Light is the most important energy source for the phototrophic microbial mat at Mushroom Spring. Although it has often been referred to as a “cyanobacterial mat” (Doemel and Brock, 1976; Brock, 1995; Ward et al., 1998; Ramsing et al., 2000; Klatt et al., 2011) highly diverse phototrophic bacteria inhabit the mat community. Sixteen different chlorophototrophic bacteria have been identified by a combination of molecular and isolation- and enrichment-based studies (Table 2; Klatt et al., 2011; Thiel et al., 2016; see Figure 4 in Tank et al., 2017). Genes encoding type-1 photosynthetic reaction center (RCs; psaA/psaB and pscA) and type-2 RCs (pufLM, psbA, and, psbD) of 10 chlorophototrophs were identified in the undermat metagenome at 60°C (Table S2). Taxa containing type-2 RCs are the dominant oxygenic chlorophototrophic Cyanobacteria: Synechococcus spp. Types A and B' (OTUs 22 and 5, respectively); members of the Chloroflexi: Roseiflexus spp. (OTU-1), “Ca. Roseilinea gracile” (OTU-6), Chloroflexus sp. (OTU-11) and “Ca. Chloranaerofilum corporosum” (OTU-15); as well as two less abundant chlorophototrophic Alphaproteobacteria: “Ca. Elioraea thermophila” (OTU-46) and “Ca. Roseovibrio tepidum” (OTU-121; Table S2; Thiel et al., 2016; Tank et al., 2017).

Aside from Synechococcus spp. Types A and B' (OTUs 22 and 5, respectively), taxa containing Type-1 RCs, Chloracidobacterium thermophilum (Bryant et al., 2007; Garcia Costas et al., 2012b; Tank and Bryant, 2015a,b) and “Ca. Thermochlorobacter aerophilum” (Liu et al., 2012), which were first identified in the upper layer of the mat, were also detected in the undermat metagenome (Table S2). Although Heliobacterium modesticaldum was previously isolated from these same mats in a region of lower temperature (Kimble et al., 1995), no pshA sequences, encoding for the P800 photosynthetic reaction center core protein of heliobacteria, were identified nor did the reference genome of this organism recruit any significant hits from the metagenome. This is presumably because H. modesticaldum does not grow at temperatures above ~56°C (Kimble et al., 1995). Similarly, no psaA and psaB genes affiliated with Thermosynechococcus sp. C1, Synechococcus sp. C9, or Leptolyngbya sp. were identified in the metagenome, although these organisms have previously been isolated from these mats (Tank et al., 2017 and references therein). This is probably explained either by their very low abundance and/or optimal growth temperatures lower than that studied here. Similarly, Thermochromatium tepidum and Blastochloris sp. have been obtained through cultivation studies from lower temperature mat samples (~50°C; Tank et al., 2017), but were not detected in the 60°C undermat metagenome.

The highest diversity in genes for type-2 reaction centers was seen for members of OTU-1, Roseiflexus spp. (16 partial pufLM genes with coverage values ranging from 71 to 1227 × Table S2). This indicates that several Roseiflexus spp. ecotypes with differences in their RC proteins are likely present in the mat community. Similarly, cyanobacterial scaffolds containing RC genes showed high diversity, suggesting the presence of different ecotypes adapted to specific light niches as shown previously (Table S2; Becraft et al., 2015; Nowack et al., 2015; Olsen et al., 2015). In both cases the high microdiversity was correlated with assembly difficulties, and only a few long scaffolds were found for these taxa. Physiological information for these two phototrophs was based on reference genomes and isolates (Bhaya et al., 2007; van der Meer et al., 2010; Nowack et al., 2015; Olsen et al., 2015).

A surprising finding is that several members of the undermat contain genes belonging to the bacteriorhodopsin superfamily (Table 2). Whether these genes are used to obtain energy from light is not clear, but this is certainly a possibility. In general, blue-green light does not penetrate deeply into the mat (see Figures 4 in Nübel et al., 2002; Becraft et al., 2015), so light wavelengths absorbed by the rhodopsin-like proteins should not be abundant in the undermat. On the other hand, light of around 450–560 nm, which includes the major absorption maxima of xanthorhodopsin (Lanyi and Balashov, 2008), is not well absorbed by Chl a, phycocyanin and carotenoids of cyanobacteria, and thus blue-green light is slightly more abundant in deeper layers than blue or red light (Nübel et al., 2002; Becraft et al., 2015). Seventeen annotated rhodopsin genes in the assembled undermat metagenome were affiliated with seven phylogenetic groups, representing an unexpected diversity of potential retinal-based phototrophy in the mat (Figure 2; Table S3). Both the diversity and the abundance of rhodopsins was considerably lower in the upper layer. Only 10 rhodopsin sequences, representing four community members, Roseiflexus sp. OTU-1, Armatimonadetes member OTU-3 (and possibly other Armatimonadetes members), “Ca. Roseilinea gracile” OTU-6 and Meiothermus sp. OTU-21, were detected in the upper layer metagenome (unpublished data). Identical rhodopsin gene sequences were also detected in the undermat metagenome. It is noteworthy that all of the members showed equal (Meiothermus sp.) or higher relative abundance in the undermat based on 16S rRNA gene amplicon analysis, indicating retinal-based phototrophy to be of unexpected importance in a layer with low irradiance (Table 2; Thiel et al., 2016). Additionally, rhodopsin gene sequences affiliated with Bellilinea sp. OTU-31, Thermus sp. OTU-70, and Isosphaera-like Planctomycetes member OTU-83 were detected exclusively in the undermat metagenome. Most rhodopsin sequences were most closely related to genes annotated as “xanthorhodopsin,” a proton-pumping rhodopsin first described in Salinibacter ruber, which contains a second carotenoid chromophore (salinaxanthin) as a light-harvesting antenna molecule (Balashov et al., 2005). The presence of an antenna chromophore in xanthorhodopsin would certainly be advantageous in niches that receive low irradiance. Additional genes were identified as Na⁺-pumping rhodopsin (Figure 2).

Xanthorhodopsin genes with phylogenetic affiliation to light-driven, xanthorhodopsin-like H⁺ pumps were found in the anoxygenic, chlorophototrophic Chloroflexi members Roseiflexus spp. OTU-1 and “Ca. R. gracile” OTU-6. This could indicate the co-existence of xanthorhodopsin and photosynthetic apparatus, as previously suggested for the thylakoid-less cyanobacterium Gloeobacter violaceus, which contains gloeorhodopsin, a rhodopsin closely related to xanthorhodopsin from Salinibacter ruber and Roseiflexus sp. RS-1 (Choi et al., 2014). Rhodopsin genes in cyanobacteria do not seem to be a rare occurrence, as a keyword search in the NCBI database reveals more than 40 entries at the time of writing (March 2017). However, the absence of a carotenoid oxygenase assigned to these organisms in all datasets, the Roseiflexus sp. RS-1 genome, “Ca. Roseilinea gracile” OTU-6 partial genome and the metagenome, suggests that either a currently unidentified protein is responsible for retinal production or retinal is derived from an external source (as discussed below).

Six of the xanthorhodopsin genes in the undermat metagenome are associated with members of the phylum Armatimonadetes (Figure 2). Due to their high sequence similarity and similar coverage values, these sequences are probably derived from different sub-populations of the same OTU, OTU-3, for which a high microdiversity has been observed (Thiel et al., 2016). Alternatively, they could be derived from different members of the Armatimonadetes that are present in the mat community (e.g., OTU-3, OTU-12, and OTU-18). Whether the low-abundance xanthorhodopsin genes that cluster with sequences for Meiothermus cerbereus and Thermus aquaticus in the phylogenetic tree, really represent H⁺-pumping xanthorhodopsins is uncertain, because the rhodopsin genes in these organisms have not been studied to our knowledge nor has their functionality been verified.

The possibility of light energy usage by members of the microbial mats was further supported by a rhodopsin gene, which is phylogenetically affiliated with a less abundant member of the Chloroflexi, Bellilinea sp. OTU-31. Phylogenetic affiliation placed this gene within a clade of light-driven sodium pumps (Figure 2) and a similar sequence is also present in the genome of the closest relative, Bellilinea caldifistulae, which is a chemotrophic member of the phylum Chloroflexi. Although no specific function has been demonstrated for the rhodopsin gene in the type strain, when the gene obtained from the metagenome was heterologously expressed in Escherichia coli, the resulting strain produced a purple-colored protein and showed sodium-pumping activity (Y. Nakajima, S. Yoshizawa, V. Thiel, D.A. Bryant, unpublished results). The presence of six putative, sodium-dependent transporters in the partial genome of the Bellilinea sp. member in the undermat further strengthens the hypothesis that this rhodopsin has sodium-pumping activity. The evidence suggests that light-driven nutrient uptake/transport using a secondary sodium gradient, similar to the light-driven uptake of vitamin B₁ in a marine flavobacterium (Gómez-Consarnau et al., 2016), might be important in this member of the Chloroflexi.

Although it is likely that the xanthorhodopsin genes described above are functional, some uncertainty exists because the partial genomes of organisms with these xanthorhodopsin genes do not generally encode a β-carotene 15,15′-monooxygenase. Xanthorhodopsin in S. ruber was shown to contain retinal and carotenoid salinixanthin (Balashov et al., 2005). Retinal is an oxidative cleavage product of carotenoids, most often β-carotene, which is catalyzed by a β-carotene 15,15′-monooxygenase, sometimes called β-carotene 15,15′-dioxygenase, in eukaryotes (Woggon, 2002; Kim et al., 2009). In bacteria proteins cleaving β-carotene to produce retinal include bacteriopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh; (Kim et al., 2009) and references therein). Although they have the same function, mammalian β-carotene 15,15′-dioxygenase (BCD) and prokaryotic Brp/Blh family β-carotene 15,15′-monooxygenases are quite different and share only 20% amino acid sequence similarity (Sabehi et al., 2005; Kim et al., 2009). Only low-abundance sequences in the metagenome, none of which was affiliated with members containing highly abundant rhodopsin genes, showed similarity to β-carotene 15,15′-monooxygenase from Oscillatoria nigroviridis PCC 7112 (Table S4). Only one combination of a gene encoding an enzyme for possible cleavage of β-carotene and a xanthorhodopsin-like gene was identified for an Isosphaera pallida-like organism (OTU-83), a less abundant member of the undermat. This indicates the possibility of a functional xanthorhodopsin and thus light utilization for this organism. However, the failure to detect highly abundant β-carotene 15,15′-monooxygenases affiliated with more abundant rhodopsin-containing complete or partial genomes does not necessarily invalidate the hypothesis that these xanthorhodopsins are used for light energy capture. Firstly, sequences encoding enzymes similar to β-carotene 15,15′-monooxygenases, which could not be identified taxonomically, were present in the unassembled part of the metagenome. Secondly, other oxygenases could substitute for this function in different bacteria, or carotenoids other than retinal could possibly be bound to the rhodopsins. Thirdly, a still unknown family of β-carotene cleaving enzymes/genes with low sequence similarity to known carotenoid oxygenases but exhibiting the same function in other xanthorhodopsin-containing microbes might still be discovered. Finally, β-carotene 15,15′-monooxygenases that produce retinal in some of the major mat organisms (e.g., Synechococcus spp., Cab. thermophilum) might be used as a community resource by other mat members. This last possibility would be of particular ecological interest, as it would emphasize the close relationships and interactions that are connecting the mat members and stabilizing the community. Indications for this kind of co-factor dependency from the environment were recently suggested for the freshwater actinobacterium Rhodoluna lacicola, which expresses a functional rhodopsin gene but exhibits proton pumping activity only when exogenous retinal is provided (Keffer et al., 2015).

The key enzyme for the CBB cycle is the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Almost all sequences encoding type-I RubisCO that were identified in the metagenome were affiliated with the dominant cyanobacteria, Synechococcus spp. Types A and B'; consistent with their known ability to fix inorganic carbon via the CBB cycle. Only one low-abundance partial sequence appeared to be affiliated with the proteobacterial phylum (JGI24185J35167_11464881; Table S5). Two low-abundance anoxygenic phototrophic Alphaproteobacteria were found in the undermat metagenome, “Ca. Elioraea thermophila” and “Ca. Roseovibrio tepidum” (see above). Because the genome of the closely related Elioraea tepidum is available and does not contain any autotrophic pathway genes, this could be an indication that a CBB cycle occurs in “Ca. Roseovibrio tepidum” represented by OTU-121, which is further supported by the presence of a gene for a type-1 RubisCO in the non-phototrophic but closely related Rhodospirillales/Acetobacteraceae member Roseomonas gilardii subsp. rosea ATCC BAA-691 (Acc. No. NZ_JADY01000025; L882_RS0115610).

The 3-HP bi-cycle is a complex autotrophic carbon fixation pathway that has been fully elucidated in Chloroflexus aurantiacus and most likely also operates in other autotrophic members of the Chloroflexaceae (Klatt et al., 2007, 2013b; Zarzycki et al., 2009; van der Meer et al., 2010). The multifunctional enzymes malonyl-CoA reductase, propionyl-CoA synthase, and malyl-CoA/β-methylmalyl-CoA/citramalyl-CoA (MMC) lyase are considered to be the key and diagnostic enzymes of the 3-HP bi-cycle (Fuchs, 2011; Hügler and Sievert, 2011 and references therein). The incorporation of CO₂ into biomass is catalyzed by the CO₂-fixing enzymes acetyl-CoA and propionyl-CoA carboxylase. Other characteristic enzymes are CoA-transferases and hydratases. The complete pathway is shown in Figure S1. Apart from serving as a carbon fixation pathway for autotrophic growth, the 3-HP bi-cycle may be utilized together with the TCA cycle as mixotrophic pathways for the simultaneous incorporation of organic and inorganic carbon (Klatt et al., 2007, 2013b; Zarzycki and Fuchs, 2011; Bryant et al., 2012). In nature, mixotrophy is most likely the predominant lifestyle for these organisms (also see below).

Sequences for all three of the diagnostic enzymes were detected in the metagenome for Chloroflexi members, the highly abundant Roseiflexus sp. (OTU-1), Chloroflexus sp. MS-G (OTU-11), and “Ca. Chloranaerofilum corporosum”(OTU-15), which indicates that these undermat organisms probably have the capacity to fix inorganic carbon via the 3-HP bi-cycle and grow either mixotrophically or autotrophically (Table S6).

The high microdiversity for malonyl-CoA reductase gene sequences associated with Roseiflexus spp. OTU-1 (Table S7) indicates the presence of putative ecotypes potentially with differences in carbon metabolism—more specifically in enzymes involved in carbon fixation. Several genes encoding important enzymes of the 3-HP bi-cycle, including acetyl-CoA carboxylase, malonyl-CoA reductase and propionyl-CoA synthase, are arranged in an apparent operon in Roseiflexus sp. RS-1 (RoseRS_3199–RoseRS_3203; see Klatt et al., 2007; van der Meer et al., 2010). Transcript levels for the genes in this operon increased during the day in the Roseiflexus spp. population in the upper green layer (Klatt et al., 2013b). A similar increase during day-time hours was observed when RoseRS_3199–3203 were used as mapping targets for RNA-seq data for the undermat (V. Thiel and D. A. Bryant, unpublished results). These data indicate that the 3-HP bi-cycle is probably active during the day when light is available and the mat is highly oxic in the upper layer.

For other members of the Chloroflexi, OTU-6 (“Ca. Roseilinea gracile”) and OTU-9 we could not detect any genes for key enzymes of the 3-HP bi-cycle (Table S6). OTU-9 instead has the genes for the reductive acetyl-CoA pathway (see below), while OTU-6 seems to have a photoheterotrophic lifestyle.

Key enzymes of the reductive or reverse TCA cycle include 2-oxoglutarate synthase (2-oxoglutarate:ferredoxin oxidoreductase), fumarate reductase and especially the citrate-cleaving enzyme. The ATP-dependent cleavage of citrate into acetyl-CoA and oxaloacetate is a complex reaction, that can be accomplished either by the combined action of the enzymes citryl-CoA synthetase (CCS) and citryl-CoA lyase (CCL) or by the heterodimeric enzyme ATP-citrate lyase (ACL; Aoshima, 2007; Hügler et al., 2007; Hügler and Sievert, 2011). Sequences coding for ACL [either the conventional type I, or the novel proposed type II (Hügler and Sievert, 2011; Thiel et al., 2012)] were not found in the metagenome. However, genes encoding the two-enzyme variant of citrate cleavage were present in the metagenome and could be assigned to two mat members. Two copies each of the genes encoding CCS and CCL were affiliated with a highly abundant mat member, Thermocrinis sp. OTU-4, which supports the presence of at least two different populations for this taxon in the mat as previously discussed (Thiel et al., 2016). Chemolithoautotrophic growth has been shown to occur for the closest relative, Thermocrinis ruber with H₂, thiosulfate and S⁰ as electron donors and oxygen as electron acceptor (Huber et al., 1998; Hügler et al., 2007). In addition to CCS and CCL, all other genes necessary for a functional reductive citric acid cycle in Thermocrinis sp. OTU-4 could be identified in the metagenome (see Figure S2). In addition to the two-step citrate cleavage mechanism, Thermocrinis sp. OTU-4 also accomplishes a two-step carboxylation of 2-oxoglutarate to isocitrate using the enzymes 2-oxoglutarate carboxylase and oxalosuccinate reductase instead of the conventional isocitrate dehydrogenase. Both of these two-step reactions, the two-step citrate cleavage, as well as the two-step carboxylation of 2-oxoglutarate, might be relics of an ancient variant of the reductive TCA cycle, that has been shown to occur in other members of the family Aquificaceae, e.g., Hydrogenobacter thermophilus or Aquifex aeolicus (Aoshima, 2007; Hügler et al., 2007).

Another set of genes potentially encoding the citrate-cleaving enzymes CCS and CCL were found on scaffold JGI24185J35167_1000715, which is part of a metagenomic bin identified as OTU-14, a member of the Planctomycetes. The genes were annotated as “succinyl-CoA synthetase alpha and beta subunit” and “citrate synthase” (JGI24185J35167_10007157-9). BLASTp searches against the nr database identified hypothetical proteins from Spirochaetes, Phycisphaerae and Plantomycetes as closest relatives with amino acid sequence identity values of 52–54%. A CDD search identified the genes as “CS_ACL-C_CCL superfamily” citrate synthase as well as “succinyl CoA synthetase alpha and beta subunits.” A phylogenetic analysis using the amino acid sequences of both subunits of ACL and the amino acid sequencing of the concatenated enzymes CCS and CCL is shown in Figure 3. The putative citrate cleaving genes of OTU-14 and related genes from other Plantomycetes cluster between the CCS-CCL of the Aquificae and Leptospirillum and ACL type II sequences. Thus, the genes for citrate-cleaving enzymes of these Planctomycetes bacteria give important insights into the evolution of the reductive TCA cycle and ACL. Phylogenetically, these enzymes are intermediates between the ancient variant that is still present within the Aquificaceae and the type II ACL (Figure 3). Interestingly, only OTU-14 shows separate genes for CCS and CCL, while the closest sequences from the database (e.g., Planctomycetes bacterium DG20) has a heterodimeric ACL; the gene for the second subunit of CCS (ccsB) and ccl are fused to form one gene, aclA (Figure S3). A possible explanation for this difference would be temperature. While OTU-14 is a thermophile (water temperature above the mat was 60°C; Thiel et al., 2016), the closely related Planctomycetes DG20 was detected in a metagenomics study of estuary sediments from North Carolina (Baker et al., 2015), and thus lives in a mesophilic environment. Another surprising feature of the bin assigned to Planctomycetes sp. OTU-14 is, that in addition to the key genes of the reductive TCA cycle, it also harbors genes encoding carbon monoxide dehydrogenase/acetyl CoA synthase and other enzymes of the reductive acetyl-CoA pathway. Thus, this organism has the genomic potential to use two different carbon fixation pathways for autotrophic growth (see below).

The reductive carboxylation of either succinyl-CoA or acetyl-CoA to 2-oxoglutarate or pyruvate, respectively, is carried out by the enzymes 2-oxoglutarate or pyruvate:ferredoxin oxidoreductases. Although necessary for autotrophic bacteria using the reductive TCA cycle for carbon fixation, these enzymes can also occur in heterotrophic or mixotrophic microbes. These enzymes perform anaplerotic CO₂ fixation reactions that may play an important role within the undermat portion of the community, as genes encoding several versions of these enzymes are found in the metagenome. Thus, several undermat organisms possibly require an ample supply of HCO3−/CO₂ for biosynthetic purposes as has been shown for Cab. thermophilum (Tank and Bryant, 2015a,b) and hypothesized for “Ca. T. aerophilum” (Liu et al., 2012).

The reductive acetyl-CoA or Wood-Ljungdahl (WL) pathway is the only known carbon fixation pathway used by Bacteria as well as by Archaea, consistent with the hypothesis that it is the most ancient pathway (e.g., Weiss et al., 2016). The key enzyme of the pathway is CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), which catalyzes the reduction of CO₂ to CO as well as the synthesis of acetyl-CoA from the methyl- and carbonyl moieties. The reduction of CO₂ to the methyl group is accomplished by enzymes that are also characteristic for this pathway (Figure S4; see Ragsdale and Pierce, 2008 for a review).

Four potentially bi-functional CODH/ACS subunit genes were identified in the metagenome (Table S8). The most abundant putative CODH/ACS gene (124 × coverage) belonged to the metagenomic bin for OTU-9, which has been associated with an Anaerolineae-like chemotrophic member of the Chloroflexi (Thiel et al., 2016). The protein shows highest similarity (55%) to CODH/ACS genes from Deltaproteobacteria and Clostridia (e.g., Desulfonatronospira sp., Ammonifex degensii) for which autotrophic growth has been observed, and enzyme activity measurements strongly suggest an active reductive acetyl-CoA pathway (Sorokin et al., 2008). Thus, the metagenomic data suggests that the Anaerolineae-like member of the Chloroflexi OTU-9 has the potential to grow autotrophically via the WL pathway. The next most abundant CODH/ACS sequences had read depths of 68× and 24×; these sequences belonged to two different potential members of the Planctomycetes, one representing OTU-14 (see below), and the other a less abundant member of the undermat layer that was not identified by 16S rRNA (Thiel et al., 2016). The least abundant CODH/ACS gene belonged to a member of the Thermodesulfobacteriaceae (OTU-26), a relative of Caldimicrobium thiodismutans, for which autotrophic growth by hydrogen oxidation or disproportionation of sulfur compounds has been demonstrated (Kojima et al., 2016). Two copies of CODH/ACS sequences were affiliated with Thermodesulfovibrio sp. OTU-8, a member of the phylum Nitrospirae. In addition to that, the genes for all other enzymes of the reductive acetyl-CoA pathway were found in the metagenomic bin representing OTU-8. Similarly, the genes for the reductive acetyl-CoA pathway were also present in the genome of the closely related species Thermodesulfovibrio yellowstonii (Table S8). Many acetotrophic, sulfate-reducing bacteria employ the WL pathway to cleave acetyl-CoA. Yet, the presence of a bifunctional CODH/ACS suggests, that Thermodesulfovibrio spp. are at least facultative autotrophs, although autotrophic growth has not been demonstrated for T. yellowstonii (Henry et al., 1994).

The most intriguing finding is the presence of genes encoding a bifunctional CODH/ACS together with genes for other enzymes of the reductive acetyl-CoA pathway in the Planctomycetes sp. OTU-14, in addition to genes of the reductive TCA cycle. We checked the metagenome of the closely related Planctomycetes sp. DG20 and also found the genes for the bifunctional CODH/ACS. Thus, these uncultured members of the Planctomycetes encode the genes for enzymes of two carbon fixation pathways, the reductive TCA cycle and the reductive acetyl-CoA pathway. So far, the presence of two different carbon fixation pathways in one bacterium has been only been described for the endosymbionts of vestimentiferan tubeworms living near hydrothermal vents and seeps (Markert et al., 2007; Thiel et al., 2012). This endosymbiotic gammaproteobacterium uses the reductive TCA cycle in addition to the CBB cycle for carbon fixation. As mentioned above, the reductive acetyl-CoA is most likely the most ancient autotrophic carbon fixation pathway (Sojo et al., 2016; Weiss et al., 2016). Braakman and Smith (2012) proposed that a hybrid reductive TCA/ reductive acetyl-CoA pathway is the basis of all extant carbon fixation pathways. A more detailed study of the genomes of these members of the Planctomycetes might shed more light on the early evolution of carbon fixation.

Lactate is present in the microbial mat in the late afternoon, and it has been suggested that it is exchanged between Synechococcus and Roseiflexus spp. (Kim et al., 2015). Lactate permease genes are present in the genome(s) of Roseiflexus spp.; their relative transcript levels become more abundant during the early evening hours, and lactate levels decline after this occurs. The ability to take up and utilize lactate, as indicated by the presence of genes encoding lactate permease and lactate dehydrogenase genes, is indicated for at least four and up to 7 of the 15 most abundant undermat members (Table S9). Additionally, genes encoding lactate permease are affiliated with some less abundant members, e.g., Thermodesulfobacteria sp. OTU-26, Bellilinea sp. OTU-31, Solibacter-like Acidobacteria OTU-36, and “Ca. E. thermophila” OTU-46.

Due to the high abundance and activity of cyanobacterial cells in the upper green layer of the mat, the measured and available lactate has mostly been attributed to Synechococcus spp. Types A and B' (Kim et al., 2015). Metagenomic data from the undermat layer, however, indicates additional putative producers of lactate as a fermentation end-product: e.g., Armatimonadetes (OP10) member OTU-3, fermentative Atribacteria (OP9) member OTU-7 and Aminicenantes (OP8) member OTU-13 (Table S9).

A saprophytic life style, based on cellular components made available by cell lysis of the major primary producer, Synechococcus spp., can be assumed for the majority of the heterotrophic mat community. Excreted proteases, secretion systems, and pathogenesis islands were not observed, so there was little evidence for an active predatory life style for any of the most abundant undermat members, neither in the metagenome nor in the reference genomes employed in this study (data not shown). The presence of viruses in the mat and their probable involvement in cell lysis processes can be assumed (e.g., on the basis of CRISPR systems in genomes), but this has not yet been analyzed and must be shown by future studies.

The presence of genes for transporters for oligopeptides and dipeptides, as well as generic amino acid and/or branched chain amino acid (BCAAs) transporters, were identified in the majority of the abundant mat members. This suggests that most organisms in the undermat have the potential to utilize peptides and amino acids as sources of carbon and nitrogen. Nearly complete degradation pathways for BCAAs were found for all four abundant Choroflexi members (Table S10). This indicates the ability to utilize BCAAs as putative carbon sources for these organisms, which is further supported by the ability of the chlorophototrophs to grow in CTM-medium that contains BCAAs (Tank and Bryant, 2015a,b; Tank et al., 2017). However, complete dependency on BCAAs, as demonstrated for Cab. thermophilum (Tank and Bryant, 2015a,b) and postulated for “Ca. Thermochlorobacter aerophilum,” was generally not found except for perhaps Chloroflexi member OTU-9, for which the partial genome so far only indicates biosynthesis of BCAAs from the precursor 2-isopropylmalate. Degradation of BCAAs is also likely to occur for Armatimonadetes member OTU-3 and EM3 member OTU-10 (Table S10).

Utilization of nucleic acids as a nutrient and energy source is suggested to occur in these mats. For example, at least 13 of the 15 most abundant members should be able to utilize ribose, as indicated by the presence of genes for both ribokinase and ribose-5-phosphate isomerase in metagenomic bins and/or reference genomes (Table S11). The presence of ABC-type ribose transporter genes in at least six of undermat members further supports the uptake and utilization of ribose from the environment by these organisms. Furthermore, 23 predicted extracellular nucleases were present in the metagenome, of which 10 were affiliated with 4 of the most abundant members (Table S11). Two highly abundant genes could not be affiliated with a specific taxon (JGI24185J35167_10498681, JGI24185J35167_10496441). Thus, up to eight highly abundant mat members may be able to degrade extracellular nucleic acids and utilization the resulting nucleotides, phosphate, and/or ribose.

Genes encoding putative nitrogenases (nifHDK) were found in four members of the mat: Synechococcus spp., Roseiflexus spp., “Ca. Chloranaerofilum corporosum” and Thermodesulfovibrio sp. (Table S12). However, active nitrogen fixation has only been shown for one of these organisms, Synechococcus spp. (Steunou et al., 2006, 2008), which are believed to provide the mat community with bio-available fixed nitrogen. It is not clear what the function of the nifHDK-like genes in Roseiflexus spp. might be, because most other accessory proteins required for maturation of nitrogenase are not present in any available Roseiflexus spp. genome (only nifB is present), and e.g., Roseiflexus castenholzii is unable to grow on dinitrogen as sole nitrogen source (D.A. Bryant, unpublished results). “Ca. Chloranaerofilum corporosum” contains the same set of genes as Roseiflexus spp. and is similarly not expected to grow on dinitrogen as sole nitrogen source. Frank et al. (2016) have reported that three Thermodesulfovibrio spp., T. yellowstonii, T. aggregans, and T. islandicus, genomes have the complete set of genes for nitrogenase, strongly suggesting that these organsims use dinitrogen as a nitrogen source; however, diazotrophic growth has not been reported for any of these isolates. The presence of additional nif genes (nifENXB) as well as a transcriptional repressor of the nif operon, suggests that this Thermodesulfovibrio sp. is also likely to be a nitrogen-fixing organism. This suggestion is strengthened by the observation that transcription of the nif genes in Thermodesulfovibrio sp. occurs and varies as a function of the diel cycle (unpublished observations). The presence of similar organisms in other hot spring microbial mats, especially in those dominated by non-nitrogen fixing cyanobacteria (Everroad et al., 2012), supports the possibility that they have the ability to fix nitrogen in situ and thus contribute to nitrogen input to the microbial mat community.

Despite the low concentration of ammonium in the hot spring water (values ranging from <3 to 35 μM have been measured over the years; Doemel and Brock, 1976; Papke et al., 2003; Ball et al., 2004), ammonium seems to be the preferred nitrogen source, along with organic nitrogen compounds such as amino acids (see above). Ammonium probably becomes available through ammonification and through the degradation of organic compounds, specifically of cyanobacterial proteins and nucleic acids. In accordance with this, numerous annotated genes for ammonium permease (amtB) are found in the undermat metagenome (95 partial genes in total). Putative ammonium permease genes were identified in 10 of the 15 most abundant mat members (Table S12). Genes for the key enzymes utilized in both ammonification (amino acid catabolism) and use of ammonium in (amino acid) anabolism, glutamine synthetase (GS), and glutamate:2-oxoglutarate amidotransferase (GOGAT; glutamate synthase) are present in high numbers in the metagenome.

Based on the metagenomic data, nitrate reduction appears to be uncommon in the microbial undermat layer. Although genes encoding nitrite reductase (nirB) and nitrate reductase (narB) in combination with genes for a nitrate ABC transporter (nrtABC), are present in 2 of the 15 most abundant mat members (Table S12), assimilatory nitrate reduction is only assumed to occur in Thermocrinis sp. OTU-4.

Based on the presence of nirA and narB genes in the genomes of isolates as well as the metagenome, assimilatory nitrate reduction has long been assumed to occur for the dominant Synechococcus spp. Moreover, nitrate has traditionally been the only nitrogen source used in cultivation experiments of uni-cyanobacterial mixed cultures of Synechococcus spp. Types A and B' obtained from the mats (Nowack et al., 2015; Olsen et al., 2015). However, in experiments with Synechococcus spp. isolates on agar plates aiming to produce axenic cultures, nitrate did not support growth under constant light and oxygen conditions nor did it enhance growth under conditions that favor nitrogen fixation. Thus, nitrate may not be assimilated by Synechococcus spp. under laboratory conditions (M. Tank, N. Soulier, D.A. Bryant, unpublished). These observations might be explained by micro-niche conditions favoring nitrogen fixation or a nitrate-reducing “helper” bacterium present in these mixed cultures. In contrast, assimilatory nitrate reduction can be assumed for Thermocrinis sp. OTU-4, for which nirA and narB genes showing closest similarity to Thermocrinis ruber were identified in the metagenome. Assimilatory nitrate reduction for this organism is further supported by chemolithoautotrophic growth of its closest relative, Thermocrinis ruber, in media containing nitrate as sole nitrogen source (Härtig et al., 2014). Similarly, the absence of narGHI genes in the genome of the type strain, as well as the metagenome sequences, correlate with the observation that nitrate is not an electron acceptor that can support the growth of Thermocrinis ruber strain OC1/4 (Huber et al., 1998).

Nitrification does not seem to occur in any of the abundant microbial undermat members. Ammonia monooxygenase (amoA), the key enzyme responsible for the ammonium oxidation step in nitrification, was not identified in the metagenome. Similarly, dentrification genes were not detected in the undermat metagenome. Therefore, denitrification does not appear to occur in any abundant mat organism. This is consistent with the low concentration of nitrate in the spring water (<0.1 mg/L; ~ <1.6 μM; see Thiel et al., 2016).

Dissimilatory sulfate reduction is likely to occur in at least three members of the mat community. Previous studies revealed four distinct clusters of dsrAB sequences by a PCR-based cloning survey (Dillon et al., 2007). Two complete sets of sulfate reduction genes (sat, aprBA, dsrAB, dsrTMKJOP, qmoABC) were present in the metagenomic dataset. The more abundant set of genes (~127 × coverage for the dsrAB genes) belonged to Thermodesulfovibrio sp. OTU-8, which was associated with measured sulfate reduction activity in previous studies (“clade-1,” Dillon et al., 2007). A second set of less abundant dsrAB genes (63 × coverage) were the same as unidentified drsAB clone sequences that were previously retrieved from the mats (“clade-2,” Dillon et al., 2007). These sequences were affiliated with a metagenomic bin (bin-24, Thiel et al., 2016), with an uncertain phylogenetic affiliation within the Bacteroidetes-Chlorobi group. Due to the lack of a 16S rRNA gene sequence in the metagenome bin, phylogenetic affiliation was based on phylogenetic marker genes, most of which were affiliated with either the Bacteroidetes or the Chlorobi group. Phylogenetic analyses of 17 concatenated phylogenetic marker genes placed the unidentified novel uncultured organism in a cluster with Bacteroidetes and both heterotrophic as well as phototrophic Chlorobi representatives (Thiel et al., in preparation). Furthermore, the presence of a 1023-bp partial 23S rRNA gene sequence with closest relationship to Bacteroidetes-Chlorobi group members strengthens the affiliation. “Clade-3” sequences were also found in the metagenome at very low abundance (6–8×) (Table S13). A previous phylogenetic analysis by Müller et al. (2015), as well as this study, places these sequences into an unclassified cluster within a group of Firmicutes-like dsrAB sequences (dsrAB ARB database available at http://www.microbial-ecology.net/download). “Clade-4” sequences reported by Dillon et al. (2007) were not found in the undermat metagenome.

In addition to the previously known dsrAB phylotypes, sequences encoding dissimilatory sulfite reductase from a Thermodesulfobacteria member, putatively OTU-26, were recovered from the metagenome. Three low abundance partial dsrAB sequences (16–26 × coverage) showed closest similarity to Caldimicrobium thiodismutans with 87–93% amino acid identity and 96–98% similarity (Table S13). C. thiodismutans has recently been isolated from a hot spring in Japan and displays sulfur disproportionating activity (Kojima et al., 2016). A similar sulfur-disproportionating metabolism is hypothesized for the Mushroom Spring mat member (Figure 6).

Assimilatory sulfate reduction does not seem to be prevalent in the undermat metagenome. This indicates that the majority of mat members rely on reduced sulfur sources, as has been shown for Chloracidobacterium thermophilum (Tank and Bryant, 2015a,b). Genes for ferredoxin-dependent sulfite reductase (sir) were only identified for 3 of the 15 most abundant undermat members (OTUs-5, 11, and 14; Table S14). Additional low-coverage gene sequences were affiliated with less abundant members, e.g., Armatimonadetes member OTU-18, Acidobacterium member OTU-36 (bin-20, Thiel et al., 2016) as well as two unidentified Planctomycetes members (bins-23 and 29; Thiel et al., 2016), while some remained unidentified. Roseiflexus spp. strains do not contain complete reduction pathways and presumably cannot perform assimilatory sulfate reduction (Bryant et al., 2012). Among members of the phylum Chloroflexi, genes for sulfite reductase were only identified in Chloroflexus sp. OTU-11.

Although the concentration of sulfide present in the hot spring source water is at the limit of detection (0.003 mg/L = ~0.1 μM, Ball et al., 2004), sulfide accumulates in the mats as a result of biological sulfate reduction (Dillon et al., 2007). Little is known about the oxidative part of the sulfur cycle in the mats. Sulfide can be used as an electron donor by some anoxygenic phototrophic bacteria, but it is also toxic to many organisms, and detoxification mechanisms have evolved. Chloroflexi are known to contain sulfide:quinone oxidoreductase (sqr) of the type II family (Bryant et al., 2012) and have the ability to oxidize sulfide to polysulfides (Madigan and Brock, 1975), which sometimes even support autotrophic growth (Madigan et al., 1974; Madigan and Brock, 1977; Keppen et al., 2000; Gich et al., 2003; Klappenbach and Pierson, 2004; Thiel et al., 2014). SQR genes were also found in the metagenome and were affiliated with chlorophototrophic and chemotrophic members of the Chloroflexi, Thermotogae, Armatimonadetes, and Nitrospirae (Table S14).

When sulfide is oxidized by Chloroflexus and Oscillochloris spp., globules of elemental sulfur are deposited outside the cells (Madigan and Brock, 1975; Keppen et al., 2000). Elemental sulfur was shown to be a sulfur source for the growth of Chloracidobacterium thermophilum (Tank and Bryant, 2015a,b). It might also be a sulfur source for other mat members that lack the genes for assimilatory sulfate reduction (e.g., OTU-7, see Table S14). Furthermore, elemental sulfur is known to be an electron donor for Thermocrinis ruber (Huber et al., 1998), which is the closest type-strain relative to the microbial mat member Thermocrinis sp. OTU-4 (Thiel et al., 2016), for which a complete set of sox genes was identified in the metagenome. It is thus expected that this mat member can oxidize elemental sulfur to sulfate, which would close the sulfur cycle and provide sulfate that can act as electron acceptor for biological sulfate-reduction by Thermodesulfovibrio sp. OTU-8 and any other putative sulfate-reducers in the mat (Figure 6).

There is evidence that Synechococcus spp. in the mats can also oxidize sulfide. In situ studies showed that sulfide addition strongly stimulated ¹³C-bicarbonate incorporation into Synechococcus spp. lipid biomarkers (van der Meer et al., 2005). Some cyanobacteria are known to possess sulfide quinone reductase, but this gene is not present in the genomes of Synechococcus spp. that occur in the mats. Cyanobacteria that do not produce polysulfides from sulfide oxidation produce thiosulfate as the product (De Wit and van Gemerden, 1987; Rabenstein et al., 1995); however, the mechanism of thiosulfate production by such organisms is currently unknown.

Microbes can produce molecular hydrogen (H₂) via fermentation and dinitrogen fixation (Nielsen et al., 2015; Revsbech et al., 2016). Hydrogen accumulation has been detected in these mats following a diel pattern. In the evening, H₂ accumulated rapidly after the onset of darkness, reaching peak values of up to 30 μmol H₂ L⁻¹ at about 1-mm depth below the mat surface, and slowly decreasing to about 11 μmol H₂ L⁻¹ just before sunrise (Revsbech et al., 2016). Another pulse of H₂ production, reaching a peak concentration of 46 μmol H₂ L⁻¹, was found in the early morning under dim light conditions that were too low to cause the accumulation of O₂ in the mat, indicating that the nitrogenase activity of the dominant cyanobacteria Synechococcus spp. Types A and B' is of greatest importance for this morning peak. During the night, fermentation has been proposed to contribute to the formation of H₂ (Revsbech et al., 2016).

In this study, we identified several microbial community members that could possibly contribute to fermentative H₂ evolution. The anaerobic fermenter, Pseudothermotoga sp. OTU-2, is probably the most abundant hydrogen-producing mat member. The partial genome of this bacterium contains genes encoding a [FeFe]-hydrogenase (H₂ase; Table 3, Table S15) enabling fermentative H₂ production, as has been shown in the next closest relative (Pseudo)thermotoga hypogea strain NBRC 106472 (Fardeau et al., 1997; Yang et al., 2015).

The microbial mat also contains other fermentative bacteria: e.g., an uncultured member of the Atribacteria (Candidate phylum OP9), OTU-7 and a member (OTU-13) of the phylum Aminicenantes (Candidate phylum OP8; Table 3, Table S15). Analysis of the OTU-7 metagenomic bin indicates an anaerobic, fermentative lifestyle for this mat member, similar to that deduced from single-cell genomics previously obtained for members of the phylum Atribacteria that occur in hot springs in California and Nevada, USA (Dodsworth et al., 2013). The presence of a gene for a monomeric, periplasmic [FeFe]-H₂ase in the partial genome further suggests the ability of this organism to produce H₂ fermentatively. For OTU-13 a H₂-evolving group 4 H₂ase (formate hydrogenlyase complex) and a bidirectional [NiFe]-H₂ase III could be responsible for fermentative H₂ evolution, as shown in Thiocapsa roseopersicina (Rákhely et al., 2007). Thermodesulfovibrio sp. OTU-8 may grow on fumarate and acetate, as observed for Thermodesulfovibrio yellowstonii (Henry et al., 1994), and may subsequently produce H₂ via a type-IV hydrogenase (fumarate hydrogenlyase complex; Table S15).

Several members of the Chloroflexi (OTUs-1, 11, 15), Thermocrinis sp. OTU-4, but also the sulfate reducing Thermodesulfovibrio sp. OTU-8, presumably the Planctomycetes member OTU-14 as well as a less abundant member of the Thermodesulfobacteria, OTU-26, were identified as putative H₂ consumers due to the presence of genes encoding [NiFe]-H₂ases (Table S15). Because it is the most abundant member of the undermat, Roseiflexus sp. OTU-1 is expected to be the major consumer of H₂. The genome of Roseiflexus sp. strain RS-1 contains a type-I uptake [NiFe]-H₂ase, encoded by genes hydAB (hyaAB), as well as a complete suite of hyp genes potentially involved in the biosynthesis and maturation of this H₂ase. Accordingly, this organism has been suggested to utilize H₂ (van der Meer et al., 2010). All of these genes were also present in the undermat metagenome. A high diversity (between 1 and 14 sequences) of different (partial) H₂ases with 66–100% amino acid similarity to the Roseiflexus sp. RS-1 reflects the high microdiversity for this taxon (Ferris and Ward, 1997; Thiel et al., 2016) and might indicate the presence of putative ecotypes with different H₂ases gene sequences. [NiFe]-H₂ase genes (hydABCD, RoseRS_2319–RoseRS_2322) were shown to be expressed, and the relative transcript abundances for these genes exhibited a nocturnal expression pattern during the evening and night hours in the upper green layer of the microbial mat. This pattern led to the suggestion that H₂ is an important source of electrons for hypothesized light-driven CO₂ fixation by filamentous anoxygenic phototrophs (FAPs) in the morning, when light is available but while the mat remains anoxic (van der Meer et al., 2005; Klatt et al., 2013b).

Thermocrinis sp. OTU-4 is likely to be the second most abundant H₂ consumer in the mat. Although no H₂ase genes affiliated with this organism were detected in the assembled metagenome, their presence was confirmed in the unbinned portion of the sequences (Table S15). The closest relative of the organism(s) represented in this bin, Thermocrinis ruber, which was isolated from Octopus Spring from temperatures above 80°C, grows on H₂, elemental sulfur, and thiosulfate (Huber et al., 1998; Jahnke et al., 2001; Härtig et al., 2014) and a similar metabolism can be assumed for the undermat member at 60°C based on the corresponding metagenomic bin (Table 3). The presence of affiliated H₂ase genes only in the unassembled part of the metagenome, as well as the low number of scaffolds in the metagenome bin are indicative of assembly difficulties probably arising from the presence of closely related populations with highly similar but non-identical genome sequences. Another mat member, Thermodesulfovibrio sp. OTU-8, is assumed to grow on H₂ plus acetate, as shown for its closest relative, T. yellowstonii (Henry et al., 1994). This suggestion is supported by the presence of genes encoding a [FeFe]-H₂ase in the genome of the type strain and the metagenomic bin. However, genes for additional group-4 H₂ases (formate hydrogen lyase) are present in both the isolate genome (Lim et al., 2010; Bhatnagar et al., 2015) and metagenomic bin-8 (Table S15). These genes indicate an ability to produce H₂, e.g., when growing on formate and acetate as described by Henry et al. (1994). A less abundant putatively H₂-utilizing member of the mat is the Thermodesulfobacteria member OTU-26. Genes encoding a [NiFe]-H₂ase and an [NiFe]-H₂ase maturation protease, closely related to sequences from Caldimicrobium thiodismutans have been identified in the undermat metagenome (scaffold JGI24185J35167_1006546). Additionally, H₂ase maturation genes indicating the presence of [NiFe]-H₂ases with unknown function and identity were identified in the novel chlorophototrophic FAP “Ca. R. gracile” OTU-6 and the chemotrophic Chloroflexi member OTU-9, as well as the unidentified Planctomycetes member OTU-14 (Table S15).