Microbiological procedures. Cultures of M. barkeri wild-type strain MS (ATCC 51582; obtained from T. Kral, Univ. of Arkansas, Fayetteville, AK USA) were grown in DSMZ 120a media supplemented with 10.0 mg L− 1 of ethylenediaminetetraacetic acid (EDTA), 10 ml L− 1 of ATCC Trace Mineral Supplement solution (#MD-TMS, ATCC, Manassas, VA USA), and 10 ml L− 1 of ATCC Vitamin Supplement solution (#MD-VS; ATCC). All chemicals were obtained from Sigma-Aldrich, Co. (St. Louis, MO USA) unless otherwise noted.
Cells of M. barkeri were grown in crimp-sealed balch anaerobic tubes in 10 mL of DSMZ 120a media and headspaces pressurized to 1500 mbar with an 80:20 mixture of hydrogen (H2) and carbon dioxide (CO2). Sodium resazurin (0.5 ml L− 1, 0.1% w/v) was used as a low pO2 indictor reagent. All balch tubes were sparged as required with either 0.2 µm filter-sterilized anoxic N2 or 80:20 H2:CO2 gases. Cultures were grown for 4 weeks at 30°C before use. Cell densities were estimated by aseptically and anaerobically withdrawing 100 µL of suspended cells in 120a media from each culture, staining the cells with 100 µM aqueous acridine orange, and counting cells in a hemacytometer. Cell densities per culture were approximations because cells of M. barkeri formed medium to large aggregates in the 120a media (Fig. 1C; Table S3). Protocols for preparing M. barkeri cells for SEM imaging are described elsewhere (74). All anaerobic media and cultures were prepared and handled in a Coy Anaerobic Chamber (Coy Laboratory Products, Grass Lake, MI USA).
Methane Assay Chambers (MAC). Two methane assay chambers (MAC units) were designed in-house and manufactured by EMF, Inc. (Merritt Island, FL USA). Two MAC units were installed into a previously described Planetary Atmospheric Chamber (PAC) (75, 76), as shown in Fig. 1A. Each MAC unit was plumbed with gas supply and sampling lines as shown in Fig. 1B.
Four-week-old cultures of M. barkeri were harvested, centrifuged, washed thrice with fresh 120a media (i.e., to purge dissolved CH4 from the culture media), combined, and otherwise aseptically and anaerobically processed such that 10 mL of 120a media with ~ 5.0E + 08 cells were available for each MAC unit. The MAC units were precooled to ~ 7–8°C with a liquid nitrogen (LN2) cryogenic system. With filter-sterilized anoxic N2 gas flowing into the MAC units, the tops were briefly opened and 10 mL of M. barkeri cells placed within sterile 6-cm glass petri dishes. The MAC units were fitted with new copper gaskets, sealed, and chilled to 4°C. Each MAC unit was loaded separately with anoxic and viable cultures of M. barkeri to maintain cultures in an oxygen-free state. All cultures showing pink coloration after the setup protocol were discarded and the process restarted (i.e., resazurin dye indicated absorption of trace O2 into the media).
After both MAC units were anaerobically loaded with viable cultures of M. barkeri, the temperature of the MAC and PAC systems was lowered to 0°C (\(\pm\) 0.5°C) and 7.0 mbar (\(\pm\) 0.2 mbar) of N2. While the internal MAC units were stabilizing at Martian temperature and pressure, the N2 gases within the headspaces of the units were purged three times with fresh N2 (over approx. 5 min), and then purged thrice more with either the 80:20 H2:CO2 or Mars gas + 2.9% pH2 mixtures (over approx. 5 min). An Opto-22 datalogging system (Opto-22, Inc., Temecula, CA USA) was used to record the internal temperatures, pressures, and relative humidities (RH) in headspaces above M. barkeri cultures within both MAC units. Cultures of M. barkeri cells in the MAC units were then maintained for 14 days at 0°C and 7–12 mbar and then sampled for the accumulation of CH4 in the headspace gases.
Over the course of the 14-day experiments (described below), liquid water evaporated from the DSMZ 120a medium and increased the headspace pressures within the MAC units to 12 mbar. The system could not maintain the MAC headspace pressures at lower than 12 mbar without evacuating accumulated CH4. Therefore, we report that these experiments were conducted between 7–12 mbar.
Methane concentrations within the MAC unit headspaces were collected by pumping the headspace gases from the MAC units directly into 10 cc Tedlar bags using a low-pressure 2-stage pump (model MPU3920-N813, KNF Manufacturing, Trenton, NJ USA). The gases within the Tedlar bags were immediately sampled and injected into a gas chromatograph (Trace 1310, Thermo-Fisher Scientific, Waltham, MA USA) fitted with a flame ionization detector (FID). Preliminary trials with gas mixtures doped with known concentrations of CH4 indicated that removing the headspace gases from the MAC units at 7 mbar, collecting them outside the Mars chamber at 1015 mbar (i.e., average atmospheric pressure on the Florida coast where the PAC is located), injecting them into the GC-FID system had a minimum detection level of 10 ppm CH4 in the MAC atmospheres. Thus, all readings below 10 ppm were considered below the detection limit and treated as zeroes in the data spreadsheets. Standard curves for measuring CH4 in the MAC units are given in Fig. S1.
Two separate experiments were conducted in the PAC at 7–12 mbar. In the first experiment, the headspace gas mixtures within the MAC units were comprised of 80:20 H2:CO2 gas mixtures (n = 6 replicates); i.e., at Mars surface pressure but under a stoichiometrically-balanced atmosphere to support hydrogenotrophic (H2:CO2-based) methanogenesis. The second experiment also tested for hydrogenotrophic methanogenesis at 7–12 mbar, but under a Mars gas mixture(42) supplemented with H2 (n = 6 replicates). The Mars gas mixture was composed of 92.5% CO2, 2.9% H2, 2.59% N2, 1.94% Ar, 0.16% O2, 0.03% H20. The second experiment more accurately represented conditions in the present-day Martian shallow subsurface at 7–12 mbar.
Statistical analysis of CH 4 evolution data. Methane evolution data for both experiments were analyzed with the statistical analysis software package SAS, v9.4 (SAS Institute, Inc., Cary, NC USA). Data were log-transformed to induce homogeneity of treatment variances. Transformed data were subjected to ANOVA (PROC GLM) and protected least-squares mean (LSM) separation tests (P ≤ 0.01; n = 6 replicates for the first experiment and n = 4 replicates for the second experiment). Data in Fig. 1D are presented as untransformed values. Both within-group and between-group statistical differences in methane production were assessed with respect to atmospheric gas mix condition (e.g., H2:CO2 across assayed temperatures and pressures, and H2:CO2 vs. Mars gas mix for a given temperature and pressure condition).
Culture preservation for downstream transcriptomics analyses. After collecting gas samples from individual MAC units, the units were repressurized with the headspace gases described above for each experiment. As soon as the MAC units and the bulk Mars chamber headspace were equilibrated to 1015 mbar, the PAC system was opened, the MAC tops unbolted, and 10 mL of RNAlater (Invitrogen, Thermo-Fisher Scientific, Waltham, MA USA) were immediately added to each culture. The process was completed as quickly as possible to faithfully capture gene expression profiles at the ends of both experiments, with timed extractions accomplished within 12–15 min from the initial re-pressurization step. The LN2 cold plate, and thus, the M. barkeri cultures, were maintained at 0°C during the extraction process.
Once the RNAlater was injected into the M. barkeri cultures, the cells were transferred to sterile 15 cc Falcon tubes (Corning Inc., Corning, NY USA), pelleted by centrifugation, decanted, and refreshed with pre-chilled RNAlater added to the tubes. The cells were washed thrice with RNAlater while holding the temperature at 0°C. Cultures were stored at -80°C and shipped overnight on dry ice to Harvard University, where they were maintained at -80˚C until further processing.
RNA isolation, library prep, and sequencing. RNA was isolated and purified as previously described (38) with parallel extraction blanks and thorough RNAse decontamination at each step to ensure cleanliness of the extraction procedure. An aliquot of RNA from each sample was quantified using a Qubit hs RNA assay kit coupled to a Qubit 2.0 fluoremeter (ThermoFisher Scientific). RNA concentrations were subsequently used to calculate first order approximations of cell counts per sample at the termination of each experiment according to Eq. 1:
$$\text{Cell counts}\text{ =}\frac{{\text{RNA}}_{\text{conc}}}{{\text{a}}_{\text{r_M}\text{. barkeri}}}\text{× V}$$
1
where RNAconc was equal to the RNA concentration of the sample in ng/µl, ar_M.barkeri was estimated as the relative RNA content of M. barkeri as a function of total cellular nucleic acid content (RNA/(RNA + DNA), conservatively estimated to be ~ 0.5 for the total cell population (77), and V was the volume of the sample culture. Complementary DNA (cDNA) was subsequently synthesized from purified RNA templates using an iScript gDNA Clear cDNA Synthesis Kit following the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA USA). Resulting cDNA was diluted in nuclease-free H2O, quantified using a Qubit ssDNA assay (Thermo-Fisher Scientific), and quality-checked on an Agilent 4200 TapeStation System using High Sensitivity D5000 ScreenTape (Agilent Technologies, Santa Clara, CA USA).
A two-directional library preparation was performed for each experiment using the Nextera XT DNA Library Prep Kit according to the manufacturer’s instructions (Illumina, Inc., San Diego, CA USA). The resulting libraries were quality checked via TapeStation as described above, and final qPCR of pooled libraries were performed on a Bio-Rad CFX96 system (Bio-Rad Laboratories, Hercules, CA USA) following the standard Illumina qPCR protocol (Illumina, Inc.). Two-directional (2 x 150 bp) sequencing was performed on one lane of a NovaSeq S4 flowcell (Illumina) at the Harvard University Bauer Core Facility.
Bioinformatic and statistical pipeline. Quality filtering, mapping, and annotation of paired-end reads to M. barkeri coding sequences (CDS) were performed as previously described (39). Single variable differential expression analyses were performed using the ‘DESeq2’ package in R (78), considering 30˚C as the reference temperature condition, 1500 mbar as the reference pressure condition, and 80:20 H2:CO2 as the reference atmospheric condition. Because of the significant reduction in library size and quality for the Mars gas mix-incubated samples, a highly conservative statistical approach was taken to minimize the risk of false discovery rates.
Transcripts were indexed by gene assignment into a count table, and library size factors were estimated using the ‘poscounts’ option to address instances of zero inflation in low-biomass libraries. Fragments per million mapped reads (FPM) were calculated using the robust median ratio method to account for sequencing differences in library size and RNA composition of samples. Gene-wise dispersion and mean-dispersion relationships were estimated using negative binomial generalized linear model with a local fit type and Wald significance tests. Resulting log2-fold changes in expression (LFC) were then shrunk using the ‘apeglm’ shrinkage estimator (79). Resulting model count data were then variance-stabilized using a regularized log transformation to minimize differences between samples containing genes with zero and near-zero transcript counts. The resulting dataset was scaled and centered, and a two-dimensional non-metric multiscale dimensional analysis (NMDS) solution was determined using a Bray-Curtis dissimilarity index iterated over 2000 random starts using function ‘metaMDS’ in the ‘vegan’ package (80).