Transcriptome assembly statistics
M. barkeri possesses the second largest described genome amongst the methanogenic Archaea48. 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-filtered 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 cells49. 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(ClO4)2, Mg(ClO4)2, and Na(ClO4) reduced total net CH4 production by 48%, 32%, and 24%, respectively, relative to the perchlorate-free control (Fig. 2). Significant reduction in CH4 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 significant decrease in CH4 for at least one time point relative to the 0˚C control (Fig. 2). No CH4 production was observed in the media blank controls (data not shown). Cultures were monitored via optical density measurements at 600 nm (OD600), but perchlorate-amended media experienced precipitate formation which made obtaining reliable growth data difficult (Fig. S3).
When incubated at 0˚C, the perchlorate-free control demonstrated significant up-regulation of several genes in the hydrogenotrophic pathway relative to the 30˚C perchlorate-free control (Table 1, Fig. 3), including log2-fold changes (LFC) in molybdenum (Mo)-formylmethanofuran dehydrogenase subunit B (fmdB), methenyl-tetrahydrosarcinapterin (H4SPT) cyclohydrolase (mch), and periplasmic heterodisulphide reductase (hdrDE). Likewise, significant down-regulation was observed in the sodium ion (Na+) transporter methyl-H4SPT:coenzyme M methyltransferase complex (mtrA), as well as the F420-reducing subunit of the periplasmic energy conserving hydrogenase (echF). All subunits of both coenzyme F420 hydrogenases (frhabg) were significantly up-regulated in perchlorate-free 0˚C incubations (Table 1, Fig. 3). In the perchlorate-treated incubations at 0˚C, we only observed down-regulation of frhabg in the presence of Ca(ClO4)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 CO2 reduction step of the hydrogenotrophic pathway (Fig. 3). In the presence of both Mg(ClO4)2 and Na(ClO4), one copy of (Mo)-fmdE exhibited significant up-regulation, while the other was significantly down-regulated (Table 1). Unique to the Mg(ClO4)2 incubations was the up-regulation of tungsten (W)-fmdA. In contrast, perchlorate-supplemented treatments incubated at 0˚C demonstrated statistically significant down-regulation of Mo-fmd genes, but up-regulation of the (W)-fmd operon (Table 2).
Energy conserving hydrogenase subunit F, echF, which supplies reduced ferredoxin to fmd, was down-regulated in the presence of Mg(ClO4)2 and Na(ClO4) at 30˚C, whereas most subunits of ech were significantly up-regulated in 30˚C Ca(ClO4)2 conditions (Fig. 3). Among other hydrogenases demonstrating significant up-regulation was methanophenazine-dependent hydrogenase, vht, specifically, 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 H2 being the only reducing equivalent provided for the production of CH4 in our incubations, the addition of Ca(ClO4)2, Mg(ClO4)2, and Na(ClO4) resulted in significant 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 (CH3-CoM) reductase subunit alpha (mcrA) was down-regulated at 30˚C in Mg(ClO4)2 and Na(ClO4) enrichments (Fig. 3, Table 1). Although this reduction in expression is consistent with the decreased CH4 production rates observed in these treatments (Fig. 2), the Ca(ClO4)2-amended M. barkeri MS, which generated the least CH4, showed no significant 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 significantly differentially expressed at 0˚C in the perchlorate-free control relative to the 30˚C perchlorate-free control (Fig. 3) despite its far lower CH4 production rate (Fig. 2). Therefore, the decreased expression of mcrA does not appear to be sufficient to explain the associated decrease in CH4 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 fixation via the Wood-Ljungdahl pathway, demonstrated significant regulatory changes as a function of perchlorate exposure and temperature. The cooS subunit of CODH, which reversibly converts CO and CO2, was up-regulated in 30˚C Mg(ClO4)2 and Na(ClO4) treatments (Fig. 3, Table 1). Carbon monoxide dehydrogenase subunit epsilon (cdhε), which recycles ferredoxin in the reversible conversion between CO and CO2, was up-regulated with Ca(ClO4)2 at 30˚C (Fig. 3, Table 1), but was down-regulated in the 0˚C Mg(ClO4)2 and Na(ClO4) treatments (Fig. 3, Table 2). The 30˚C Ca(ClO4)2 treatment also demonstrated significant up-regulation of 5-H4SPT:corrinoid Fe-S protein methyltransferase (cdhγ), which plays a key role in the generation H4SPT and acetyl-CoA for biomass synthesis in the Wood-Ljundal pathway (Fig. 3, Table 1). Both copies of this gene were down-regulated at 0˚C in the Mg(ClO4)2 and Na(ClO4) 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(ClO4) treatment with respect to the 0˚C perchlorate-free control, while only cdha was down-regulated in the 0˚C Mg(ClO4)2 treatment (Fig. 3, Table 2). Cdhd, which encodes an iron-sulfur corrinoid protein, was also down-regulated at 0˚C in Mg(ClO4)2 and Na(ClO4)-supplemented incubations (Fig. 3, Table 2). We observed no significant differential expression of CODH/ACS complex genes in the 0˚C Ca(ClO4)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(ClO4)2 and Na(ClO4)-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 a2b2 N2 binding site (encoded by nifD and nifK, respectively) via ATP hydrolysis, as well as the P-II regulatory repressors, nifI, which shut off N2 fixation when ammonia is bioavailable50–53. Biosynthesis and assembly proteins for (Mo)-Nase, nifE and nifN, were also upregulated at 30˚C with Mg(ClO4)2 and Na(ClO4). At 30˚C, only nifH demonstrated significant up-regulation in both the Ca(ClO4)2 and perchlorate-free control, but the 0˚C Ca(ClO4)2 treatment also showed significant up-regulation of nifI and nifE (Fig. 4).
We observed substantial up-regulation of ammonium transporters (amt) in the 30˚C perchlorate treatments, but no significant differences in amt expression were observed in any 0˚C treatments (Fig. 4).
Up-regulation of osmostress response genes
In the 30˚C Mg(ClO4)2 treatment, the complete operon for osmostress protectants uptake A, opuA, observed positive LFC of 0.64 ± 0.17 (opuAA, P < 0.001), 0.73 ± 0.20 (opuAB, P < 0.001), and 0.66 ± 0.30 (opuAC, P = 0.02). In the 30˚C Na(ClO4) treatment, significant up-regulation was also observed for opuAA (LFC = 0.54 ± 0.17, P = 4 10-3) and opuAB (LFC = 0.59 ± 0.20, P = 7 10-3). OpuA is responsible for the uptake of extracellular glycine betaine, belonging to a family of ABC transporters that hydrolyze ATP to import glycine betaine and other osmoprotectants such as proline54–56. The opu family also includes uptake systems for choline, a glycine betaine precursor56, but M. barkeri lacks the cellular machinery for de novo glycine betaine synthesis57. Relative to the 0˚C perchlorate-free control, significant down-regulation of opuAA in 0˚C Mg(ClO4)2 (LFC = -0.94 ± 0.30, P = 8.02 10-3) and Na(ClO4) (LFC = -0.69 ± 0.31, P = 3.14 10-2) treatments was observed. OpuAB was also down-regulated in the 0˚C Mg(ClO4)2 treatment (LFC = -1.03 ± 0.38, P = 1.56 10-2).
Evidence for osmotic stress can also be observed in the regulation of cell surface protein synthesis. Methanochondroitin is the primary constituent of the extracellular matrix that clumps M. barkeri cells into multicellular aggregates under optimal growth conditions58. Glucuronic acid, generated from glucose degradation via UDP-glucose dehydrogenase (UGDH), is a major component of methanochondroitin58,59. We observed significant down-regulation of UGDH under Mg(ClO4)2 and Na(ClO4) conditions at 30˚C (LFCMg = -0.55 ± 0.17, P = 3 10-3; LFCNa = -0.51 ± 0.17, P = 7 10-3).
Down-regulation of sulfur-containing amino acids
In addition to the 20 common amino acids, M. barkeri also encodes a 21st residue, pyrrolysine, via the ‘amber’ stop codon UAG60. A comparison of the genes encoding amino acid synthesis proteins showed large negative log2-fold changes at 30˚C in Mg(ClO4)2 and Na(ClO4)-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).