Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph 1 2

Zoë R. Harrold1, Mark L. Skidmore1, Trinity L. Hamilton2, Libby Desch3, Kirina Amada3, Will van 3 Gelder1, Kevin Glover1, Eric E. Roden4,5, and Eric S. Boyd3,5 * 4

5 1Department of Earth Sciences, Montana State University, Bozeman, MT 59717 6 2Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 7 3Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717 8 4Department of Geosciences, University of Wisconsin, Madison, WI 53706 9 5NASA Astrobiology Institute 10 11

12 13

*Correspondence: Eric Boyd (eboyd@montana.edu) 14 15 Keywords: Pyrite, autotroph, cold adapted, thiosulfate, glacier, anoxic, thermodynamics, cell yield 16 17 18 19 20 21 22 23 24 25 26

AEM Accepted Manuscript Posted Online 28 December 2015Appl. Environ. Microbiol. doi:10.1128/AEM.03398-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT (239 words) 27 28

Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important 29 drivers of mineral weathering in oxic and anoxic zones of many aquatic environments including those 30 beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the 31 metastable intermediate thiosulfate (S2O3

2-), which represents an electron donor capable of supporting 32 microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada over a 33 seasonal melt cycle reveal concentrations of S2O3

2- that are typically below detection despite the presence 34 of available pyrite and several orders of magnitude higher concentrations of the FeS2 oxidation product 35 sulfate (SO4

2-). Here we report the physiological and genomic characterization of the 36 chemolithoautotrophic facultative anaerobe Thiobacillus sp. RG5 isolated from the subglacial 37 environment at RG. The RG5 genome encodes pathways for the complete oxidation of S2O3

2-, CO2 38 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicate that the 39 energy required to synthesize a cell under oxygen or nitrate reducing conditions with S2O3

2- as electron 40 donor was lower at 5.1 °C than 14.4 °C, indicating that this organism is cold-adapted. RG sediment-41 associated soxB transcripts, which encode a component of the S2O3

2--oxidizing complex, were closely 42 affiliated to soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial 43 environment at RG mediated in part by populations closely affiliated with RG5. Microbial consumption 44 of S2O3

2- by RG5-like populations may accelerate abiotic FeS2 oxidation thereby enhancing mineral 45 weathering in the subglacial environment. 46 47

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INTRODUCTION 48 49

The communition of bedrock in subglacial systems promotes weathering processes by exposing 50 fresh minerals with high surface area (1-5). Subglacial water chemical profiles (e.g. 5, 6), field- and 51 laboratory-based microcosm experiments (e.g. 7, 8, 9), and molecular analyses (e.g. 6, 10, 11) indicate the 52 presence of an active and diverse subglacial microbiome founded on chemical energy that functions to 53 enhance rates of mineral weathering (8). Given ice covers approximately 10% of the present day 54 continental landmass, this makes the subglacial environment a widespread habitat for microbial life and 55 for mineral weathering. 56

57 Aqueous geochemical data collected from the melt waters of numerous glaciers suggests pyrite 58

(FeS2) oxidation and the concomitant production of hydrogen ions are key drivers of subglacial bedrock 59 weathering (5, 6, 9, 12, 13). It has also been inferred that FeS2 weathering in the subglacial environment 60 may be microbially mediated (3, 6, 9). This inference is supported by DNA-based molecular data that 61 show the presence of a number of taxa in subglacial systems closely related to organisms capable of Fe 62 and S oxidation (6, 7, 9, 11, 14, 15). Moreover, Mitchell et al. (7) showed that microbial communities 63 colonizing FeS2 incubated in situ in a subglacial meltwater stream at Robertson Glacier (RG), Canada 64 were phylogenetically more similar at the level of 16S rRNA genes to those associated with native 65 subglacial sediments and suspended sediments than communities colonizing other iron bearing (i.e. 66 magnetite, hematite and olivine) and carbonate (i.e., calcite) minerals (7). These data suggest a 67 relationship between microbial community structure and bedrock mineralogy and imply that bedrock 68 minerals serve as a source of energy for subglacial microbial communities. Further evidence for the role 69 of FeS2 in supporting subglacial microbial communities comes from the recovery of 16S rRNA transcripts 70 from RG sediments that exhibit close affiliation with known Fe and S oxidizing taxa (11). However, FeS2 71 oxidation pathways in the subglacial system and the role of microbes in these geochemical 72 transformations are poorly understood; especially those FeS2 oxidation processes that occur under 73

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hypoxic or anoxic conditions thought to characterize significant sectors of subglacial drainage networks 74 (16). 75

76 In acidic (pH < 4) environments, abiotic FeS2 oxidation is known to occur through both oxic and 77

anoxic processes (17-20). Anoxic FeS2 oxidation in these systems is achieved through surface oxidation 78 with aqueous Fe3+ (e.g. 18, 20). Geochemical and isotopic measurements of subglacial waters suggest that 79 FeS2 oxidation under anoxic conditions occurs in several subglacial environments (13, 21). However, 80 abiotic, anoxic FeS2 oxidation via aqueous Fe3+ is not possible at the circumneutral to alkaline pH (22, 23) 81 that characterize many subglacial outflow waters due to the rapid precipitation of ferric iron as iron 82 hydroxide (Fe(OH)3), which does not promote FeS2 oxidation (24). Consequently, FeS2 oxidation in 83 circumneutral to alkaline pH systems is thought to be driven primarily by oxidation with O2. Unlike in 84 acidic systems, however, reduced sulfur intermediates released during oxic FeS2 oxidation (Table 1, R1), 85 such as thiosulfate (S2O3

2-), are metastable in O2-rich waters at circumneutral to alkaline pH (20). 86 Metastable S2O3

2- produced in oxic, circumneutral subglacial waters may be oxidized by aerobic 87 microorganisms resulting in the production of SO4

2- (Table 1, R2.2) or be transported to anoxic zones 88 where anaerobic microbial processes may drive its oxidation to SO4

2- (25). Previous work has 89

demonstrated the importance of nitrate as an oxidant capable of supporting microbial metabolism in a 90 variety of subglacial environments (26-28), including RG (29). 91

92 Here we describe the isolation of strain RG5 from RG subglacial sediments using S2O3

2- as an 93 electron donor and NO3

- as an electron acceptor. Strain RG5 is a chemoautotrophic facultative anaerobe. 94 Genomic data indicate it is closely affiliated with Thiobacillus denitrificans. For the remainder of this 95 communication, this strain will be referred to as Thiobacillus sp. RG5 or as strain RG5. We demonstrate 96 Thiobacillus sp. RG5 is 1) capable of catalyzing the oxidation of S2O3

2- with O2, NO2- or NO3

- as electron 97 acceptors at circumneutral pH and 2) adapted to the low temperatures that characterize subglacial habitats 98 based on physiological and energetic data. Consistent with these findings, soxB transcripts, which encode 99

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a component of the S2O32--oxidizing complex, were recovered from RG subglacial sediments. They 100

exhibit close affiliation with soxB encoded in the genome of Thiobacillus sp. RG5. Considering the 101 widespread distribution of thiobacilli in contemporary subglacial environments (e.g. 6, 11, 30-32), these 102 results are suggestive of a key role for autotrophic organisms like Thiobacillus sp. RG5 in driving oxic 103 and anoxic subglacial mineral weathering on a global scale. 104

105 METHODS 106 107

Site Description. Robertson Glacier (RG) is located in Peter Lougheed Provincial Park, Alberta, 108 Canada. The RG catchment bedrock is sedimentary, part of a Late Devonian marine carbonate sequence, 109 with minor clastic input, and is primarily composed of impure carbonates, including limestones and 110 dolostones, with silicic interbeds of siltstone, shale and sandstone (33, 34). Trace amounts of FeS2 (1-2 wt 111 %) form nodules within glacial till and bedrock and provide the only substantive source of reduced sulfur 112 (S) to the subglacial environment (33). Sediments contain mineral abundances similar to those in bedrock, 113 with low (<1-2 wt %) amounts of FeS2 (35). Gypsum and other sulfate bearing minerals are not observed 114 in RG bedrock (33). Bassinite (2CaSO4 • H2O) has been identified as a minor component in a single RG 115 till sample, however, its absence from the bedrock suggests it may be a secondary precipitate (33). One, 116 predominant subglacial outflow stream drains from the western edge of the glacier terminus. The 117 subglacial outflow waters, at temperatures < 1 °C, form an ice cave-like incision beneath the glacier, 118 exposing basal sediments. SO4

2- concentrations in supraglacial waters that only have ice melt as their 119 source have been measured at 1-2 M (Boyd et al., 2014). These input waters to the subglacial system 120 have sulfate concentrations that are one to two orders of magnitude lower than measured in the subglacial 121 outflow stream, 23-571 M, indicating that there is a sulfate source in the subglacial environment (9). 122 123

Sample Collection. Fine-grained basal sediments were collected, using aseptic techniques, on 124 October 10, 2010 from within an ice cave that formed at the terminus of RG where the subglacial melt 125

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stream discharges. The samples were collected for RNA-based analysis and enrichment culturing as 126 previously described (11). Briefly, triplicate samples of sediment submerged beneath the melt stream 127 were collected from a 0.5 m2 area of the sediment surface within the ice cave and these were pooled in 128 order to minimize the influence of spatial heterogeneity on culture- and molecular-based analyses. 129 Sediments were collected from this 0.5 m2 area using a flame sterilized spatula or spoon. Sediment 130 aliquots (~1 g) for RNA-based analysis were collected in sterile 2-mL tubes containing 0.5 mL RNALater 131 (Qiagen, Valencia, CA, USA) and flash-frozen in a dry ice–ethanol slurry. Samples were stored on dry ice 132 during transport to the field station and back to Montana State University where they were stored at -80 133 °C until further processing. Sediments for enrichment and culture isolations (described below) were 134 collected from the same location as those for RNA analyses using a flame sterilized spoon. These 135 sediments were placed in a sterile 500 mL screw cap container and transported and maintained at < 4 °C 136 until used for enrichment and isolation. Water samples were collected from the subglacial stream outlet 137 every 6 hours over diurnal cycles in July (17 & 18), August (5 & 6) and September (26 & 27) 2014 and 138 filtered using a 0.4 µm Nuclepore membrane for analysis of major anion and cation concentrations as 139 previously described (30). 140 141

Enrichment and Isolation. A basal medium containing 5.1 mM NaCl, 10 mM K2HPO4, 5 mM 142 NH4Cl, 0.25 mM MgCl2 • 6H2O, and 6 mM Na2S2O3 was prepared as previously described (36). Thirty 143 mL of medium were dispensed into 70 mL serum bottles and their contents were degassed with N2 passed 144 over H2-reduced and heated (210 °C) copper shavings, sealed with butyl rubber stoppers, and autoclaved 145 at 121 °C for 20 minutes. All media was subsequently amended with anoxic, filter-sterilized solutions to 146 achieve final concentrations of 20 mM NaHCO3, 0.1 % v/v SL 10 trace metal solution (37), and 0.1 % v/v 147 Wolfe’s Vitamin solution. Anoxic medium was then amended with KNO3 as the sole electron acceptor to 148 a final concentration of 5 mM. 149

150

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Inoculum was prepared by mixing five grams of fine-grained basal sediment with 10 mL of 151 sterile, anoxic medium to create a slurry. Three mL of slurry was added to 70 mL serum bottles 152 containing 30 mL of prepared medium. The enrichment was immediately subjected to a series of ten-fold 153 dilutions (6 in total). Following a month of incubation at 5 °C, a 2 mL aliquot of each dilution culture was 154 removed aseptically, filter-sterilized, and assayed for sulfate (SO4

2-), nitrate (NO3-), and nitrite (NO2

-) 155 using ion chromatography (described below). Analyte concentrations in inoculated medium were 156 compared to those in un-inoculated medium to quantify S2O3

- oxidation and NO3- reduction activities. The 157

most dilute culture from the first dilution to extinction series exhibiting an increase in SO42- (proxy for 158

S2O3- oxidation) and a decrease in NO3- concentration relative to un-inoculated controls was used as 159

inoculum for a second additional dilution to extinction sequence following the same procedure as above. 160 This same process was repeated for a third and final dilution to extinction series. The most dilute culture 161 of the third sequence was used for further physiological experimentation as well as DNA extraction, 162 quantification, and genome sequencing, as described below. 163 164

DNA Extraction, Quantification, and Sequencing. Two mL of the most dilute culture from the 165 third dilution to extinction series were concentrated via centrifugation (14,000 x g, 15 min., 5 166 oC).Genomic DNA was extracted, purified, and quantified as previously described (8). Total genomic 167 DNA was sequenced at the Genomics Core Facility at the University of Wisconsin-Madison using the 168 paired end Illumina MiSeq platform (Illumina, San Diego, CA). DNA fragments were prepared according 169 to the manufacturer’s protocol. Quality of the reads was checked with FastQC 170 (bioinformatics.babraham.ac.uk/projects/fastqc). The reads were quality trimmed from both ends using 171 Trimmomatic (38). Reads containing more than 3 N’s were also removed and reads with an average 172 quality score of less than Q20 or a sequence length less than 50 bps were removed. The trimmed paired-173 end reads were assembled using Velvet (39) as previously described (40) specifying a kmer length of 49. 174 Contigs were annotated using the RAST server (41). Raw sequencing reads from this research have been 175

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deposited at DDBJ/EMBL/GenBank under the accession LDUG00000000. The version described in this 176 paper is LDUG01000000. 177

178 RNA Extraction, Quantification, and Sequencing. RNA extraction and initial purification 179

were performed using the FastRNA Pro Soil-Direct kit (MP Biomedical, Solon, OH). RNA was extracted 180 in triplicate from approximately 400 mg of wet weight sediment. After initial purification, RNA was 181 subjected to DNAse I digestion (Roche, Indianapolis, IN) for 1 hr. at room temperature (~ 22 °C). 182 Following digestion, RNA was further purified using a High Pure RNA Isolation Kit (Roche, 183 Indianapolis, IN) and was stored at -80 °C in a solution of 100% ethanol and 0.3 M sodium acetate until 184 further processed. Subglacial sediment dry mass was determined by drying (80 °C, 24 hr.) and weighing 185 the residue after RNA extraction. 186

187 The concentration of RNA was determined using the Qubit RNA Assay kit (Molecular Probes, 188 Eugene, OR) and a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). RNA extracts were screened for 189 the presence of contaminating genomic DNA by performing a PCR using ~ 1 ng of RNA as template and 190 archaeal and bacterial 16S rRNA gene primers, as described previously (11). Equal volumes of each 191 DNA-free RNA extract were pooled and subjected to cDNA synthesis. cDNA was synthesized from 20 192 ng of purified RNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and the following 193 reaction cycling conditions: 5 min at 25 °C, 30 min at 42 °C, and 5 min at 85 °C. Following synthesis of 194 cDNA, samples were purified by ethanol precipitation and re-suspended in nuclease-free water. 195 196 Amplification of soxB transcript fragments from cDNA was performed using primers soxB693F 197 (5'-ATCGGNCARGCNTTYCCNTA-3') and soxB1446R (5'-CATGTCNCCNCCRTGYTG-3') (42). 198 Approximately 10 ng of purified cDNA was subjected to PCR in triplicate using reaction and cycling 199 conditions as described previously (42), with the following exceptions: an initial 10 cycles of PCR was 200 conducted at an annealing temperature of 55 °C followed by 25 cycles of PCR at an annealing 201

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temperature of 47 °C. Equal volumes of each replicate reaction were combined, purified, cloned, and 202 sequenced as described previously (43). 203 204

Phylogenetic Analysis. soxB transcript sequences obtained from RG, the soxB gene sequence 205 from strain RG5, as well as soxB sequences from related strains were compiled and then translated using 206 the ExPASy server (http://web.expasy.org/translate/). SoxB sequences were aligned with the program 207 ClustalW (vers. 1.8.1) (44) specifying default alignment parameters. A Maximum-Likelihood 208 phylogenetic reconstruction was inferred using PhyML version 3.0 (45) specifying the LG substitution 209 matrix and a four category gamma substitution model. The phylogeny was projected from 100 bootstrap 210 replicates using the program FigTree (ver. 1.4.2; http://tree.bio.ed.ac.uk/software/figtree/). SoxB 211 sequences from Thermus thermophilus strains HB8 and HB27 were used to root the tree. 212 213

Characterization of Thiobacillus sp. RG5 Growth. Growth of strain RG5 was characterized 214 under oxic (O2-dependent) and anoxic (NO3

-- and NO2--dependent) conditions at 5.1 and 14.4 °C. Sixty 215

mL cultures were prepared in 120 mL serum bottles as described above (see Enrichment and Isolation). 216 Medium was inoculated to an initial cell concentration of 4×106 ± 3×106 (1σ) cells mL-1. Triplicate biotic 217 and abiotic (un-inoculated) cultures were incubated at 5.1 ± 0.8 °C and 14.4 ± 0.7 °C (1σ). Temperatures 218 below 5°C were not tested to avoid freeze-thaw cycles that could occur due to the observed ± 1 °C 219 fluctuation in incubator temperature. The average pH of the medium for all assays at the start of 220 incubation was 8.3 ± 0.1 (1σ). Samples (1 mL) were removed from the cultures every 24 to 72 hours. 221 Culture bottle headspace was periodically pressurized with filter-sterilized air or N2 gas, for aerobic and 222 anaerobic cultures, respectively, to prevent contamination, and oxidation via the development of a 223 vacuum, as samples were removed over time. This additional headspace pressure also allowed for the 224 removal of 10 mL gas samples from culture bottles without pulling a vacuum. Filter-sterilized, 225 concentrated formaldehyde was added to aliquots for cell counting to achieve a final concentration of 5 % 226 (v/v). Samples were refrigerated at 5 °C until used for analyses. Aliquots (2-100 μL; contingent on 227

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predicted cell concentration) of formaldehyde-fixed samples were vortexed lightly and adjusted to 1 mL 228 with filter sterilized H2O. Cell suspensions were amended with 0.01 % v/v SYTO 9 (ThermoFisher, 229 Waltham, MA) and incubated in the dark for 1 h prior to filtration onto 0.2 μm black polycarbonate filters 230 (Millipore Billerica, MA). Cells were counted using fluorescence microscopy on an Axioskop 2 Plus 231 microscope with a Zeiss 100 x oil immersion objective. Sample aliquots for major anion and cation 232 analysis were filtered immediately through 0.2 μm nylon filters and diluted gravimetrically (i.e. by mass) 233 with 18.2 MΩ cm-1 H2O prior to analysis via ion chromatography. Ten mL gas samples were taken from 234 the headspace of each anaerobic culture following the plateau of growth curves and stored under a 235 positive pressure in 20 or 50 mL vials containing a saturated NaCl solution for subsequent N2O analysis. 236

237 Geochemical Analyses. Concentrations of major anions and cations (i.e. NO3

-, NO2-, SO4

2-, 238 S2O3

2- and NH4+) in field and laboratory samples were determined using a Metrohm Ion Chromatograph 239

(Riverview, FL) equipped with 250 mm Metrosep A Supp 5 and C4 anion and cation columns with 13 240 and 20 μL sample injection volumes, respectively. Total run times were 50 minutes. Linear calibration 241 curves (R2 = 0.99 or better) were fitted to standard data spanning from 1.2, 1.6, 0.8, 0.7 and 4.2 µM, the 242 lower detection limit, to 483.8, 652.1, 312.3, 265.2, and 1108.6 uM, the upper detection limit, for NO3

-, 243 NO2

-, SO42-, S2O3

2- and NH4+, respectively. Corresponding anion and cation concentration detection limits 244

for samples from RG5 cultures were 0.06, 0.08, 0.04, 0.03 and 0.21 mM to 24.19, 32.60, 15.62, 13.26, 245 55.43 mM for NO3

-, NO2-, SO4

2-, S2O32- and NH4

+, respectively, because of 50 x sample dilutions. A 246 lower range of standards was used when analyzing field samples with the lowest standard for S2O3

2- at 247 0.01 ppm. Nitrous oxide (N2O) concentrations were measured in the headspace of anaerobic cultures 248 provided NO3

- as an oxidant on an HP 5890 Series II gas chromatograph outfitted with an electron 249 capture detector (ECD) and a 1 cm3 sample loop. Two analytical columns (both 183 cm x 0.32 cm OD, 250 packed with Chromosorb 102 80/100 mesh and Porapak-Q 80/100 mesh, respectively) were used in series 251 for gas separation. Certified standard mixtures of N2O in N2 (Scott Specialty Gases, Houston, TX) were 252

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used for instrument calibration (either directly or following volumetric dilution into carrier N2). The limit 253 of quantification for N2O was approximately 0.003 ppmv. pH was monitored during the course of the 254 incubation. Despite the strong buffering capacity of the medium (10 mM HCO3

-, 5 mM PO43-), the pH in 255

biotic incubations dropped by 1.1 to 1.4 units in aerobic cultures incubated at 5.1 and 14.4 ᵒC but did not 256 change substantially in anaerobic incubations or abiotic assays (data not shown) 257

258 Geochemical and Thermodynamic Modeling. SO4

2- was chosen for comparative geochemical 259 modeling to enable more accurate detection of reaction progress, because S2O3

2- concentrations approach 260 the analytical detection limit during growth of Thiobacillus sp. RG5 (described below). Best-fit logistic 261 curves were developed for the average SO4

2- and cell concentrations from triplicate assays as a function of 262 time (t) for each temperature and geochemical condition (Eq. 1) 263 Equation 1: = ( ) 264 where is the concentration of SO4

2- or cells at time t, is the predicted maximum concentration of 265 SO4

2- or cells produced, is the maximum growth rate, or slope of the curve at the point of inflection, and 266 is the time at which the sigmoid midpoint occurs. 267

268 The amount of energy released as a function of time for aerobic S2O3

- oxidation was determined 269 based on logistic curve models describing SO4

2- production and assuming stoichiometric conversion of 270 S2O3

2- to SO42- in a ratio of 1:2. The energy resulting from S2O3

- oxidation coupled to NO3- reduction was 271

determined based on a nitrogen (N) mass balance for time points during which NO3- was consumed, 272

assuming the formation of NO2- and then N2. Activities of redox active analytes (α, i.e. S2O3

2-, SO42-, 273

O2(aq), NO3-, NO2

-, N2(aq)) were determined with PHREEQC and the Lawrence Livermore National Lab 274 thermodynamics database (http://wwwbrr.cr.usgs.gov/projects/GWC_coupled/phreeqc/). The standard 275 state Gibbs free energy of each reaction (∆ °) and reaction quotient at each time step ( , ) was 276 calculated from the activities of reactants and products using the CHNOSZ thermodynamics database 277

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(http://www.chnosz.net/) and subcrt function. Gibbs free energy of the reaction at each time point (∆ , ) 278 was calculated according to Eq. 2. 279 Equation 2: ∆ , = ∆ ° + 2.303 , 280 Where R is the gas constant, T is temperature in Kelvin and Q , is the reaction quotient at time t. 281 Stepwise energy release ( , Eq. 3) was calculated based on reaction progress, where ( ) and ( − 1) 282 are the activities of sulfate at time t and t-1, and is the stoichiometric coefficient of sulfate in each 283 respective, metabolic reaction. 284 Equation 3. = ∆ , × ( ) ( ) 285 Energy requirements per cell produced ( ) during exponential growth phase for S2O3

2- oxidation coupled 286 to O2 and NO3

- reduction were calculated based on the average of model derived and average cell 287 concentrations ( ), respectively, according to Eq. 4. 288 Equation 4. = ( ) ( ) 289 290 RESULTS AND DISCUSSION 291 292

Enrichment, Isolation and Genomic Characterization of Thiobacillus sp. RG5. Three rounds 293 of dilution to extinction in cultures incubated at 5.1 °C with thiosulfate and NO3

- resulted in a culture 294 comprising a single morphotype (RG5) that exhibited close affiliation with Thiobacillus denitrificans, 295 based on genomic characterization. The Thiobacillus sp. RG5 draft genome, which consists of 3.29 Mbp, 296 is predicted to be 95% complete based on the presence of conserved phylogenetic marker genes (Table 2; 297 Table S1). This genome size is slightly larger than that of T. denitrificans, which consists of 2.90 Mbp 298 (46). The average GC content of the RG isolate draft genome, 62.7%, is less than the T. denitrificans 299 genome, which has an average GC content of 66.0%. 300

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Thiobacillus denitrificans is a facultative anaerobe and chemolithoautotroph capable of coupling 302 the complete oxidation of inorganic sulfur compounds such as S2O3

2- to the reduction of oxidized nitrogen 303 compounds (e.g. NO3

-, NO2-) or O2 (46). Similar to the T. denitrificans genome (46), the Thiobacillus sp. 304

RG5 draft genome encodes for a number of enzymes necessary for aerobic and anaerobic respiration. The 305 genome also encodes for proteins involved in the oxidation of reduced sulfur compounds including the 306 Sox enzyme complex (SoxB, SoxAX, and SoxYZ), which is a component of a widely distributed pathway 307 for the oxidation of S2O3

- to elemental sulfur (S80) (25). The genome also encodes the reverse 308

dissimilatory sulfite reductase (rDsr) protein complex in a gene cluster (dsrABEFHCMKLJOPNR) that is 309 similar to other lithotrophic sulfur oxidizing organisms including T. denitrificans (46, 47). The rDsr 310 enzyme complex is involved in the oxidation of intracellular S8

0 to SO4

2-. The presence of genes encoding 311 both Sox and rDsr suggests that Thiobacillus sp. RG5 is capable of catalyzing the complete oxidation of 312 S2O3

- to SO42-. Other genes encoding proteins with putative roles in sulfur oxidation are also present in the 313

genome. These include genes that encode a thiosulfate sulfurtransferase (rhodanese), sulfate 314 adenylyltransferase, adenylylsulfate reductase, tetrathionate reductase, dimethyl sulfoxide reductase, and 315 a sulfide:quinone oxidoreductase. 316

317 The Thiobacillus sp. RG5 genome encodes enzymatic machinery for dissimilatory nitrate 318

reduction, including nitrate reductase (NarGHIJ), and nitrite reductase (NirBD), as well as the enzymes 319 necessary for complete denitrification of nitrate to nitrogen which includes nitrate reductase (NarGHIJ), 320 nitrite reductase (NirS), nitric oxide reductase (NorBC), and nitrous oxide reductase (NosZ). Also 321 encoded in the genome is enzymatic machinery necessary for aerobic respiration. Similar to T. 322 denitrificans, the genome encodes an NADH:ubiquinone oxidoreductase, a succinate dehydrogenase, and 323 a cytochrome b/c1 ubiquinol oxidoreductase complex. The genome also encodes aa3- and cbb3-type 324 cytochrome c oxidases and an F-type ATPase. 325

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Characterized members of the Thiobacillus genus are chemoautotrophic, capable of fixing CO2 327 using the Calvin-Benson-Bassham cycle (46). A cluster of genes encoding for homologs of the ribulose 328 bisphosphate carboxylase (cbbRLQO) is encoded in the draft genome of Thiobacillus sp. RG5 in the same 329 orientation as in the genome of T. dentrificans (46), providing a putative mechanism allowing for 330 autotrophic growth in this organism. 331

332 Molecular and Chemical Evidence for S2O3

- Oxidation at RG: A Putative Role for 333 Thiobacillus sp. RG5. SoxB from Thiobacillus sp. RG5 exhibited 90% amino acid sequence identities 334 with Thiobacillus thioparus and 89% sequence identities with T. denitrificans strain 25259. Intriguingly, 335 SoxAYXZ, all of which are located on the same contig in an apparent operon (organized as soxBAZYX) in 336 the partial genome of Thiobacillus sp. RG5, were more closely affiliated with those proteins encoded by 337 T. dentrificans (81-90% sequence identities) than to proteins encoded by T. thioparus (76-86% sequence 338 identities). Thus, although SoxB from Thiobacillus sp RG5 is more closely related to SoxB from T. 339 thioparus, the rest of the proteins forming the Sox complex exhibit closer phylogenetic affiliations with T. 340 denitrificans. 341

342 We extracted RNA from RG sediments and amplified soxB from cDNA in an effort to determine 343

the potential for S2O32- oxidation by Thiobacillus sp. RG5 in the subglacial environment. A total of 31 344

soxB transcripts were sequenced from RG subglacial sediments sampled in October 2010. Phylogenetic 345 reconstruction of translated soxB transcripts revealed 5 distinct sequence clusters (Fig. 1). One of the five 346 clusters was most closely related to T. thioparus (~90 % sequence identities). SoxB from T. denitrificans 347 formed a sister group to this cluster. This cluster also included the SoxB sequence from Thiobacillus sp. 348 RG5, suggesting that the strain isolated in the present study is representative of several Thiobacillus-like 349 populations that may be involved in S2O3

- oxidation in the RG subglacial environment. Despite not being 350 collected on the same dates, evidence for a diverse community of putative S2O3

2- oxidizers in the RG 351 subglacial environment in 2010 is consistent with geochemical data collected in the outflowing stream at 352

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RG over a summer melt season in 2014. Only one sample, 5.00 AM 27th September 2014 contained S2O32- 353

at a concentration of 0.3 µM; all other samples were below detection limit (data not shown). This early 354 morning sample should contain a high proportion of subglacially derived water because supraglacial input 355 from surficial melt would be negligible given that it was late in the melt season, it was dark (i.e. no 356 insolation to drive surface melt), and the air temperature was close to freezing (48). This sample also had 357 a SO4

2- concentration of 332 µM, the second highest measured in 2014 (data not shown). 358 359

The recovery of soxB transcripts affiliated with Thiobacillus in RG subglacial sediments is 360 consistent with previous molecular data indicating the presence of members of the genus in this 361 environment. For example, translated RuBisCO form II (cbbL) transcripts recovered from the same 362 aliquot of subglacial sediments analyzed here included a sequence closely affiliated (92% sequence 363 identities) to CbbL from T. denitrificans (9). CbbL from Thiobacillus sp. RG5 exhibits 92% sequence 364 identities to CbbL from T. denitrificans. This provides further support that the strain isolated in the 365 present study is likely representative of thiobacilli in the subglacial environment at RG. Mitchell et al. (7) 366 also recovered 16S rRNA genes that exhibited close affiliation to Thiobacillus-like (> 94% sequence 367 identities) populations within RG subglacial sediments. Based on 16S rRNA gene sequence similarity, 368 thiobacilli have also been detected in a wide range of subglacial systems including Bench Glacier, Alaska 369 (6), John Evans Glacier, Ellesmere Island (6), and the Kamb (31) and Whillans (30) Ice Streams, West 370 Antarctica. Sattley and Madigan (49) isolated a sulfur-oxidizing Thiobacillus strain, most closely related 371 to the mesophile Thiobacillus thioparus, from the cold (< 2 oC) water column of ice-covered Lake 372 Fryxell, Antarctica. This organism was shown to grow optimally at 18 oC, consistent with it being 373 psychrotolerant (49). Collectively, these data reveal a widespread occurrence of thiobacilli in both polar 374 and alpine subglacial systems and indicate a role for these cold-adapted populations in aerobic and 375 anaerobic oxidation of sulfur compounds in these systems. 376 377

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Influence of Oxidants on Growth of Thiobacillus sp. RG5. We generated growth curves for 378 cultures grown at 5.1 and 14.4 °C with S2O3

- as the sole electron donor and O2, NO3- or NO2

- as electron 379 acceptors (Figs. 2, 3, and 4). The composition of bulk medium was monitored in each culture until S2O3

2- 380 oxidation and SO4

2- production ceased (i.e. concentrations reached an asymptote), indicating that the 381 culture had reached stationary phase. Cell production was observed in cultures of Thiobacillus sp. RG5 382 under all growth conditions except anaerobic growth at 14.4 °C with NO2

- as the sole oxidant. SO42- 383

production in abiotic controls was less than 0.1 mM for all oxidants and temperatures studied, suggesting 384 abiotic S2O3

2- oxidation was minimal or absent over the duration of the incubation. 385 386 Cultures provided S2O3

2- and O2 (Table 1, R2.2) offer the most available energy with an initial 387 ∆ of -871 and -837 kJ mol-1 in cultures incubated at 5.1 and 14.4 °C, respectively. Nearly complete 388 oxidation of S2O3

2- to SO42- (Fig. 2, Table 3) was observed in cultures exposed to atmospheric (~21%) 389

levels of O2. Rates of SO42- production, and by inference S2O3

2- oxidation, were more rapid when coupled 390 to O2 as an electron acceptor, than when coupled to to NO2

- or NO3- in cultures incubated at 5.1 or 14.4 °C 391

(Table S2). It is important to note that cells in anoxic cultures, provided with NO2- or NO3

- as the sole 392 electron acceptor, were visibly smaller than cells in oxic cultures. It is possible that numerous, 393 interconnected factors contribute to more rapid S2O3

2- oxidation in oxic versus anoxic cultures including, 394 but not limited to, cell size, cell concentration, and cell growth efficiency. 395

396 NO3

- and NO2- supported nearly stoichiometric oxidation of S2O3

2- to SO42- (Figs. 3 and 4, Table 397

3). S2O32- oxidation coupled to NO3

- reduction resulted in the release of NO2- into bulk solution. Once the 398

concentration of NO3- dropped below the analytical detection limit (0.04 mM) in the cultivation medium, 399

Thiobacillus sp. RG5 utilized the NO3- reduction product NO2

- to further oxidize S2O32- to SO4

2-. 400 Intermediate production of NO2

- within Thiobacillus sp. RG5 cultures was not, however, stoichiometric 401 with respect to NO3

- (Fig. 3). Claus and Kutzner (50) observed the same phenomenon of aqueous NO2- 402

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accumulation and subsequent reduction during anaerobic growth of a T. denitrificans isolate in batch 403 culture with S2O3

2- oxidation coupled to NO3- reduction at 30 °C. 404

405 After a plateau in SO4

2- production was reached, neither ammonium (NH4+) nor nitrous oxide 406

(N2O) were detected above the initial background concentration (data not shown) in the anaerobic 407 cultures incubated at 5.1 and 14.4 °C and supplied with NO3

- as the sole oxidant (Fig. 3). This suggests 408 NO3

- and NO2- were likely reduced to N2 in these cultures (Table 1, R2.3 and 2.4). These findings are 409

consistent with previous results from other T. denitrificans strains in which neither NH4+ or N2O were 410

detected during NO3--dependent growth (50). The non-stoichiometric production of NO2

- in cultures 411 supplied with NO3

- as the sole oxidant, and the lack of N2O and NH4+ accumulation, suggests a range of 412

possible explanations, including (1) some NO3- may have been completely reduced to N2 without the 413

formation of intermediates; (2) NO3- and NO2

- reduction steps may have occurred simultaneously in 414 addition to the observed, subsequent NO2

- reduction step; (3) an unmeasured, reduced nitrogen 415 intermediate, such as NO, may have been produced and was potentially utilized as an oxidant; (4) a 416 portion of the NO3

- or NO2- may have been assimilated into biomass. 417

418 Incomplete, abiotic FeS2 oxidation (Table 1, R1) can produce metastable reduced S intermediates, 419

such as S2O32-, in aquatic environments with circumneutral pH (20) such as the subglacial environment at 420

RG (e.g. 33). Characterization of strain RG5 (Figs. 2-4) suggests that aerobic S2O32- oxidation and 421

anaerobic S2O32- oxidation coupled to NO3

- and NO2- reduction are plausible metabolic strategies 422

supporting chemoautotrophic life at low-temperatures in the RG subglacial system. Additional work is 423 required, however, to determine if populations such as Thiobacillus sp. RG5 are capable of enhancing the 424 rate or extent of FeS2 oxidation by oxidizing S2O3

2- under such conditions (Table 1, R2.1). 425 426 Effect of Temperature on Growth and Metabolic Efficiency of Thiobacillus sp. RG5. Cell 427

concentration was chosen as a metric for evaluating cold-adaptation since the differences in the efficiency 428

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of cell synthesis and cell concentration integrate the cumulative enzymatic and physiological factors that 429 enhance or inhibit low-temperature growth. There was no visual difference in cell size between cultures 430 containing the same electron donor but incubated at different temperatures. We therefore restrict cell 431 concentration and energy per cell comparisons to cultures with the same electron donor, but different 432 incubation temperatures. This limitation takes into account large differences in cell biomass due to 433 differences in cell size between oxic and anoxic cultures. 434

435 Maximum cell concentrations in aerobic cultures incubated at 5.1°C were higher when compared 436

to 14.4 °C (Fig. 2), suggesting that the coupling of energy generation to cellular synthesis is more 437 efficient at low temperature. Indeed, normalization of best-fit model-derived maximum cell 438 concentrations to the maximum concentrations of oxidized S2O3

2- in aerobic cultures incubated at 5.1 and 439 14.4 °C revealed that 9.9 x 107 ± 1.0 x 107 (1σ) and 3.7 x 107 ± 0.3 x 107 (1σ) cells were produced per 440 mM S2O3

2- oxidized, respectively (Table S3). Moreover, the doubling times for O2-dependent, RG5 441 growth when incubated at 5.1 and 14.4 °C were 140 and 294 h, respectively (Table S3). While these 442 metrics strongly suggest that this organism is cold-adapted, it is possible that the increased solubility of 443 O2(g) and therefore higher activity of O2(aq) at 5.1 °C is partially responsible for these observations. 444

445 We calculated the energy requirements for aerobic S2O3

2--dependent growth (Ec) to account for 446 the influence of solution chemistry and temperature variation on cellular synthesis in cultures of RG5 447 incubated at 5.1 versus 14.4 °C. This thermodynamic assessment determines the Gibbs free energy of the 448 reaction at each sample time point during exponential growth (∆ , ) based on incubation temperature 449 and calculated activities of model-derived reactants and products, including deviations in O2(aq) activity as 450 a function of incubation temperature. The results of this analysis indicate that cellular synthesis of 451 Thiobacillus sp. RG5 by aerobic S2O3

2- oxidation at 5.1 and 14.4 °C requires an Ec of 3.2 ± 0.6 and 7.7 ± 452 2.4 µJ cell-1 (1σ, Table S3), respectively, over the duration of incubation. Comparing Ec values via the t-453

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test shows that the energy requirements per synthesis of a cell at 5.1 and 14.4 °C are significantly 454 different (p < 0.01). These findings indicate that Thiobacillus sp. RG5 is more efficient at coupling energy 455 generation to cell production at 5.1 than 14.4 °C, and is evidence that the organism is cold-adapted. 456

457 A similar pattern in cellular growth, including higher cell concentrations (Fig. 2) and shorter 458

generation times (Table S3) for cultures incubated at 5.1 versus 14.4 °C, was observed in anaerobic 459 cultures provided NO3

- as the sole oxidant. The Ec for synthesis of a cell under NO3--dependent conditions 460

was calculated from data prior to the onset of NO2- consumption and assumed production of NO2

-. The 461 resulting Ec values for synthesis of a cell at 5.1 and 14.4 °C were 2.3 ± 1.7 and 7.6 ± 3.4 µJ cell-1 (1σ, 462 Table S2), respectively. Despite a t-test analysis suggesting the Ec values for NO3

--dependent synthesis of 463 a cell at 5.1 and 14.4 °C are not significantly different (p = 0.07), these data and the temperature-464 dependent differences in cell concentration during NO3

- reduction (Fig. 3) are consistent with cell 465 synthesis data in aerobic cultures and together suggest that Thiobacillus sp. RG5 is cold-adapted. It 466 should be noted that Ec values for the synthesis of a cell under O2 and NO3

- reducing conditions cannot be 467 directly compared, because of the noticeably smaller size of cells when grown with NO3

- when compared 468 to O2, regardless of incubation temperature (data not shown). 469

470 Growth was not detected at 14.4 °C with NO2

- as the sole oxidant. Cultures of the isolate 471 Thiobacillus sp. RG5 provided with NO2

- as an oxidant and incubated at 14.4 °C produced only 0.06 mM 472 of SO4

2- after 1345 h incubation (data not shown) versus approximately 4 mM SO42- production for 473

cultures incubated at 5.1 °C (Fig. 4). As a result, we did not calculate the energetics associated with cell 474 production under the NO2

- reducing growth condition. 475 476 Numerous adaptations can contribute to more energy-efficient cellular synthesis at low-477

temperature. These include enzyme variations that result in increased substrate conversion efficiencies or 478 lower activation energies for reactions at low-temperature (e.g. 51, 52). The same adaptations can, 479

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however, impart a high degree of temperature sensitivity to an organism. Exposing cold-adapted 480 microorganisms to elevated temperatures can hinder growth due to enzyme denaturation, morphological 481 and membrane modifications, and inhibition of RNA synthesis and cell division (51). It is possible that 482 multiple factors, including both adaptations to low temperature and sensitivity at high temperature, are 483 responsible for the enhanced growth rates and more efficient production of cells observed at 5.1 versus 484 14.4 °C in cultures of Thiobacillus sp. RG5. The use of cell concentration, generation time, and the 485 energetics of cellular production as metrics to describe cold-adaptation as opposed to the more widely 486 used approach of normalizing to protein synthesis may provide a more robust and integrated assessment 487 of cold-adaptation by taking into account growth efficiency at the level of the entire cell. 488

489 CONCLUSIONS 490

491 Thiobacillus sp. RG5, isolated from RG subglacial sediments and closely related to T. 492

denitrificans, is a relevant member of the subglacial community based on soxB transcript analysis. An 493 evaluation of its genome reveals two enzyme complexes associated with S2O3

2- oxidation to S80 (sox) and 494

ultimately SO42- (rDsr). Additional genes were identified that encode enzyme complexes capable of 495

complete dissimilatory denitrification from NO3- to N2 and the Calvin-Benson-Bassham cycle, which 496

provides a putative mechanism for NO3--dependent autotrophic growth. 497

498 Microbially-mediated anaerobic S2O3

2- oxidation has never been demonstrated, to our knowledge, 499 under conditions relevant to subglacial environments such as RG, which has a temperature of < 1 °C (10). 500 The suite of putative enzymes encoded in the Thiobacillus sp. RG5 genomes is consistent with the 501 activity and cellular production data presented here which indicates that Thiobacillus sp. RG5 is capable 502 of aerobic (O2) and anaerobic (NO3

- and NO2-) S2O3

- oxidation at low temperatures (5.1 °C) and solution 503 pH conditions relevant to the RG subglacial system. Shorter generation times and more energy efficient 504

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cellular production in cultures grown under O2- or NO3--dependent conditions at 5.1 °C when compared 505

to 14.4 °C indicate that this Thiobacillus isolate is cold-adapted. 506 507 The recovery of soxB transcripts from RG subglacial sediments closely affiliated with strain RG5 508

and the physiological characterization of the isolate reported here, coupled with previously reported cbbL 509 transcript data from RG that also exhibit close affiliation with RG5 (9), suggest that aerobic and anaerobic 510 S2O3

2- oxidation by the autotrophic Thiobacillus sp. RG5 likely contributes to primary production within 511 the subglacial system at RG. Given that the most probable source of SO4

2- in this system is FeS2 (7, 35) 512 these results indicate a role for Thiobacillus sp. RG5 in catalyzing the oxidation of S2O3

2- in situ thereby 513 maintaining its low concentration (< 0.3 µM) in RG meltwaters. The widespread distribution of 514 Thiobacillus-like populations in subglacial environments beneath both valley glaciers (6) and continental 515 ice sheets, where sediments have been isolated from surficial input for tens of thousands of years (30, 31), 516 are suggestive of a key role for autotrophic organisms such as Thiobacillus sp. RG5 in driving mineral 517 weathering in subglacial systems both today and in Earth’s past. The activity of chemolithoautotrophs, 518 such as Thiobacillus sp. RG5, would contribute to the local microbial food web by generating 519 chemosynthate and biomass (9). This biomass would then be available to support numerous heterotrophic 520 taxa, including bacterivorous eukaryotes, that have been previously detected in subglacial environments 521 (11). 522 523 ACKNOWLEDGEMENTS 524 525

This work was supported by NASA grants NNX10AT31G (MLS and ESB) and NNA15BB02A 526 (ESB). T.L.H. acknowledges support for this work from the NASA Postdoctoral Program. We thank the 527 Biogeosciences Institute at University of Calgary’s Kananaskis Field Station for the use of field and 528 laboratory facilities. We thank Chris Allen for assistance with IC analyses and Dr. John Dore for 529 assistance with N2O measurements. 530

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REFERENCES 531 1. Sharp, M., J. Parkes, B. Cragg, I. J. Fairchild, H. Lamb, and M. Tranter. 1999. 532

Widespread bacterial populations at glacier beds and their relationship to rock weathering 533 and carbon cycling. Geology 27:107-110. 534

2. Hodson, A., A. M. Anesio, M. Tranter, A. Fountain, M. Osborn, J. Priscu, J. 535 Laybourn-Parry, and B. Sattler. 2008. Glacial ecosystems. Ecological Monographs 536 78:41-67. 537

3. Wadham, J., M. Tranter, M. Skidmore, A. Hodson, J. Priscu, W. Lyons, M. Sharp, 538 P. Wynn, and M. Jackson. 2010. Biogeochemical weathering under ice: Size matters. 539 Global Biogeochemical Cycles 24:GB3025. 540

4. Statham, P. J., M. Skidmore, and M. Tranter. 2008. Inputs of glacially derived 541 dissolved and colloidal iron to the coastal ocean and implications for primary 542 productivity. Global Biogeochemical Cycles 22:GB3013. 543

5. Tranter, M., M. Sharp, H. Lamb, G. Brown, B. Hubbard, and I. Willis. 2002. 544 Geochemical weathering at the bed of Haut Glacier d'Arolla, Switzerland‚ a new model. 545 Hydrological Processes 16:959-993. 546

6. Skidmore, M., S. P. Anderson, M. Sharp, J. Foght, and B. D. Lanoil. 2005. 547 Comparison of microbial community compositions of two subglacial environments 548 reveals a possible role for microbes in chemical weathering processes. Applied and 549 Environmental Microbiology 71:6986-6997. 550

7. Mitchell, A. C., M. J. Lafreniere, M. L. Skidmore, and E. S. Boyd. 2013. Influence of 551 bedrock mineral composition on microbial diversity in a subglacial environment. 552 Geology 41:855-858. 553

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

8. Montross, S. N., M. Skidmore, M. Tranter, A.-L. Kivimäki, and R. J. Parkes. 2013. 554 A microbial driver of chemical weathering in glaciated systems. Geology 41:215-218. 555

9. Boyd, E. S., T. L. Hamilton, J. R. Havig, M. L. Skidmore, and E. L. Shock. 2014. 556 Chemolithotrophic primary production in a subglacial ecosystem. Applied and 557 Environmental Microbiology 80:6146-6153. 558

10. Boyd, E. S., M. Skidmore, A. C. Mitchell, C. Bakermans, and J. W. Peters. 2010. 559 Methanogenesis in subglacial sediments. Environmental Microbiology Reports 2:685-560 692. 561

11. Hamilton, T. L., J. W. Peters, M. L. Skidmore, and E. S. Boyd. 2013. Molecular 562 evidence for an active endogenous microbiome beneath glacial ice. The ISME journal 563 7:1402–1412. 564

12. Tranter, M., and R. Raiswell. 1991. The composition of the englacial and subglacial 565 component in bulk meltwaters draining the Gornergletscher, Switzerland. Journal of 566 Glaciology 37:59-66. 567

13. Tranter, M., P. Huybrechts, G. Munhoven, M. J. Sharp, G. H. Brown, I. W. Jones, 568 A. J. Hodson, R. Hodgkins, and J. L. Wadham. 2002. Direct effect of ice sheets on 569 terrestrial bicarbonate, sulphate and base cation fluxes during the last glacial cycle: 570 minimal impact on atmospheric CO2 concentrations. Chemical Geology 190:33-44. 571

14. Skidmore, M. 2011. Microbial communities in Antarctic subglacial aquatic 572 environments. Geophysical Monograph Series 192:61-81. 573

15. Christner, B. C., M. L. Skidmore, J. C. Priscu, M. Tranter, and C. M. Foreman. 574 2008. Bacteria in subglacial environments, p. 51-71, Psychrophiles: from biodiversity to 575 biotechnology. Springer. 576

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

16. Tranter, M., M. Skidmore, and J. Wadham. 2005. Hydrological controls on microbial 577 communities in subglacial environments. Hydrological Processes 19:995-998. 578

17. Edwards, K. J., B. M. Goebel, T. M. Rodgers, M. O. Schrenk, T. M. Gihring, M. M. 579 Cardona, M. M. Mcguire, R. J. Hamers, N. R. Pace, and J. F. Banfield. 1999. 580 Geomicrobiology of pyrite (FeS2) dissolution: Case study at Iron Mountain, California. 581 Geomicrobiology Journal 16:155-179. 582

18. Moses, C. O., D. Kirk Nordstrom, J. S. Herman, and A. L. Mills. 1987. Aqueous 583 pyrite oxidation by dissolved oxygen and by ferric iron. Geochimica et Cosmochimica 584 Acta 51:1561-1571. 585

19. Rimstidt, J. D., and D. J. Vaughan. 2003. Pyrite oxidation: a state-of-the-art assessment 586 of the reaction mechanism. Geochimica et Cosmochimica Acta 67:873-880. 587

20. Chandra, A. P., and A. R. Gerson. 2010. The mechanisms of pyrite oxidation and 588 leaching: A fundamental perspective. Surface Science Reports 65:293-315. 589

21. Bottrell, S. H., and M. Tranter. 2002. Sulphide oxidation under partially anoxic 590 conditions at the bed of the Haut Glacier d'Arolla, Switzerland. Hydrological Processes 591 16:2363-2368. 592

22. Moses, C. O., and J. S. Herman. 1991. Pyrite oxidation at circumneutral pH. 593 Geochimica et Cosmochimica Acta 55:471-482. 594

23. Jorgensen, C., O. S. Jacobsen, B. Elberling, and J. Aamand. 2009. Microbial 595 oxidation of pyrite coupled to nitrate reduction in anoxic groundwater sediment. 596 Environmental Science & Technology 43:4851-4857. 597

24. Schippers, A., and B. B. Jorgensen. 2002. Biogeochemistry of pyrite and iron sulfide 598 oxidation in marine sediments. Geochimica et Cosmochimica Acta 66:85-92. 599

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

25. Friedrich, C. G., D. Rother, F. Bardischewsky, A. Quentmeier, and J. R. Fischer. 600 2001. Oxidation of reduced inorganic sulfur compounds by Bacteria: Emergence of a 601 common mechanism? Applied and Environmental Microbiology 67:2873-2882. 602

26. Skidmore, M. L., J. M. Foght, and M. J. Sharp. 2000. Microbial life beneath a high 603 arctic glacier. Applied and Environmental Microbiology 66:3214–3220. 604

27. Wynn, P. M., A. J. Hodson, T. H. E. Heaton, and S. R. Chenery. 2007. Nitrate 605 production beneath a High Arctic glacier, Svalbard. Chemical Geology 244 88-102. 606

28. Hodson, A., T. J. Roberts, A.-C. Engvall, K. Holmén, and P. Mumford. 2010. Glacier 607 ecosystem response to episodic nitrogen enrichment in Svalbard, European High Arctic. 608 Biogeochemistry 98:171–184. 609

29. Boyd, E. S., R. K. Lange, A. C. Mitchell, J. R. Havig, T. L. Hamilton, M. J. 610 Lafrenière, E. L. Shock, J. W. Peters, and M. Skidmore. 2011. Diversity, abundance, 611 and potential activity of nitrifying and nitrate-reducing microbial assemblages in a 612 subglacial ecosystem. Applied and Environmental Microbiology 77:4778-4787. 613

30. Christner, B. C., J. C. Priscu, A. M. Achberger, C. Barbante, S. P. Carter, K. 614 Christianson, A. B. Michaud, J. A. Mikucki, A. C. Mitchell, M. L. Skidmore, T. J. 615 Vick-Majors, and a. t. W. S. Team. 2014. A microbial ecosystem beneath the West 616 Antarctic ice sheet. Nature 512:310-313. 617

31. Lanoil, B., M. Skidmore, J. C. Priscu, S. Han, W. Foo, S. W. Vogel, S. Tulaczyk, and 618 H. Engelhardt. 2009. Bacteria beneath the West Antarctic ice sheet. Environmental 619 Microbiology 11:609-615. 620

32. Skidmore, M., M. Tranter, S. Tulaczyk, and B. Lanoil. 2010. Hydrochemistry of ice 621 stream beds -evaporitic or microbial effects? Hydrological Processes 24:517-523. 622

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

33. Sharp, M., R. A. Creaser, and M. Skidmore. 2002. Strontium isotope composition of 623 runoff from a glaciated carbonate terrain. Geochimica et Cosmochimica Acta 66:595-624 614. 625

34. McMechan, M. 1988. Geology of Peter Lougheed Provincial Park, Rocky Mountain 626 Front Ranges, Alberta. Geological Survey of Canada. 627

35. Griggs, R. 2013. Characterization of subglacial till from Robertson Glacier, Alberta, 628 Canada: implications for biogeochemical weathering. MS thesis, Department of Earth 629 Sciences, Montana State University:115 pp. 630

36. Sorokin, D. Y., T. P. Tourova, G. Braker, and G. Muyzer. 2007. Thiohalomonas 631 denitrificans gen. nov., sp. nov. and Thiohalomonas nitratireducens sp. nov., novel 632 obligately chemolithoautotrophic, moderately halophilic, thiodenitrifying 633 Gammaproteobacteria from hypersaline habitats. International Journal of Systematic and 634 Evolutionary Microbiology 57:1582-1589. 635

37. Widdel, F., G. Kohring, and F. Mayer. 1983. Studies on the dissimilatory sulfate that 636 decomposes fatty acids. III. Characterization of the filamentous gliding Desulfonema 637 limicola gen. nov., sp. nov., and Desulfonema magnum sp. nov. Arch Microbiol 134:286-638 294. 639

38. Lohse, M., Bolger, A., Nagel, A., Fernie, A. R., Lunn, J. E., Stitt, M. and Usadel, B. . 640 2012. RobiNA: a user-friendly, integrated software solution for RNA-Seq-based 641 transcriptomics. Nucleic Acids Research 40:W622-W6271. 642

39. Zerbino, D. R., and E. Birney. 2008. Velvet: algorithms for de novo short read 643 assembly using de Bruijn graphs. Genome Research 18:821-829. 644

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

40. Hug, L. A., Castelle, C. J., Wrighton, K. C., Thomas, B. C., Sharon, I., Frischkorn, 645 K. R., Williams, K. H., Tringe, S. G. and Banfield, J. F. 2013. Community genomic 646 analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and 647 indicate roles in sediment carbon cycling. Microbiome 1:1-17. 648

41. Aziz, R. K., Bartels, D., Best, A. A., DeJongh, M., Disz, T., Edwards, R. A., 649 Formsma, K., Gerdes, S., Glass, E. M. and Kubal, M. 2008. The RAST Server: rapid 650 annotations using subsystems technology. BMC Genomics 9:75. 651

42. Petri, R., L. Podgorsek, and J. F. Imhoff. 2001. Phylogeny and distribution of the soxB 652 gene among thiosulfate-oxidizing bacteria. FEMS Microbiology Letters 197:171-178. 653

43. Boyd, E. S., R. A. Jackson, G. Encarnacion, J. A. Zahn, T. Beard, W. D. Leavitt, Y. 654 Pi, C. L. Zhang, A. Pearson, and G. G. Geesey. 2007. Isolation, characterization, and 655 ecology of sulfur-respiring Crenarchaea inhabiting acid-sulfate-chloride-containing 656 geothermal springs in Yellowstone National Park. Applied and Environmental 657 Microbiology 73:6669-6677. 658

44. Larkin, M. A., G. Blackshields, N. Brown, R. Chenna, P. A. McGettigan, H. 659 McWilliam, F. Valentin, I. M. Wallace, A. Wilm, and R. Lopez. 2007. Clustal W and 660 Clustal X version 2.0. Bioinformatics 23:2947-2948. 661

45. Guindon, S., and O. Gascuel. 2003. A simple, fast, and accurate algorithm to estimate 662 large phylogenies by maximum likelihood. Systematic Biology 52:696-704. 663

46. Beller, H. R., P. S. G. Chain, T. E. Letain, A. Chakicherla, F. W. Larimer, P. M. 664 Richardson, M. A. Coleman, A. P. Wood, and D. P. Kelly. 2006. The genome 665 sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium 666 Thiobacillus denitrificans. Journal of Bacteriology 188:1473-1488. 667

on April 11, 2020 by guest

http://aem.asm

.org/D

ownloaded from

47. Dahl, C., S. Engels, A. S. Pott-Sperling, A. Schulte, J. Sander, Y. Lübbe, O. Deuster, 668 and D. C. Brune. 2005. Novel genes of the dsr gene cluster and evidence for close 669 interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium 670 Allochromatium vinosum. Journal of Bacteriology 187:1392-1404. 671

48. Gurnell, A. M., and M. J. Clark. 1987. Glacio-fluvial sediment transfer: an alpine 672 perspective. Wiley, Chichester. 673

49. Sattley, W. M., and M. T. Madigan. 2006. Isolation, Characterization, and Ecology of 674 Cold-Active, Chemolithotrophic, Sulfur-Oxidizing Bacteria from Perennially Ice-675 Covered Lake Fryxell, Antarctica. Applied and Environmental Microbiology 72:5562-676 5568. 677

50. Claus, G., and H. J. Kutzner. 1985. Autotrophic denitrification by Thiobacillus 678 denitrificans in a packed bed reactor. Applied Microbiology and Biotechnology 22:289-679 296. 680

51. Margesin, R., and F. Schinner. 1994. Properties of cold-adapted microorganisms and 681 their potential role in biotechnology. Journal of Biotechnology 33:1-14. 682

52. Gerday, C., M. Aittaleb, M. Bentahir, J.-P. Chessa, P. Claverie, T. Collins, S. 683 D'Amico, J. Dumont, G. Garsoux, D. Georlette, A. Hoyoux, T. Lonhienne, M.-A. 684 Meuwis, and G. Feller. 2000. Cold-adapted enzymes: from fundamentals to 685 biotechnology. Trends in Biotechnology 18:103-107. 686

687 688

689 690

on April 11, 2020 by guest

http://aem.asm

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691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713

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FIGURE LEGENDS 714 715 FIGURE 1. Maximum-likelihood phylogenetic reconstruction of translated SoxB protein sequences 716 recovered from RG sediment cDNA, the genome of Thiobacillus sp. RG5, and from reference sequences 717 obtained from the GenBank database. Numbers in parentheses indicate the number of times a given OTU 718 (defined at 3% protein sequence identities) was detected in RG sediment cDNA. 719 720 FIGURE 2. Concentrations of SO4

2- () and S2O32- () and cells () during the growth of Thiobacillus 721

sp. RG5 when incubated at 5.1 °C (A) or 14.4 °C (B) with S2O32- provided as the electron donor and O2 722

provided as the electron acceptor. Values represent the average of triplicate biotic assays. Error bars are 723 1σ. Best-fit logistic models describe average SO4

2- (– –) and cell count (⋅⋅⋅) data. 724 725 FIGURE 3. Concentration of SO4

2- (), S2O32- (), NO3

- (), NO2- (), and cells() during growth of 726

Thiobacillus sp. RG5, incubated at 5.1 °C (A and B) and 14.4 °C (C and D) with S2O32- provided as the 727

sole electron donor and NO3- provided as the sole electron acceptor. Values represent the average of 728

triplicate biotic assays with error bars showing 1σ. NO3- reduction results in the intermediate production 729

of aqueous NO2-, which is subsequently utilized as an electron acceptor (B and D). Dashed line (– –) 730

shows best-fit logistic models describing SO42- production. 731

732 FIGURE 4. Concentration of SO4

2- (), S2O32- (), NO2

- (), and cells () during growth of 733 Thiobacillus sp. RG5 when incubated at 5.1 °C with S2O3

2- provided as the electron donor and NO2- 734

provided as the electron acceptor. Values represent the average of triplicate biotic assays. Error bars are 735 1σ. Dashed line (– –) and (⋅⋅⋅) shows best-fit logistic model describing SO4

2- production and cell formation 736 data, respectively. 737 738 739

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Table 1: Proposed mechanisms of FeS2 and S2O3

2- oxidation in oxic and anoxic environments at circumneutral to alkaline pH, as observed in meltwaters emanating from RG. Reaction

(R1) + . + → ,ℎ , → + +

(R2.1)

(R2.2)

(R2.3)a

(R2.4)a

+ + +

+ + +

+ + + +

+ + + + a Potential reduced, nitrogenous intermediates not shown.

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Table 2. Statistics associated with the assembly of the Thiobacillus sp. RG5 draft genome.

Genome features

Total base pairs (bp) 3293652

Contigs 86

N50 (bp) 146048

%GC 62.71

Longest (bp) 312422

tRNA 44

Protein coding genes 3259

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Table 3: Net production and consumption of oxidized and reduced chemical species during growth of Thiobacillus sp. RG5 on S2O3

2- with O2 or NO3- at 5 and 15 °C. The stoichiometry of the production or

consumption of a given chemical species is based on measured values and was normalized to net S2O32-

consumption. The data presented represent the difference in the average of triplicate measurements taken on triplicate biological controls.

Reaction Stoichiometry

Oxidant Δ[NO2-]

(mM) Δ[NO3

-] (mM)

Δ[SO42-]

(mM) Δ[S2O3

2-] (mM) S2O3

2- oxidant SO42-

5 oC O2 -- -- 10.9 -6.0 1.0 -- 1.8

NO3- -- -4.0 7.0 -3.0 1.0 1.3 2.3

NO2- -4.5 -- 4.2 -1.8 1.0 2.5 2.4

15 oC

O2 -- -- 13.5 -6.5 1.0 -- 2.1 NO3

- -- -4.6 7.0 -3.9 1.0 1.2 1.8 NO2

- n.a. -- n.a. n.a. n.a. n.a. n.a

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