Regulatory responses of Methanosarcina barkeri to freezing temperatures and perchlorates: Transcriptomic insights into the potential for biological Martian methanogenesis

Observations of trace methane (CH4) in the Martian atmosphere are signicant to the astrobiology community given the overwhelming contribution of biological methanogenesis to atmospheric CH4 on Earth. Previous studies have shown that methanogenic Archaea can generate CH4 when incubated with perchlorates, highly oxidizing chaotropic salts which have been found across the Martian surface. However, the regulatory mechanisms behind this remain completely unexplored. In this study we performed comparative transcriptomics on the methanogen Methanosarcina barkeri, which was incubated at 30˚C and 0˚C with 10 mM calcium-, magnesium-, or sodium perchlorate. Consistent with prior studies, we observed decreased CH4 production and apparent perchlorate reduction, with the latter process proceeding by heretofore essentially unknown mechanisms. Transcriptomic responses of M. barkeri to perchlorates include up-regulation of osmoprotectant transporters and selection against redox-sensitive amino acids. Regulatory switches to methylamines for methanogenesis suggest competition for H2 with perchlorate reduction, which we propose is catalyzed by up-regulated molybdenum-containing enzymes and maintained by siphoning diffused H2 from energy-conserving hydrogenases. Methanogenesis regulatory patterns suggest Mars’ freezing temperatures alone pose greater constraints to CH4 production than perchlorates. These ndings increase our understanding of potential methanogen survival beyond Earth and a biological contribution to Martian CH4. Annotation and comparative transcriptomics. Quality ltering of single-end reads was performed using fastp v.0.12.6 95 , removing reads <50 nt, containing >1 Ns, Phred quality scores < 30, and sample barcode sequences. Using Bowtie2 v.2.3.2 96 , retained, quality-ltered reads from each experiment were mapped to coding sequence (CDS) regions subset from the complete M. barkeri MS reference genome obtained from NCBI GenBank (NZ_CP009528.1). CDS-mapped reads were then sorted, indexed, and processed for extraction from the sequenced transcriptome using Samtools v.1.5 97 and BEDTools v.2.17.0 98 . Gene annotation was performed using NCBI BLASTn v.2.2.29+ 99 against a reference M. barkeri MS CDS assembly database generated using option -makeblastdb. Protein assignment was determined as the entry with the greatest sequence identity alignment with the query sequence, the lowest E-value, and largest bit score. Differential expression analysis of investigated treatments relative to the 30˚C and 0˚C perchlorate-free controls, within-group (i.e. biological replicate) variance estimation, and FPM were performed using DESeq2 100 . Metabolic pathway involvement of identied genes was determined by referencing the Kyoto Encyclopedia of Genes and Genomes (KEGG) 101 .


Introduction
The story of Martian atmospheric methane (CH 4 ) remains enigmatic and under intense debate. In the past 15 years, a growing body of evidence has unfolded to suggest episodic appearances (and disappearances) of ppbv-level CH 4 1-7 . Myriad abiotic mechanisms have been suggested as potential CH 4 sources, including cometary impacts 8 , UV degradation of meteoritic and interplanetary dust particle organics [9][10][11] , and Fischer-Tropsch-type synthesis in the subsurface coupled to either serpentinization of ultrama c silicates 12,13 or radiolysis of H 2 O 14 , which subsequently releases CH 4 to the surface through seeps and salinity-induced hydrate dissociation 15 (Fig. 1). On Earth, however, nearly 70% of CH 4 is of biological origin, generated by methanogenic Archaea 16 . This has led to an extensive debate considering the biological origin of Martian CH 4 . Understandably, the quest to comprehend the nature of CH 4 cycling on Mars is a fervent one, as it may be the most conspicuous biosignature detected on Mars to date. In the absence of returned Martian samples or stable isotopic data of Martian atmospheric CH 4 , presently the best approach to constrain this debate is to experimentally test the ability of methanogenic Archaea to metabolize under Martian conditions.
Perchlorates are of great interest to Martian habitability studies for their hygroscopicity and low eutectic temperatures, allowing for the formation of stable liquid water brines at temperatures as low as -74.6˚C and 55% relative humidity [42][43][44][45][46][47] . Previous work has reported decreased CH 4 production in methanogenic cultures supplemented with increasing concentrations of perchlorate salts 25,26 . This suggests that this aspect of the Martian environment may be debilitating to biological methanogenesis, but the mechanisms resulting in this apparent inhibition have not been identi ed. Here we utilize transcriptomics to evaluate regulatory responses of the methanogenic archaeon Methanosarcina barkeri strain MS during a 28-day exposure to high concentrations of sodium-, magnesium-, and calcium perchlorate salts at 30˚C and 0˚C to assess if perchlorate salts are inhibitory to methanogenesis -and if so, how -in order to appraise the potential for methanogens' survival on Mars.

Results
Transcriptome assembly statistics M. barkeri possesses the second largest described genome amongst the methanogenic Archaea 48 . This genome comprises a 4.53 megabase (Mb) circular chromosome and a 40 kilobase (kb) plasmid, which collectively encode 3,760 genes, 3,470 of which are protein-encoding coding sequence (CDS) regions (3.17 Mb). RNA sequencing yielded a total of 1,500,716,043 quality paired end reads across 24 libraries (8 conditions × 3 replicates/condition) with a mean Phred (sequence quality) score of 36. On average 1.6 ± 0.7% of 30˚C and 1.5 ± 0.5% of 0˚C quality-ltered reads mapped back to CDS regions (n = 12 libraries/temperature condition; Table S1), consistent with expectations that mRNA typically comprises 1-5% of total RNA in prokaryotic cells 49 . Average fragment counts per million mapped reads (FPM) are organized by gene position in Table S2 and visualized in Figs. S1-S2.

Methanogenesis and associated regulatory responses
At 30˚C, the addition of Ca(ClO 4 ) 2 , Mg(ClO 4 ) 2 , and Na(ClO 4 ) reduced total net CH 4 production by 48%, 32%, and 24%, respectively, relative to the perchlorate-free control (Fig. 2). Signi cant reduction in CH 4 production rates were observed across all conditions at 0˚C with respect to their 30˚C counterparts, and each perchlorate condition at 0˚C showed a statistically signi cant decrease in CH 4 for at least one time point relative to the 0˚C control (Fig. 2). No CH 4 production was observed in the media blank controls (data not shown). Cultures were monitored via optical density measurements at 600 nm (OD 600 ), but perchlorate-amended media experienced precipitate formation which made obtaining reliable growth data di cult (Fig. S3).
When incubated at 0˚C, the perchlorate-free control demonstrated signi cant up-regulation of several genes in the hydrogenotrophic pathway relative to the 30˚C perchlorate-free control (  Fig. 3). In the perchlorate-treated incubations at 0˚C, we only observed down-regulation of frhabg in the presence of Ca(ClO 4 ) 2 with respect to the 0˚C perchlorate-free control ( Table 2, Fig. 3).
At 30˚C, methanogenesis-associated regulatory responses were shared across all perchlorate conditions (Table 1). This included up-regulation of B, C, and D subunits of (Mo)-formylmethanofuran dehydrogenase, fmd, which catalyzes the CO 2 reduction step of the hydrogenotrophic pathway (Fig. 3 Table 2).
Energy conserving hydrogenase subunit F, echF, which supplies reduced ferredoxin to fmd, was downregulated in the presence of Mg(ClO 4 ) 2 and Na(ClO 4 ) at 30˚C, whereas most subunits of ech were signi cantly up-regulated in 30˚C Ca(ClO 4 ) 2 conditions (Fig. 3). Among other hydrogenases demonstrating signi cant up-regulation was methanophenazine-dependent hydrogenase, vht, speci cally, the large subunit vhtA and cytochrome b subunit vhtC ( Fig. 3). At 0˚C, ech hydrogenases were not differentially regulated in the presence of perchlorates relative to the perchlorate-free control (Fig. 3).
Despite H 2 being the only reducing equivalent provided for the production of CH 4 in our incubations, the addition of Ca(ClO 4 ) 2 , Mg(ClO 4 ) 2 , and Na(ClO 4 ) resulted in signi cant up-regulation of all genes in the mono-, di-, and trimethylamine pathways and associated membrane permeases in the 30˚C perchlorate treatments (Fig. 3, Tables S6, S8, S10).
The terminal step encoding methyl-coenzyme M (CH 3 -CoM) reductase subunit alpha (mcrA) was downregulated at 30˚C in Mg(ClO 4 ) 2 and Na(ClO 4 ) enrichments (Fig. 3, Table 1). Although this reduction in expression is consistent with the decreased CH 4 production rates observed in these treatments (Fig. 2), the Ca(ClO 4 ) 2 -amended M. barkeri MS, which generated the least CH 4 , showed no signi cant difference in expression of the mcr complex relative to the 30˚C perchlorate-free control (Fig. 3, Table 1). Furthermore, no elements of mcr were signi cantly differentially expressed at 0˚C in the perchlorate-free control relative to the 30˚C perchlorate-free control (Fig. 3) despite its far lower CH 4 production rate (Fig. 2). Therefore, the decreased expression of mcrA does not appear to be su cient to explain the associated decrease in CH 4 production rates with perchlorate exposure at 30˚C or incubation at 0˚C.
The carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS), which plays a key role in both energy conversation and carbon xation via the Wood-Ljungdahl pathway, demonstrated signi cant regulatory changes as a function of perchlorate exposure and temperature. The cooS subunit of CODH, which reversibly converts CO and CO 2 , was up-regulated in 30˚C Mg(ClO 4 ) 2 and Na(ClO 4 ) treatments (Fig. 3, Table 1). Carbon monoxide dehydrogenase subunit epsilon (cdhε), which recycles ferredoxin in the reversible conversion between CO and CO 2 , was up-regulated with Ca(ClO 4 ) 2 at 30˚C (Fig.   3, Table 1), but was down-regulated in the 0˚C Mg(ClO 4 ) 2 and Na(ClO 4 ) treatments (Fig. 3, Table 2). The  Table 1). Both copies of this gene were down-regulated at 0˚C in the Mg(ClO 4 ) 2 and Na(ClO 4 ) treatments ( Fig. 3, Table 2).
Both alpha and beta chains of ACS, respectively encoded by cdha and cdhb, were down-regulated in the 0˚C Na(ClO 4 ) treatment with respect to the 0˚C perchlorate-free control, while only cdha was downregulated in the 0˚C Mg(ClO 4 ) 2 treatment (Fig. 3, Table 2). Cdhd, which encodes an iron-sulfur corrinoid protein, was also down-regulated at 0˚C in Mg(ClO 4 ) 2 and Na(ClO 4 )-supplemented incubations (Fig. 3, Table 2). We observed no signi cant differential expression of CODH/ACS complex genes in the 0˚C Ca(ClO 4 ) 2 treatment (Fig. 3).
Concurrent up-regulation of ammonium transporters, Mo-nitrogenases, and P-II repressors The most substantial up-regulation patterns were observed in genes relating to nitrogen cycling in perchlorate-incubated treatments. The 30˚C Mg(ClO 4 ) 2 and Na(ClO 4 )-amended conditions showed positive differential expression of 6 genes in the (Mo)-nitrogenase (hereafter (Mo)-Nase) complex ( Fig. 4) including nifH, the MoFe-dinitrogen reductase that is responsible for electron transfer to the a 2 b 2 N 2 binding site (encoded by nifD and nifK, respectively) via ATP hydrolysis, as well as the P-II regulatory repressors, nifI, which shut off N 2 xation when ammonia is bioavailable [50][51][52][53]  We observed substantial up-regulation of ammonium transporters (amt) in the 30˚C perchlorate treatments, but no signi cant differences in amt expression were observed in any 0˚C treatments (Fig. 4).

Down-regulation of sulfur-containing amino acids
In addition to the 20 common amino acids, M. barkeri also encodes a 21 st residue, pyrrolysine, via the 'amber' stop codon UAG 60 . A comparison of the genes encoding amino acid synthesis proteins showed large negative log 2 -fold changes at 30˚C in Mg(ClO 4 ) 2 and Na(ClO 4 )-amended treatments with respect to cysteine-producing proteins cysteine synthase (cysK) and serine acetyltransferase (cysE) (Fig. 5a).
Further examination of complete amino acid metabolic pathways (Figs. S4 -S16) revealed that this pattern of substantial gene down-regulation was characteristic of not only cysteine, but also the other sulfur-containing amino acid methionine (Fig. 5b).

Discussion
In this study we took our inspiration from nearly two decades of research into Martian methane to investigate how methanogens may survive under challenging shallow subsurface conditions of Mars, speci cally under freezing temperatures and in the presence of chaotropic perchlorate salts. We tracked CH 4 production and changes in global gene expression in M. barkeri MS via RNA-Seq following 28 days' incubation at 30˚C or 0˚C, with and without 10 mM of dissolved Na-, Mg-, or Ca-perchlorate. In accordance with prior ndings 25,26 we observed quanti able but inhibited methanogenesis in cultures under supplemented with perchlorates (Fig. 2). For the rst time we present transcriptomic evidence of the underlying biochemistry. Regulatory responses point to unambiguous shifts in amino acid synthesis and recycling, as well as mechanisms to combat increased osmotic stress -not unexpected reactions for an obligate anaerobe exposed to strongly oxidizing and chaotropic perchlorate salts. A surprising regulatory switch was observed in the methanogenesis pathway, with a signi cant up-regulation of methylamineutilizing genes, despite these substrates not being present in the media ab initio. Metalloenzymes with molybdenum active sites, including (Mo)-formylmethanofuran dehydrogenase and (Mo)-Nase were among the most signi cantly up-regulated genes in perchlorate-amended incubations at 30˚C. We contextualize these revelations with a precedent of perchlorate reduction in methanogenic cultures 25,26 , offering new insight into Mo active sites as hydrogenation catalysts for these reactions to proceed.
Evidence for selection against redox-sensitive amino acids under temperate conditions Cysteine and methionine are exceptionally sensitive to oxidation by reactive radical species 61 . Both residues have shown strong binding a nities to both perchlorate and perchloric acid, resulting in the oxidation of methionine to methionine sulfoxide and cysteine to sulfonic acid 62 . The susceptibility of cysteine and methionine to react with perchlorate risks degradation of protein structure and function. The extensive and substantial down-regulation of cysteine and methionine metabolic pathways we observed in the presence of Na-and Mg-perchlorates at 30°C (Fig. 5) suggests a concerted effort by M. barkeri to reduce the synthesis and repair of these residues in the presence of these perchlorate species. A dearth of signi cant differential expression of amino acid-recycling genes (including those speci c for cysteine and methionine) in both 0˚C and 0˚C perchlorate treatments demonstrates that freezing conditions are not su cient to confer a decrease in transcriptional activity. It is clear that the synthesis and processing of essential amino acids in M. barkeri must proceed despite the metabolic stresses of frigid temperatures and strong oxidants.
A role for glycine betaine in cryo-and osmoprotection, and as a methylamine precursor The regulatory patterns of enzyme complexes protecting against osmotic stress like opuAABC coincide with temperature and redox conditions. This operon is up-regulated at 30˚C with Na(ClO 4 ) and Mg(ClO 4 ) 2 , implying an increased need for cellular machinery to import glycine betaine to combat osmotic stress. In addition to being an osmoprotectant, glycine betaine is also cryoprotective 63 . While perchlorate salts are highly oxidizing, they also depress the freezing point of water. The down-regulation of opuAB in Mg(ClO 4 ) 2 -amended cultures at 0˚C might suggest a decreased cold shock response in M. barkeri, wherein glycine betaine uptake for its cryoprotective properties was not needed under freezing conditions.
The up-regulation of methylamine-speci c methanogenesis pathway genes in all three 30°C perchlorate treatments was unexpected. Methylamines were not present in the media ab initio and free energy yields of methylamine methanogenesis reactions at 30°C range from -91 to -143 kJ/mol CH 4 -signi cantly less than the -158 kJ/mol CH 4 of the hydrogenotrophic pathway (Table S3). However, it has been suggested that glycine betaine may be a potential precursor of trimethylamine (TMA) 64,65 . Although an exact mechanism for their formation has not been reported, the observed up-regulation of monomethylamine and dimethylamine permeases mtmP and mtbP (Fig. 3) suggest possible interactions between perchlorates and complex media constituents (e.g., perhaps yeast extract or casitone) that might yield methylamines for utilization by M. barkeri.
Up-regulation of Mo-containing enzymes suggest mechanism for H 2 -dependent perchlorate reduction.
Fixed nitrogen was replete under initial incubation conditions (9.3 mM NH 4 Cl plus additional N from complex ingredients like yeast extract and casitone). The signi cant up-regulation of ammonium transporters (amt, Fig. 4), methylamine permeases (mtmP and mtbP, Fig. 3), and P-II repressors (nifI, Fig.  4) in 30˚C perchlorate treatments imply that xed N was still available and actively utilized at the time of sample preservation for RNA-Seq. It is therefore surprising to observe a simultaneous and substantial upregulation of nitrogenase genes, an overwhelming proportion of which being associated with the molybdenum isoform, (e.g., nifDEHK, Fig. 4). Nitrogen xation is an energetically expensive process, consuming at least 16 (and, by one calculation for methanogens 66 , perhaps more than 50) moles of ATP  26 . In this study, preliminary measurements of perchlorate concentrations in sterile 120a media yielded inconclusive evidence of perchlorate reducation (data not shown). More work is necessary to fully explore the composition and role of yeast extract as a potential reducing agent.
The free energy yield of H 2 oxidation coupled to perchlorate reduction (Equation 2) is substantial (∆G˚' = -289 kJ/mol H 2 ) but the reaction is kinetically sluggish at ambient conditions and requires a metallic catalyst to overcome its large activation energy 79 .
Nickel (Ni) has long been known as an excellent hydrogenation catalyst 80 and is an essential cofactor for hydrogenase activity in methanogens 81 , but an essential role in microbial perchlorate reduction has never been suggested and it is not obvious from our transcriptomic data that perchlorates elicit a universal response by Ni active sites in hydrogenases (e.g. echE, vhtA, and frha in Fig. 3). Instead, given perchlorate reduction's known Mo dependency 68,82 and the additional empirical evidence presented here, we cannot ignore the most parsimonious explanation: the substantial up-regulation of (Mo)-Nase (Fig. 4) and (Mo)formylmethanofuran dehydrogenase (Fig. 3, Table 1) at 30˚C must be associated with perchlorate reduction. Certainly, a thorough investigation is warranted in order to elucidate ner details of the biochemistry.
Methylamine methanogenesis as a mechanism for energy conservation In methylamine methanogenesis, H 2 is recycled by the partial reversal of the methanogenesis pathway, generated via the oxidation of F 420 H 2 by frha and the oxidation of ferredoxin by echF (green arrows in Figs. 3). H 2 generated by the oxidation of F 420 H 2 and ferredoxin diffuses across the membrane to be oxidized by vhtA in an energy conserving scheme to recycle methanophenazine (MP) 83-86 . Ferredoxin oxidation by echF also results in the translocation of 2H + through ech, contributing to the production of a proton gradient (high outside the cell) 86 . Evidence for the generation of this proton gradient might be indicated in our incubations based on drops in pH (up to 0.77 pH units) (Table S4). Based on upregulation of the genes for methylamine methanogenesis, we attribute the differential expression of ech, vht, and frh hydrogenases (Fig. 3) and ferredoxin (Tables S9, S11, S13) as putative sources of a proton motive force in the 30˚C Ca(ClO 4 ) 2 -supplemented incubations.
We theorize that the thermodynamic spontaneity of  Tables 1-2). Transcriptomes from additional time points can help clarify whether these discrepancies in expression are re ective of distinct metabolic responses to Ca-perchlorate versus Mg-and Na-perchlorate, or simply different stages of metabolism following prolonged exposure to perchlorates in general. We infer the latter is more likely given the novel and ubiquitous response of genes in the methylamine methanogenesis pathway.

Implications for Martian methane
These ndings better constrain our growing understanding of how microbial life responds to strong oxidants, freezing temperatures, osmotic stress, and nutrient limitation -i.e., conditions characteristic of any habitable Martian environment where we may hope to nd extant life. Notably, our study shows that major metabolic disruption by perchlorates at 30˚C is not re ected to the same degree at 0˚C, which is autoclave-sterilized media blank for 28 days (Fig. S17). For each experimental treatment (perchlorate-free control, NaClO 4 , Mg(ClO 4 ) 2 , or Ca(ClO 4 ) 2 ), optical density measurements (OD) and CH 4 production were monitored weekly in parallel experiments incubated at 30˚C or 0˚C (Fig. 2).
OD measurements were performed spectrophotometrically at 600 nm using a Genesys 30 Visible Spectrometer (Thermo-Scienti c Corp., Madison, WI USA). At the end of the incubation experiment, direct cell counts were performed on enrichment aliquots uorescently stained with Acridine Orange (AO) dye (catalogue #318337, Sigma-Aldrich Chemical, Co., Milwaukee, WI USA) (50 µM nal concentration) following an established procedure 94  RNA isolation and puri cation. After 28 days' incubation, culture and blank volume contents were brie y vortexed and aseptically transferred into sterile 15 mL Falcon ® tubes (Corning Inc., Corning, NY USA) at incubation temperature inside a Coy anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI USA). Tubes were centrifuged at 3,000 ´ g for 1 minute using an IEC Centra CL2 centrifuge (Thermo Electron Company, Milford, MA USA) to pellet cells. Media was poured off for pH and electrical conductivity measurements, leaving behind 1 mL. To ensure quanti able RNA yields downstream, the nine biological replicates from each condition were consolidated into three sets of three samples each for extraction (3 mL pelleted cell suspension/tube). RNAlater solution (ThermoFisher Scienti c, Waltham, MA USA) was added to a nal volume of ~12 mL. Samples were left to equilibrate at incubation temperature for 3 hours, transferred to 4˚C for 24 hours, and then stored at -80˚C until overnight shipment on dry ice to Princeton University for RNA extraction.
Samples preserved at -80 C in RNAlater were thawed on ice in a sealed container before contents were transferred to 50 mL Falcon ® tubes. An equal volume (12 mL) of nuclease-free water (Qiagen, Hilden, Germany) was added to RNAlater-preserved samples, brie y vortexed, and centrifuged at 5,000 g for 10 minutes using a Sorvall Legend XI centrifuge (ThermoFisher Scienti c, Waltham, MA USA). The supernatant was subsequently discarded, and RNA was extracted following a modi ed protocol from a Zymo Quick-RNA Miniprep Plus Kit (Zymo Research, Irvine, CA USA). RNA lysis buffer and nuclease-free water were added to each sample in a 5:1 ratio, and sterile 0.7 mm garnet bashing beads (Qiagen, Hilden, Germany) were added to facilitate mechanical lysis during subsequent vortexing. Samples were then vortexed for 1 minute and centrifuged at 10,000 g at 4˚C for 1 minute using an Eppendorf 5810R (Eppendorf, Hamburg, Germany) to pellet cell debris. The supernatant containing total nucleic acids was transferred to a yellow Spin-Away™ column (Zymo Research, Irvine, CA USA) tted in a 2 ml collection tube. Samples were centrifuged at 10,000 g for 1 minute using an AccuSpin Micro 17 (ThermoFisher Scienti c, Waltham, MA USA) to separate out genomic DNA. Following centrifugation, the Spin-Away™ lter was discarded and the ltrate was collected from the column for RNA puri cation.
Total nucleic acids were then precipitated by adding 1 mL of chilled absolute ethanol. Pellets were mixed by pipetting and then transferred to green Zymo-Spin™ IIICG column lters tted in clean collection tubes.
Samples were centrifuged at 10,000 g for 30 seconds to collect precipitated nucleic acids on the column, subsequently treated with 400 µL RNA wash buffer, and centrifuged for 30 seconds at 10,000 g.     are excluded from genes exhibiting no signi cant differential expression across all conditions. Genes involved in non-hydrogenotrophic (H2/CO2) pathways are encompassed in colored boxes with pathway directionality indicated by matching arrowheads (green: methylamines; pink: acetate; yellow: methanol).
Full names of listed genes and metabolites are found in Tables S12 and S13, respectively.

Figure 4
Differential expression (Log2-fold change, LFC) of ammonium transporters (amt), Mo-containing nitrogenase (nif), and V-containing nitrogenase genes in M. barkeri MS. Perchlorate-amended 30˚C and 0˚C perchlorate-free control cultures are relative to 30˚C perchlorate-free control. 0˚C perchlorate-amended cultures are relative to 0˚C perchlorate-free control. Signi cance identi ed via Wald test (P < 0.05). Full names of proteins encoded by listed genes are found in Tables S14.

Figure 5
Differential expression (Log2-fold change, LFC) of genes involved in (a) amino acid synthesis and (b) recycling of the sulfur-containing amino acids methionine and cysteine in M. barkeri MS. Perchlorateamended 30˚C and 0˚C perchlorate-free control cultures are relative to 30˚C perchlorate-free control. 0˚C perchlorate-amended cultures are relative to 0˚C perchlorate-free control. Full names of gene and amino acid abbreviations are respectively found in Tables S15 and S16.

Figure 6
Proposed models of Mo-and H2-dependent perchlorate reduction occurring a) in direct competition with hydrogenotrophic methanogenesis, and b) via the energy-conserving partial reversal of methylamine-

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