldrich. Cell densities were determined by epifluorescence microscopy, using acridine orange stain (28), and the optical densities were measured using a Perkin-Elmer Lambda 25 UV/Vis spectrometer.
Short-time-frame, colorimetric oxidation assay.Cultures of M. sedula and M. prunae were grown in DSMZ medium 88 to mid-exponential phase (about 2 × 108 cells/ml), at which point the cells were harvested by centrifugation at 5,000 × g for 20 min. Cells were washed with fresh DSMZ medium 88 (without the addition of yeast extract) and again centrifuged, decanted, and resuspended. Approximately one-third of the resuspended culture (the whole-cell [WC] fraction) was stored at 70°C until inoculation, and the remainder was French pressed through three passes within the pressure cell at approximately 16,000 lb/in2 while on ice. The lysate was then centrifuged at 20,000 × g for 20 min. The cell-free lysate (CFL) was decanted and brought to volume with DSMZ medium 88 and the whole-cell lysate (WCL) was resuspended in DSMZ medium 88. These samples were preheated to 70°C before inoculation into the petri dishes. Petri dishes with approximately 10 mg, with or without 1 mg of divanadium trioxide (V2O3), were inoculated with 10 ml of M. sedula or M. prunae fractions containing WC, CFL, or WCL preheated to 70°C. Controls containing each fraction without divanadium trioxide were used for color comparison, as was a sample of abiotic (no biological material) containing DSMZ medium 88 with divanadium oxide. Color development was monitored at 70°C for approximately 2 h.
Determination of metal concentrations.Vanadium, copper, iron, manganese, cobalt, tin, molybdenum, chromium, titanium, and rhodium concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS). Samples sent for ICP-MS were centrifuged at 6,000 × g for 10 min, and the supernatant was then sterile filtered. The sterile-filtered supernatant was sent to North Carolina State University’s Department of Soil Science for ICP-MS analysis. A 96-well plate colorimetric assay, employing Arsenazo (III) reagent, was used to determine uranium [U(VI)] concentrations in the culture supernatant (17). The assay mixture consisted of 5 µl of the sample, 195 µl of 0.1 N HCl, and 10 µl of 0.15% (wt/vol) Arsenazo (III) reagent. The absorbance was read at 652 nm in a BioTek plate reader, and the U(VI) concentrations were determined by comparison to a uranyl acetate standard curve, prepared by serial dilutions. All assay samples were completed in triplicate.
Oxygen (O2) consumption measurements.Oxygen consumption assays were completed using an AppliSens low-drift dO2 sensor with a titanium membrane tip. The reactions were completed at 70°C with constant stirring in rubber stopper sealed bottles. The cell densities in the reaction vessel were 2.0 to 5.0 × 107 cells/ml, determined upon completion of the experiment. Cells were centrifuged (5,000 × g) for 10 min at room temperature. Pellets were washed in either DSMZ medium 88 or DSMZ medium 88 made without the addition of ferric chloride. Cells were then resuspended in 0.4 ml of the appropriate medium and added to the reaction vessel. The reaction contained no other energy source, but 50 mg of vanadium oxide or 50 mg of molybdenum oxide in the appropriate medium. For the reactions completed in ferric iron minimal media, 0.4 ml of a freshly prepared 0.056 M concentrated ferric iron, dissolved in sulfuric acid (pH 2.0), was added in order to determine whether oxygen consumption rates could be recovered. Oxygen consumption rates were determined in linear regions. All rates were determined by averaging at least three biological replicates. For molybdenum and vanadium, the background oxygen consumption was subtracted prior to calculating the oxygen consumption on a per-cell basis. The dissolved oxygen at 70°C was determined using Henry’s Law with a partial pressure for oxygen of 0.21 atm, a standard Henry’s constant of 1.0 × 10–3 mol/(liters ⋅ atm), and a temperature correction factor of 1,700K.
Transcriptional response analysis.The first whole-genome transcriptional response experiment was designed to compare the response of M. sedula to the metal oxides V2O3, MoO2, and U3O8, under mixotrophic (metal oxide and YE) conditions, to a control where no metal oxide was added. M. sedula was first grown in 200 ml of DSMZ medium 88 containing 0.04% (wt/vol) YE to an OD600 of ∼0.25 (∼2.0 × 108 cells/ml), at which point 0.05% (wt/vol) of the appropriate metal oxide was added. Cells were harvested 8 h after the metal addition.
The second whole-genome transcriptional response experiment was designed to compare the response of M. sedula, under autotrophic conditions (–YE), to the presence or absence of U3O8. M. sedula was first grown in 200 ml of DSMZ medium 88 containing 0.2% (wt/vol) YE to an OD600 of ∼0.15 (∼1.0 × 108 cells/ml). Cells were then harvested by centrifugation (6,000 × g, 20 min, 4°C) and washed in DSM88 medium. Washed cells were inoculated into 200 ml of DSM88 media (–YE) containing either no U3O8 or 0.1% (wt/vol) U3O8. Cells were harvested when U(VI) concentrations reached 0.5 mM.
For both experiments, cells were harvested by cooling to <10°C using a dry ice–95% ethanol mixture, centrifugation (6,000 × g, 20 min, 4°C), and washing of the resulting pellet with 1.5 ml of Tris-EDTA, buffer, with the final cell pellets stored at −80°C. Total RNA was isolated from cell pellets using TRIzol Reagent (Invitrogen) followed by phenol-chloroform extraction. After extraction, an RNeasy minikit (Qiagen), with on-column DNase treatment, was used to further cleanup the RNA and to remove the residual organic solvent contaminants. RNA quality was determined by separation on a 1% agarose gel. RNA from biological replicates for each condition was pooled and reverse transcribed to cDNA using SuperScript III reverse transcriptase (Invitrogen). cDNA was purified by using a QIAquick kit (Qiagen). The cDNA was labeled with either Cy3 or Cy5 dye (GE Healthcare) and hybridized onto the M. sedula array, constructed as previously described (26). Slides were scanned using a Genetix 4000B scanner, and the intensity data for all of the spots on the array were normalized using an analysis of variance mixed-effects model, as used for previous transcriptomic analysis of Metallosphaera cultures (17, 20). The normalized data were analyzed using JMP Genomics (SAS Institute, Cary, NC) with the criterion for significant differential response defined as a fold change of ≥2.0 and a P value of ≤1.0E–4. The Bonferroni corrections for the multimetal oxidation and autotrophic uranium oxidation experiments were 5.51 and 5.01, respectively.
ACKNOWLEDGMENTS
We acknowledge support from the U.S. Defense Threat Reduction Agency (HDTR-09-0300) and the U.S. Air Force Office of Sponsored Research (FA9550-13-1-0236 and FA9550-17-1-0268). G.H.W. received support from a U.S. Department of Education GAANN Fellowship (P200A070582-09). J.A.C. received support from a National Institutes of Health T32 Biotechnology Traineeship (GM008776-11).
FOOTNOTES
- Received 21 November 2018.
- Accepted 4 December 2018.
- Accepted manuscript posted online 21 December 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02805-18.
- Copyright © 2019 American Society for Microbiology.
