Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability

Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability Brian Hedlund, Toshio Alvaredo, Trevor Murphy, Amanda Blocker, and 23 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6739887/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The phylum Atribacterota is ubiquitous in anoxic environments where it plays important roles in syntrophic carbon and hydrogen metabolism; however, only two species have been isolated. Here, we report the isolation of two additional species representing a new family, Caldatribacterium saccharofermentans from a hot spring and Caldatribacterium inferamans from a deep fractured-rock aquifer. Both were co-enriched on carbohydrates with sulfate-reducing bacteria (SRB). Despite unsuccessful attempts to isolate them on the defined enrichment medium, we finally isolated both species by adding yeast extract to the medium. We show that folate was the key factor provided by the SRBs and the yeast extract and that folate provided in vitamin solutions was inadvertently removed by filter sterilization according to standard media preparation protocols. We further confirm the absence of folate biosynthesis pathways across the phylum and suggest that folate precipitation during media preparation limits cultivation of Atribacterota in toto. The two Caldatribacterium species share unusual features with other Atribacterota , including three lipid membrane-like layers (LMLs), with the inner LML that appears to surround a nucleoid, and a high percentage of transmembrane proteins. This study expands the culturability of Atribacterota and identifies conserved features, including sugar fermentation, unusual cell ultrastructure, and vitamin dependencies necessary for their cultivation. Biological sciences/Microbiology/Environmental microbiology Biological sciences/Microbiology/Bacteria/Bacterial physiology Geothermal subsurface Atribacterota Atribacteria OP9 sulfate-reducing bacteria folate Wolin’s vitamins Wolfe’s vitamins NanoSIMS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Culturability is highly uneven across the prokaryotic tree of life. Despite nearly 150 years of cultivation efforts 1 , most phyla have few or no representatives in pure culture 2 . One such poorly cultivated phylum is the Atribacterota , formerly OP9/JS1 or “Atribacteria”. Atribacterota 16S rRNA gene sequences were first obtained from Obsidian Pool, Yellowstone National Park, USA 3 and are now known to be widely distributed in anoxic environments 4,5,6,7,8 . The phylum is divided into two major classes that are physiologically and ecologically distinct. Members of the class Phoenicibacteriia (JS1) are often abundant in hydrocarbon-containing terrestrial and marine sediments, aquifers, and oil reservoirs, where they may remain active over thousands to millions of years of sedimentation 9,10,11 . One study 9 detected abundant transcripts in deep marine sediments along with corresponding enzyme activities and metabolites and suggested sugar, protein, and allantoin catabolism by Phoenicibacteriia. Another 12 suggested that some Phoenicibacteriia were suggested to ferment short-chain alkanes, likely requiring hydrogenotrophic methanogens as a H 2 sink. Jiao et al., (2024) 8 provided experimental evidence of long-chain n-alkane fermentation in an enrichment culture containing Phoenicimicrobium oleiphilum HX-OS.bin.34 Ts based on the high expression of key genes for alkane fermentation, including genes for fumarate-adding enzymes, hydrogenases, the reverse glycine pathway (RGP), and acetogenesis. These authors 8 also suggested alkane fermentation is widespread in Phoenicibacteriia based on the broad distribution of these genes. However, to date, no isolates of Phoenicibacteriia have been described. The other major class, Atribacteria (OP9), is more common in terrestrial anoxic environments. Early single-cell genomic and metagenomic studies probed the metabolic capacity of Atribacteria in hot springs and bioreactors 13,14,15 . In one of these studies, a member of the Atribacteria , Candidatus Caldatribacterium saccharofermentans, was enriched on lignocellulose substrates in Great Boiling Spring (GBS), NV, USA 16 and subsequently predicted to be a sugar fermenter based on the presence of genes encoding glycoside hydrolases, hydrogenases, acetogenesis, alcohol dehydrogenase, and a Rhodobacter nitrogen fixation (RNF) complex 13,15 . Other Atribacteria were also predicted to be sugar 9 or propionate fermenters 15 , and the presence of some genes encoding the Wood-Ljungdahl pathway (WLP) in some Atribacteria suggested the possibility for syntrophic acetate oxidation or carbon fixation 9,17,18 . Yet, a more recent analysis 8 showed that the carbonyl branch of the WLP is incomplete in Atribacteria genomes, and most also lack the carbon monoxide dehydrogenase/acetyl-CoA synthetase complex. Instead, they noted the near-universal presence of the RGP in Atribacterota , indicating that the RGP, rather than WLP, is a conserved core of Atribacterota metabolism and may enable CO 2 or formate assimilation. The first Atribacteria pure culture, Atribacter laminatus RT761 T , was isolated from a deep, methane-bearing aquifer 19 . A. laminatus is a moderately thermophilic sugar fermenter that requires yeast extract for growth and produces H 2 , acetate, carbon dioxide, and ethanol. Its slow doubling time (~5 days) and low growth yield were enhanced in co-culture with a hydrogenotrophic methanogen, suggesting a semi-syntrophic interaction involving hydrogen scavenging. A. laminatus has an unusual cell ultrastructure, described as three lipid membrane-like layers (LMLs), the innermost of which contains the nucleoid and most of the RNA 19 . These were interpreted to be (from outside to in) an outer membrane (outer LML), a cytoplasmic membrane (middle LML), and a separate intracytoplasmic membrane (inner LML). However, alternative interpretations involving a separated cytoplasmic membrane and an outer membrane with a closely associated S-layer or extensive cytoplasmic membrane invaginations have also been suggested 19,20 . More recently, Jiao et al., (2024) 8 and Kawamoto et al., (2024) 21 isolated two additional members of Atribacteria , described as Thermatribacter velox B11 T and Atrimonas thermophila M15, from a terrestrial oil well and a terrestrial gas reservoir. However, these two isolates belong to the same species and the name Thermatribacter velox has priority over Atrimonas thermophila and is used here for both strains, along with the family name Thermatribacteriaceae . Both strains ferment sugars and require yeast extract, producing H 2 and acetate as major products. They also have three LMLs, similar to A. laminatus . Together, the three isolates comprise two of the three known families of Atribacteria , with Ca. Caldatribacterium representing the third, yet-uncultivated family. Here we describe enrichment and isolation of two species of Caldatribacterium , Caldiatribacterium saccharofermentans from a hot spring (GBS) and Caldiatribacterium inferamans from a hot, fractured-rock aquifer in the discharge zone of the Death Valley Regional Flow System. We show that both Caldatribacterium species require folate and propose that folate precipitation during standard media preparation protocols is a key problem that limits isolation of all Atribacterota . We also show that both Caldatribacterium species contain three LMLs similar to A. laminatus and T. velox , but note that ribosomes are partially or fully separated from the nucleoid by the inner LML. The conserved ultrastructure and high percentage of transmembrane proteins support a previously proposed “membrane-centric” biology 19 and an important role for the unusual cell ultrastructure throughout the class. Generated using WordToHTML.net - Online Word to HTML Converter Results Enrichment of two Caldatribacterium strains with distinct SRBs Based on previous in situ enrichments of Ca . C. saccharofermentans on lignocellulose substrates 13,16 , fresh in situ enrichments containing 1 gram of ammonia fiber expansion (AFEX)-treated corn stover were incubated for five months in the outflow channel of GBS ( Supplementary Fig. 1 ). Because Ca . C. saccharofermentans was previously suggested to catabolize hemicellulose based on the presence of a gene encoding a member of GH12 family of glycoside hydrolases 13 , an anaerobic medium containing xyloglucan as the sole carbon source was inoculated with material from the in situ enrichment, transported to the lab, and incubated at 73 °C. Caldatribacterium 16S rRNA gene sequences were detected by PCR after two transfers (1/1000 vol vol -1 ), and the presence of Caldatribacterium was stable for subsequent transfers, performed every two weeks thereafter. 16S rRNA gene amplicon sequencing of the xyloglucan cultures after one and two years of lab cultivation showed that Caldatribacterium comprised 8-11% of total reads (2015 XG and 2016 XG, Fig. 1A ), and that the community was stable, consisting mainly of fermenters in the phyla Dictyoglomota (two species of Dictyoglomus ) and Thermotogota (two species of Fervidobacterium and one of Pseudothermotoga ), and the sulfate-reducing bacterium (SRB) Thermodesulfobacterium . These taxa were previously observed in similar in situ enrichments 16 . Shotgun metagenomics and binning into metagenome-assembled genomes (MAGs) after two years of maintenance yielded similar results ( Supplementary Fig. 2 ). Fluorescence in situ hybridization (FISH) using an oligonucleotide specific for the 16S rRNA of Caldatribacterium also confirmed the presence of Caldatribacterium cells at ~3.5% abundance in xyloglucan enrichments ( Supplementary Fig. 2 ). The xyloglucan culture was used to inoculate media containing a variety of mono- or disaccharides as sole carbon sources to further enrich Ca . C. saccharofermentans. After two transfers, the absolute (1.1 ×10 8 - 5.5 × 10 8 16S rRNA gene copies mL -1 ) and relative (8-53% of compared to total 16S rRNA gene copies mL -1 ) abundance of Caldatribacterium in these cultures with individual sugars was higher than in xyloglucan cultures (2.5%, 2.5 × 10 5 copies mL -1 ) ( Supplementary Fig. 3 ). Because the relative enrichment of Caldatribacterium was highest with fucose as a sole carbon source and because fucose catabolism had been previously predicted from the Ca . C. saccharofermentans MAG 13 , these cultures were used for multiple rounds of dilution-to-extinction. In each dilution series, bottles receiving ~18 cells or more showed growth after 3-week incubations, while those with ~14 cells or less did not ( Supplementary Fig. 4A ). After multiple rounds of dilution-to-extinction, the abundance of Caldatribacterium in the highest dilution showing growth was near or equal to that of total Bacteria and Archaea as assessed by qPCR ( Supplementary Fig. 4A ). The dominance of Caldatribacterium in these dilution cultures, combined with our inability to obtain pure cultures by serial dilution, suggested a metabolic dependency. FISH confirmed enrichment of Caldatribacterium to ~95% of total cells, but also revealed cells negative for the Caldatribacterium -specific probe ( Supplementary Fig. 4B ). 16S rRNA gene amplicon sequencing and shotgun metagenomics of the fucose culture showed that it was a co-culture of Caldatribacterium and a Thermodesulfobacterium species; >99.9% of 16S rRNA gene amplicon reads were either Caldatribacterium or Thermodesulfobacterium ( Fig. 1A ), and essentially all metagenome contigs were binned as either Caldatribacterium or Thermodesulfobacterium ( Fig. 1B ). Additional dilution-to-extinction did not remove Thermodesulfobacterium from the culture, underscoring that the SRB was necessary for growth of Caldatribacterium enriched from GBS. A distinct enrichment containing Caldatribacterium was obtained from a borehole, Inyo-BLM 1, which intersects the Lower Carbonate Aquifer near Death Valley National Park, CA 22,23,24 , using an approach inspired by Imachi et al ., 2011 25 . Sterile polyurethane foam plugs were suspended at regular intervals in an uncased section (depth of ~751 m) of Inyo-BLM 1 for three months ( Supplementary Fig. 5 ). The plugs were then removed from the borehole and transferred to a bottle containing reduced artificial groundwater medium (AGM) 26 , transported to the lab, and stored at 4 °C. In the lab, liquid from the plug/AGM sample was inoculated into serum bottles containing AGM amended with xylitol (10 mM) and yeast extract (0.01% mass vol -1 ), based on previous observations of sugar alcohol catabolism by some subsurface microorganisms 26 . Bottles were incubated at 60 °C and transferred (1/50 vol vol -1 ) to fresh medium every week. Although Caldatribacterium was not detected in situ, 16S rRNA gene amplicon sequencing showed that the plug enriched for Caldatribacterium (19.1% of reads), and the xylitol enrichment led to high relative abundance of Caldatribacterium after multiple transfers (97.6% of reads) ( Fig. 1C ). The most abundant non- Caldatribacterium 16S rRNA gene amplicon in the xylitol enrichment was assigned to the SRB Thermodesulfovibrio (1.1% of reads), and Thermodesulfovibrio remained in low abundance after multiple rounds of dilution, suggesting the Inyo-BLM 1 Caldatribacterium strain was also dependent on a SRB, albeit one belonging to a distinct phylum ( Nitrospirota ) from the SRB that was co-enriched with Caldatribacterium from GBS (genus Thermodesulfobacterium and phylum Desulfobacterota ). A shotgun metagenome of the xylitol enrichment culture confirmed the high abundance of a Caldatribacterium strain and Thermodesulfovibrio ( Fig. 1D ). Attempts to isolate either Caldatribacterium strain on solid media with fucose (GBS enrichment) or xylitol (Inyo-BLM 1 enrichment) as sole carbon sources were unsuccessful, yet pure cultures of the respective SRBs were readily obtained. The Thermodesulfobacterium strain in the GBS fucose enrichment was isolated on GBS salts medium plates solidified with 1% mass vol -1 Gelrite and 0.4% mass vol -1 magnesium chloride hexahydrate containing 1 mM each of sodium bicarbonate, sodium thiosulfate, and sodium acetate, with 0.66 atm H 2 in the headspace ( Supplementary Note 1 ). The Thermodesulfovibrio strain in the subsurface xylitol enrichments was isolated under similar conditions but with the addition of 5 mM lactate. For the Thermodesulfobacterium strain, autotrophic growth with H 2 as the electron donor was stimulated by acetate, and the isolate could also grow using lactate ( Supplementary Data 1 ). It represents a new species, with 75.8-80.1% average nucleotide identity (ANI) to other members of the genus Thermodesulfobacterium , herein proposed as Thermodesulfobacterium auxiliatoris for its ability to help Caldatribacterium grow (see below). The MAG of the Thermodesulfovibrio strain from the Inyo-BLM 1 xylitol enrichment had a genomic ANI of 98.55% with Thermodesulfovibrio yellowstonii DSM 11347 T and the 16S rRNA gene of the isolate was identical to that strain; it could grow using lactate or a combination of H 2 and acetate, similar to other members of this species 27 ( Supplementary Note 2 ). Neither of these SRBs were able to grow on their own under the conditions of the Caldatribacterium enrichment cultures they were isolated from (i.e., fucose or xylitol as sole carbon sources) or with other sugars as sole carbon sources. Caldatribacterium in GBS enrichments assimilates sugars and amino acids In the absence of pure cultures, we used nanometer-scale secondary-ion mass spectrometry (nanoSIMS) coupled with FISH targeting Caldatribacterium to assess incorporation of 13 C-labeled substrates in the GBS xyloglucan and fucose dilution-to-extinction cultures ( Fig. 2 ). In the xyloglucan enrichments, most cells assimilated 13 C atoms from labeled glucose, xylose, ribose, and amino acids, yet only 13 C incorporation from xylose was higher in Caldatribacterium cells than other cells, suggesting direct uptake ( Fig. 2A ). In contrast, although both Caldatribacterium cells and other cells were labeled after incubation with 13 C-bicarbonate, formate, or acetate, labeling was low ( 13 C atom percent enrichment ( 13 C APE) <0.5%). In fucose enrichments, uptake of glucose, xylose, and ribose was higher in Caldatribacterium cells compared to Thermodesulfobacterium cells, again suggesting direct uptake, yet acetate and amino acid assimilation was higher in Thermodesulfobacterium cells ( Fig. 2B ). Little or no uptake of 13 C-bicarbonate, formate, or acetate was detected in Caldatribacterium cells (APE <0.5%). These taxon-specific carbon uptake patterns suggest distinct roles for Caldatribacterium and Thermodesulfobacterium in the GBS enrichment cultures and suggest potential metabolic interactions between Caldatribacterium and the co-enriched SRBs. Isolation of Caldatribacterium strains and semi-syntrophic growth with SRBs Parallel co-enrichment of the two different Caldatribacterium cultures with unrelated hydrogenotrophic SRBs initially suggested a semi-syntrophic interaction based on interspecies electron transfer via H 2 or other fermentation products, as observed between A. laminatus and Methanothermobacter thermoautotrophicus 19 . Consortial fermentation of sugars was supported by the nanoSIMS results ( Figure 2 ). For example, the rapid uptake of acetate but not sugars by Thermodesulfobacterium suggested that Caldatribacterium and Thermodesulfobacterium work together to mineralize sugars, with acetate as a waste product of Caldatribacterium sugar fermentation, as previously predicted 13,15 , and confirmed for both A. laminatus 19 and T. velox 8,21 . However, an alternative hypothesis is that Caldatribacterium strains rely on SRBs for resources during growth on sugars as sole carbon sources. To address this alternative hypothesis and try to obtain pure cultures, dilution-to-extinction was repeated with the GBS fucose cultures, and the lowest dilution that did not exhibit visible growth was used to inoculate media in quintuplicate with fucose alone or with fucose plus yeast extract and casamino acids (0.1% mass vol -1 each). While cultures with fucose alone showed no visible growth after two weeks, all replicate cultures containing yeast extract and casamino acids (0.1% mass vol -1 each) showed robust growth (up to 10 8 cells mL -1 ) after four days of incubation. Because it appeared that complex organics, in addition to a carbohydrate fermentation substrate, enabled growth of Caldatribacterium on its own, solid media with yeast extract and casamino acids (0.1% mass vol -1 each) in addition to fucose (for GBS enrichments) or fucose and xylitol (for Inyo-BLM 1 enrichments) was used to isolate Caldatribacterium strains from the dilution-to-extinction cultures. Under these conditions, colonies were obtained and streak-purified multiple times to obtain axenic cultures. Once obtained, both Caldatribacterium pure cultures could grow in liquid medium supplemented with yeast extract, casamino acids, and either fucose or xylitol. Culture purity was confirmed by FISH ( Supplementary Fig. 6 ) in combination with total cell counts, genome sequencing (see below), and viable plate counts for both strains. Subsequent experiments determined that yeast extract alone, and not casamino acids, was necessary and sufficient for growth of the Caldatribacterium pure cultures in the presence of fucose or xylitol. The isolated Caldatribacterium strains, herein proposed as Caldatribacterium saccharofermentans GBS T and Caldatribacterium inferamans SIUC1 T under the rules of the International Code of Nomenclature of Prokaryotes (protologues below), were used to confirm that various SRBs could enable growth on defined media and to further probe the nature of semi-syntrophic growth. In the absence of yeast extract, growth of both Caldatribacterium strains with fucose was supported by the addition of pure cultures of the Thermodesulfobacterium auxiliatoris and Thermodesulfovibrio yellowstonii strains isolated in this study, as well as Thermodesulfobacterium commune DSM 2178 T and Thermodesulfobacterium hveragerdense DSM 12571 T ( Fig. 3A ); under these conditions, Caldatribacterium constituted the vast majority of cells as determined by FISH ( Supplementary Fig. 7 ). Characterization of the isolated Caldatribacterium strains showed that they could ferment a variety of sugars, with C. inferamans SIUC1 T having a somewhat wider range of fermentation substrates ( Supplementary Data 2) . However, C. saccharofermentans could not grow with xylose as a sole carbon and energy source even though xylose uptake was observed by nanoSIMS. Both strains grew best at circumneutral pH and had similar doubling times (7.6-8.3 hr), with C. saccharofermentans having a slightly higher optimal growth temperature (70 °C) than C. inferamans (60-70 °C), consistent with the environments from which they were isolated ( Supplementary Data 2, Supplementary Fig. 8) . To determine whether Caldatribacterium growth could be stimulated by the removal of fermentation products by the SRBs, we added potential fermentation products to C. saccharofermentans GBS T pure cultures in fucose- and yeast-extract-amended media ( Supplementary Fig. 9 ). Acetate and formate (1 mM) had no significant effect on growth and lactate up to 10 mM had only a mild inhibitory effect, with higher concentrations yielding a longer lag phase and slower growth. H 2 at up to 0.08 atm in the headspace slowed growth, while higher levels were inhibitory. Since T. velox M15 was not stimulated by co-cultivation with a hydrogenotrophic methanogen 21 , and Caldatribacterium inferamans SIUC1 T ( Fig. 3 ) and T. velox B11 T 8 grow to high cell densities without a hydrogenotrophic partner, we infer they are also not strongly inhibited by their fermentation products. Thus, the Caldatribacterium and Thermatribacter strains do not depend on syntrophic depletion of a fermentation product, in contrast to the much stronger stimulation of A. laminatus growth following removal of H 2 from the headspace or in co-culture with a hydrogenotrophic methanogen 19 . Distinct hydrogenase complexes may alter H 2 tolerance in different Atribacteria To probe the mechanism behind the differences in H 2 sensitivity between the Caldatribacterium strains and T. velox B11 T versus A. laminatus , and to uncover other genomic features, we sequenced the genomes of both Caldatribacterium strains with a combination of Illumina and Oxford Nanopore technologies and then assembled the data into single circular contigs that were 2,255,665 bp for C. saccharofermentans GBS T and 1,967,957 bp for C. inferamans SIUC1 T . A maximum-likelihood phylogeny based on the bac120 marker protein set 28 confirmed that the two new isolates form a monophyletic clade with the previously published Ca . C. saccharofermentans MAG and the Ca . C. californiense SAG 13 , along with MAGs from the Shengli Oilfield in China and hot springs in Tengchong, China, and Tibet (taxa 1-8) ( Fig. 4A ; Supplementary Data 3 ). Based on relative evolutionary divergence and average amino acid identity, this clade is a distinct family of Atribacterota , herein proposed as Caldatribacteriaceae ( Supplementary Note 3 ). The Caldatribacterium isolate genomes shared 92% ANI and 85% AAI ( Fig. 4 ), confirming they represent distinct species of a single genus. The C. saccharofermentans GBS T and C. inferamans SIUC1 T genomes each encoded at least two putative [FeFe] hydrogenases belonging to groups A1 (HydABC) and A3 (HndABCD) and an [FeFe] group C1 hydrogenase ( Supplementary Data 4 ), as previously noted for the Ca. C. saccharofermentans MAG and Ca. C. californiense SAG 8,13,15 . Group A3 hydrogenases are electron-bifurcating/confurcating enzymes that enable fermentative H 2 production with simultaneous oxidation of ferredoxin and NADH, and this enzyme was originally predicted to confer insensitivity to high H 2 concentrations in Caldatribacterium 13 based on the insensitivity of the characterized homolog in Thermotoga maritima 29 ; however, all of these [FeFe] hydrogenases have close orthologs in the genomes of T. velox B11 T , T. velox M15, and A. laminatus RT761 T 8 ( Supplementary Data 4 ). These cultivated Atribacteria do however differ in that all except for A. laminatus RT761 T encode a [NiFe] group 3b hydrogenase. Characterized members of this group are bidirectional sulfhydrogenases that can oxidize NADH, with protons or sulfur as electron acceptors and H 2 or sulfide as products 30 . This [NiFe] group 3b hydrogenase was also speculated to enable T. velox B11 T and other Atribacteria to participate in syntrophic anaerobic acetate oxidation by providing reducing power for the RGP 8 . We propose that it has a role in the relative H 2 -insensitivity of Caldatribacterium and Thermatribacter . Folate permits Caldatribacterium growth in defined media Since semi-syntrophic growth of the Caldatribacterium strains with SRBs was not due to the consumption of fermentation products, we hypothesized that one or more vitamin may be required for growth and could be provided either by the SRBs or by yeast extract. We initially carried out all cultivation experiments with Wolin’s vitamin solution 31,32 from a concentrated stock that was filter-sterilized, as recommended for media preparation 33 and commonly used in our labs, or with Vitamin Supplement MD-VS™ from the American Type Culture Collection (ATCC); however, these solutions did not enable Caldatribacterium growth in the experiments described above, such as the dilution-to-extinction cultures. We noted a precipitate in the concentrated Wolin’s vitamin solution that was removed during filter-sterilization, so we reasoned that one or more vitamins necessary for Caldatribacterium growth may be removed during this step. To test this, we added unfiltered Wolin’s vitamin solution that was sterilized by autoclaving rather than filtration, and showed that it restores growth to levels equal to cultures with yeast extract ( Fig. 3B ). Knowing that autoclaving did not destroy the necessary vitamins, we tested single autoclaved vitamins to determine whether they could support Caldatribacterium growth when added with the filtered Wolin’s vitamin solution. An initial analysis of Caldatribacterium genomes suggested auxotrophy for folate, riboflavin, and biotin 13 ( Supplementary Data 5 ). Although addition of autoclaved biotin or riboflavin to media with filtered vitamins failed to enable growth of C. saccharofermentans GBS T and enabled only poor growth of C. inferamans SIUC1 T , folate completely restored growth in C. saccharofermentans GBS T and substantially improved growth of C. inferamans SIUC1 T ( Fig. 3B ). Subsequent experiments focusing on C. saccharofermentans GBS T showed that folate, riboflavin, and panthothenate were necessary and sufficient for growth on fucose in the absence of yeast extract, and these three vitamins plus biotin and cobalamin allowed for growth comparable to that with the full vitamin mixture and/or yeast extract ( Supplementary Fig. 10 ). Additional experiments determined that a minimum of 0.5 ng mL -1 of folate was necessary to support growth of strain GBS, although folate concentrations lower than 20 ng mL -1 resulted in slower growth ( Supplementary Fig. S11 ). We note that folate is nearly insoluble at neutral pH and insoluble below pH ~6 34,35 , and that recipes for concentrated Wolin’s vitamin solutions are unbuffered 31,33 . We also note that Wolin’s vitamin solution is sometimes referred to as Wolfe’s vitamin solution or by other names. For example, the ATCC sells these vitamins under the name Vitamin Supplement MD-VS™ and describes them as a modification of “Wolfe’s Vitamin Solution” without citation. MD-VS™ is identical to Wolin’s vitamin solution but is phosphate-buffered (900 mg L -1 ). We measured a pH of 6.0 in Vitamin Supplement MD-VS™ and reiterate that AGW media prepared with MD-VS™ successfully co-enriched C. inferamans and Thermodesulfovibrio but did not enable isolation of C. inferamans , presumably due to folate removal during filter-sterilization of Vitamin Supplement MD-VS™ at the ATCC. To explore the possibility that the SRBs that enabled growth in co-culture could produce and secrete folate required by Caldatribacterium , filter-sterilized supernatants from the SRB cultures or uninoculated SRB medium were added to the Caldatribacterium medium in the presence of filtered vitamins. All four SRB supernatants, but not the uninoculated SRB medium, supported growth of both Caldatribacteirum strains ( Fig. 3C ). Although these results suggested that folate was present in the SRB supernatants, a targeted metabolomics analysis did not detect folate monoglutamate with a minimal level of detection of ~0.25 ng mL -1 ( Supplementary Data 6 ). This could be because the supernatants contained a form of folate that was not detected by the metabolomics assay or that they contained another factor that could substitute for folate. Folate can take on a variety of forms, including polyglutamylated and N5- and N10-substituted forms that would have evaded detection in our metabolomics analysis 36,37 . Folate auxotrophy is conserved in Atribacterota To further assess folate metabolism in Caldatribacterium and examine the broader significance of folate in Atribacterota , we examined folate-dependent enzymes, biosynthesis, and transport pathways in the two complete Caldatribacterium genomes and other Atribacterota genomes ( Supplementary Data 4 and 5 ). Folates are near-universal one-carbon donors and acceptors that can be synthesized de novo from GTP, chorismate, and glutamate by many bacteria via a pathway consisting of a pterin branch, a para-aminobenzoic acid (pABA) branch, and glutamylation reactions 38 ( Supplementary Fig. 12 ). Both Caldatribacterium isolates and most Atribacterota MAGs and SAGs encode methionyl-tRNA formyltransferase (Fmt) and the formate-dependent form of thymidylate kinase (ThyA), indicating folate dependency for both translation and purine metabolism. Yet, Atribacterota genomes lacked most known folate biosynthesis genes, including the key gene of the pterin branch hydroxymethyldihydropterin pyrophosphokinase (HPPK), the key genes of the pABA branch aminodeoxychorismate (ADC) synthase and ADC lyase, and dihydropteroate synthase (DHPS), which joins the pterin and pABA branches by condensing 6-hydroxymethyl-7,8-dihydropterin pyrophosphate and pABA. The absence of DHPS also suggests that pterin and pABA salvage would not satisfy folate needs in Atribacterota ( Supplementary Fig. 12 ). Intact folate salvage has been partially characterized in some bacteria 39 and is the basis of its inclusion in media vitamin solutions. We found that energy-coupling factor (ECF) transporters with substrate-binding domains for folate were conserved across the Atribacterota , as originally noted for Ca. Caldatribacterium genomes 13 ( Supplementary Data 4 ). Consistent with their use of added folate, Atribacterota genomes also encode folylpolyglutamyl synthase (FPGS) necessary for glutamylation reactions that would be necessary for use of transported folate. In contrast to the Atribacterota genomes, the genomes of the SRBs that enabled Caldatribacterium growth as defined co-cultures encoded complete de novo folate synthesis pathways ( Supplementary Fig. 13; Supplementary Data 5 ). We also noted the absence of biosynthetic pathways for riboflavin and biotin across the Atribacterota along with ECF substrate-binding domains for both riboflavin and biotin, although neither riboflavin nor biotin fully rescued growth of the Caldatribacterium strains in pure culture ( Fig. 3B ). Riboflavin was detected in spent medium from all four SRB pure cultures, as was niacinamide, with niacin and pantothenate detected in medium from some, but not all SRBs ( Supplementary Data 6 ). To directly test the hypothesis that folate was removed during media preparation by filter-sterilization of the concentrated Wolin’s vitamin solution, we prepared Wolin’s vitamin solution as described 33 , added it to GBS salts medium, and quantified folate and other vitamins by targeted metabolomics. This experiment showed that folate, but not other vitamins, was removed by filter sterilization of the Wolin’s vitamin solution. In contrast, folate added as part of the unfiltered Wolin’s vitamin solution added prior to autoclaving was measurable in the final medium ( Supplementary Data 6 ). Caldatribacterium shares unusual cell structure and secreted proteins with other Atribacterota Given the unusual cell ultrastructure of existing Atribacterota isolates 8,19,21 and the predicted bacterial microcompartments (BMCs) in Caldatribacterium and other Atribacterota 15,40 , we examined their cell structure. Strains GBS T and SIUC1 T were both rod-shaped or ovoid cells with somewhat pointed ends ( Fig. 5 A,B; Supplementary Fig. S14 ). The ultrastructure of strains GBS T and SIUC1 T was observed by cryo-electron microscopy (Fig. 5 A,B; Supplementary Fig. S14; Supplementary Data 7; Supplementary Data 8) . Similar to A. laminatus 19 , T. velox B11 T 8 , and T. velox M15 21 , dense cytosolic regions were observed for both strains, as well as three LMLs comprised of an internal membrane and a double layer at the cell boundary that could represent two membranes or a membrane and another structure such as an S-layer. We noted previously 13 that draft Caldatribacterium genomes encode genes for synthesis and export of lipid A ( lpxABCD ; msbA ), but lacked genes required for production and attachment of keto-deoxyoctulosonate and the liposaccharide inner core. Analysis of the GBS T and SIUC1 T genomes, along with all other Atribacterota genomes, confirmed these observations ( Supplementary Data 4 ), consistent with an outer membrane with unusual lipopolysaccharide throughout the phylum. Epifluorescence microscopy using the DNA stain DAPI and FISH with Caldatribacterium 16S rRNA-targeted probes showed that rRNA and DNA were not colocalized within the cell. DAPI fluorescence was visible only within the compartmentalized nucleoids (more condensed in strain C. saccharofermentans GBS T ) typically at or near the center of cells, yet the rRNA probe signal was distinct and concentrated near cell poles ( Fig. 5 C,D; Supplementary Figs. 15 and 16) . This contrasts with similar analyses of A. laminatus , which showed co-localization of DNA and total RNA within the central part of the cell, ribosomes in both the inner LML-bounded space and the middle LML-bounded space, and signal from rRNA-targeted FISH probes distributed throughout the middle of the cell but not at cell poles 19 . The localization of rRNA outside of the compartmentalized nucleoids is inconsistent with the interpretation that the three LMLs in Caldatribacterium are an S-layer, an outer membrane, and a separated cytoplasmic membrane 19,20 , because that model would place rRNA in the periplasm. Thus, our data favor models where the nucleoids of Atribacterota are either bound by a third membrane (inner LML) or by invaginations of the cytoplasmic membrane, as observed in some Planctomycetota 41 . However, invaginations of the second LML are not apparent in any images of any cultivated Atribacterota 8,21 , including cryo-electron tomograms of A. laminatus 19 . BMCs were not observed in either strain, but their expression could depend on growth conditions. Given the unusual ultrastructure of Caldatribacterium and since a “membrane-centric” metabolism was suggested previously for A. laminatus based on a high percentage and expression of transmembrane proteins, proteins with unique N-terminal extensions, and a unique secretome that was poorly annotated by SignalP-4.1 19,42 , we examined the proteomes of the Caldatribacterium strains and other Atribacterota . We observed a similarly high proportion of proteins with transmembrane helices encoded by GBS T (29.7% of all proteins) and SIUC1 T (30.2% of all proteins) and an abundance of unusual signal sequences that were recognized by the recurrent neural network implemented in SignalP-5.0 43 but not by Hidden-Markov Models (HMMs) implemented by SignalP-4.1 ( Supplementary Fig. 18 ). In contrast to a previous report 19 , we note that Atribacterota signal peptides recognized by SignalP-5.0 are less hydrophobic, have a lower isoelectric point, and are longer than other signal peptides in Atribacterota and other diderms with unusual cell structures ( Supplementary Fig. 19; Supplementary Fig. 20 ; Supplementary Fig. 21 ). Analysis of the signal peptidase recognition sites predicted by SignalP-5.0 and SignalP-4.1 revealed an enrichment of phenylalanine at the second position in the A-X-A consensus in Atribacterota , Dicytoglomota , and Thermotogota ( Supplementary Fig. 22 ). Discussion Although Atribacterota was designated as a candidate phylum 27 years ago (as OP9), it has continued to resist lab cultivation, with isolates representing two families being described only recently. Here, we used metagenomic data derived from a corn stover enrichment in GBS over a decade ago 13,15 to target members of a third family of Atribacterota . The annotation of pathways for sugar fermentation in the original Ca. C. saccharofermentans MAG and Ca. C. californiense SAG led us to provide sugars as sole carbon and energy substrates without terminal electron acceptors, and this approach allowed us to enrich and eventually isolate C. saccharofermentans and C. inferamans . Both species were indeed shown to be strictly anaerobic sugar and sugar alcohol fermenters that produce H 2 and acetate ( Supplementary Data 9 ). This core metabolism is conserved in the three previously described isolates representing the two other families of Atribacteria . Genes for this metabolism are conserved and ancestral in the class 8 , suggesting a common role for all Atribacteria in consortial sugar fermentation. Basic phenotypic comparisons of all five Atribacterota isolates, including membrane fatty acids, are presented in Supplementary Data 2 and Supplementary Data 10 . Sugar fermentation to H 2 and acetate is highly exergonic 44 , and the isolation of primary sugar fermenters producing H 2 and acetate as the major fermentation products was described at least 83 years ago 45 . So why have Atribacterota resisted lab cultivation? A. laminatus was reported to be sensitive to H 2 accumulation and stimulated by the hydrogenotrophic methanogen Methanothermobacter thermoautotrophicus through interspecies electron transfer. The Caldatribacterium species described here cohabitate with hydrogenotrophs and probably also engage in interspecies electron transfer. C. saccharofermentans was enriched in situ along with a newly discovered hydrogenotrophic methanogen in the Archaeoglobaceae 16,46 and with the hydrogenotrophic SRB Thermodesulfobacterium auxiliatoris . In contrast, neither methanogens nor methanogenesis activity were evident in the Inyo-BLM 1 borehole from which C. inferamans was obtained, yet hydrogenotrophic SRBs were abundant and active in situ 47 . The co-enrichment of Caldatribacterium species in GBS and Inyo-BLM 1 with hydrogenotrophic SRBs also supports this model of consortial sugar fermentation, yet we showed that both Caldatribacterium species are relatively insensitive to H 2 accumulation. However, since the Caldatribacterium isolates grew well in pure culture when supplemented with yeast extract or in defined co-culture with a variety of SRBs, we were forced to consider other mechanisms of dependency. After reexamining our genomic data, we designed experiments to test the hypothesis that vitamin auxotrophy is the basis of Caldatribacterium enrichment with SRBs and for semi-syntrophic growth with other anaerobes – a hypothesis that was supported by all of our experiments. This finding was surprising because most researchers seeking to cultivate new lineages - including ourselves - use Wolin’s vitamin solution 31,32 (also called Wolfe’s vitamin solution), which contains ten different vitamins and was developed 62 years ago for this purpose. For example, MediaDive 33 lists 362 different media using Wolin’s vitamin solution and all use filter sterilization rather than autoclaving to sterilize the stock vitamin solution. We show that filter sterilization removes precipitated folate from the stock vitamin solution and that folate and all seven other vitamins that were assayed were detectable after autoclaving. Since all Atribacterota lack pathways for folate biosynthesis, we suggest that the unintentional removal of folate during media preparation may be a key problem limiting our ability to cultivate Atribacterota for nearly 150 years of microbiology research focused on pure cultures 1 . We note that neither Wolin 31,32 nor Balch 31,32 described how this vitamin solution should be sterilized, but we strongly suggest amendment of all media preparation instructions to avoid filter-sterilization of folate. The stability and solubility of folate solutions is also affected by other vitamins, with degradation catalyzed by riboflavin, ascorbic acid, and degradation products of thiamine, and solubility promoted by nicotinamide 48 . Other factors affecting folate solubility and stability have been reviewed 48 . The Caldatribacterium isolates described here were similar in their cell ultrastructure to other isolates in the Atribacteria , with what appears to be an intracellular membrane containing a nucleoid. One difference between the Caldatribacterium isolates and the other Atribacterota isolates is the profile of DNA and rRNA in the cell. Caldatribacterium has a distinct distribution of DNA in the cell center and rRNA at the cell ends, apparently separated by the inner LML. The Atribacterota are also unique, along with other thermophiles with unusual membrane architectures - Dicytoglomota and Thermotogota - by possessing a high percentage of unusual signal peptides and a high percentage of transmembrane proteins, congruent with a membrane-centric metabolism . We suggest that the unique cell ultrastructure of these phyla should be a focus of future studies to better understand their origins and roles in ecophysiology. Protologues Description of Caldiatribacterium gen. nov. Caldiatribacterium gen. nov. (L. adj. kal -di, hot; L. adj. ater -tra -trum, black; L. neut. n. bacterium, rod or staff. N.L. neut. n., Caldiatribacterium , ‘black’ or ‘dark’ references both ‘microbial dark matter,’ referring to the poor culturability of the genus and phylum, and the dark, anoxic environments where it is found. Cells are rod-shaped or ovoid cells with pointed ends. Cells stain Gram-variable, but genomic data and cryo-EM reveal three lipid membrane-like structures including an outer membrane and a nucleoid that appears to be membrane-bound. No endospore formation is observed. Thermophilic. Obligately anaerobic chemoorganoheterotroph. Fermentation end products are acetate and H 2 . The major fatty acids (>10%) were C 13:0 iso 3OH , C 15:0 iso , C 16:0 , and C 18:0 . The type species is Caldiatribacterium saccharofermentans . Description of Caldiatribacterium saccharofermentans sp. nov. Caldiatribacterium saccharofermentans sp. nov. ( sac.cha.ro.fer.men′tans. Gr. n. sakchâr sugar; L. part. adj. fermentans fermenting; N.L. neut. n. Saccharofermentans sugar-fermenting). Caldiatribacterium saccharofermentans , “The mysterious heat-loving sugar-fermenting rod.” Cells are rod-shaped or ovoid cells with somewhat pointed ends 1.5-3.0 µm long and 0.5-0.9 µm wide, occurring singly or in pairs. Flagellar motility rarely observed, but flagella observed by cryo-EM. Flagellar biosynthesis gene clusters complete. Optimal growth temperature is 70 o C, with a maximum of 77.5 o C and minimum of 60 o C. Optimal pH is 7.0 with a range of 5.5–9.0. Obligately anaerobic chemoorganoheterotroph that ferments sugars and sugar alcohols as sole carbon sources and fermentation substrates: adonitol, cellobiose, fructose, fucose, galactose, gluconate, glucose, inositol, lyxose, maltose, mannose, N-acetylglucosamine, raffinose, rhamnose, ribose, sorbitol, sucrose, tagatose, and xylitol. Fermentation end products are acetate and H 2 . The major fatty acids (>10%) were C 13:0 iso 3OH , C 16:0 , and C 18:0 . The type strain is GBS T (DSM 110181 T and JCM C190371 T ), which was isolated from Great Boiling Spring, NV., USA. Description of Caldiatribacterium inferamans sp. nov. Caldiatribacterium inferamans sp. nov. ( L. pl. n. inferi, the underworld; L. pres. part. amans, loving; N.L. part. adj. inferamans , loving the underworld referring to the origin of the strain). Caldiatribacterium inferamans , “The mysterious heat-lover from the underworld” or “The mysterious heat-loving rod that loves the hot underworld.” Cells are rod or ovoid cells with somewhat pointed ends 1.0-2.6 µm long and 0.5-0.8 µm wide, typically occurring singly. Flagellar motility rarely observed, but flagella observed by cryo-EM. Flagellar biosynthesis gene clusters complete. Optimal growth temperature is 60-70 o C, with a maximum of 75 o C and minimum of 55 o C. Optimal pH is 7.4-8.7 with a range of 5.9–9.5. Obligately anaerobic chemoorganoheterotroph that ferments sugars, sugar alcohols, and proteins as a sole carbon source and electron donor/acceptor for growth: arabinose, cellobiose, fructose, fucose, fumarate, galactose, gluconate, glucose, lactose, maltose, mannitol, mannose, peptone, raffinose, sorbitol, starch, succinate, sucrose, trehalose, xylose, xylitol, xylan, and yeast extract. Fermentation end product is acetate and H 2 . The estimated genome size of SIUC1 T is 1,967,957 bp, with 56.5 mol% DNA G + C content. The major fatty acids (>10%) were C 13:0 iso 3OH , C 15:0 iso , C 16:0 , and C 18:0 . The type strain is SIUC1 T (DSM 110249 T and JCM 39079 T ), which was isolated from the Deep Carbonate Aquifer located in the southern hydrographic Great Basin, NV, USA. Description of Caldatribacteriaceae fam nov. Caldiatribacteriaceae (L. adj. kal -di, hot; L. adj. ater -tra -trum, black; L. neut. n. bacterium, rod or staff. N.L. masc. n. Caldatribacterium type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Caldiatribacteriaceae the family of the genus Caldatribacterium ). Belongs to the order Atribacterales , class Atribacteria , and phylum Atribacterota . Description of Thermodesulfobacterium auxiliatoris sp. nov. Thermodesulfobacterium auxiliatoris sp. nov (L. gen. masc. n. auxiliatoris, one who gives aid). Thermodesulfobacterium auxiliatoris , "The thermophilic sulfate reducing rod who provides aid," based on its ability to facilitate growth of Caldatribacterium in co-culture. Cells are regular rods averaging 1.5 μm long and 0.5 μm wide, usually occurring singly. Diderm cell envelope structure. Optimal growth temperature is 70-75 ºC with a maximum of 80 ºC and a minimum of 50 ºC. Optimal pH is 7.0 with a range of 5.5-8.0. Facultative chemolithoautotroph capable of using H 2 as the electron donor and CO 2 and the carbon source, with sulfate as the terminal electron acceptor. Acetate stimulates growth under autotrophic conditions, and formate and lactate can serve as electron donors and carbon sourcesfor heterotrophic growth ( Supplementary Data 1 ). The type strain is TA1 T (DSM 107971 T and JCM 32932 T ), isolated from Great Boiling Spring, NV, USA. Inclusion and ethics This research included local researchers as full authors, when possible, to recognize both logistical and intellectual contributions. No potential or listed authors were discriminated against on the basis of gender, race, ethnicity or any other factors not related to scientific contributions. Data availability The complete C. saccharofermentans GBS T and C. inferamans SIUC1 T genomes and are available in GenBank under accession numbers CP187957 and CP189974. Raw reads for all metagenomes and 16S rRNA gene amplicons are available from the Sequence Read Archive (SRA) within BioProjects PRJNA1250235 and BioProject PRJNA956542 at the accessions in Supplementary Data 11 . GenBank accession numbers for GBS T , SIUC1 T , and T. auxiliatoris 16S rRNA genes are MN114535, MT023787, and MH513620. All accessions are tabulated in Supplementary Data 11 . Source data are provided with this paper. Code Availability Scripts used to account for vitamin biosynthetic pathways and compare signal peptides are available on GitHub: https://github.com/Hedlund-Lab/atribacterota-2025. Methods GBS in situ enrichment and isolation of strain GBS T C. saccharofermentans GBS T was obtained from GBS, a circumneutral pH, Na–Cl spring in northwestern Nevada (40°39’41.3”N, 119°21’58.1”W) 49 . In situ enrichments consisting of 1 g of AFEX-treated corn stover sealed in 100 µm nylon mesh bags were performed essentially as described in ref 16 , because previous in situ enrichments contained populations of Ca. Caldatribacterium saccharofermentans 13 . The bags were incubated at the water/sediment interface at the "C site" in the outflow of GBS from 26 October 2013 to 28 March 2014. Immediately after removal, the corn stover was transferred to a 25 mL serum tube containing 10 mL of sterile, anaerobic GBS spring water (previously collected, sparged with N 2 , autoclaved, and reduced by addition of cysteine to 0.05%) under a stream of N 2 . The tube was sealed and shaken, and then 0.1 mL of the contents was transferred using an N 2 -flushed, sterile needle and syringe to 50 mL of anaerobic medium in a 150 mL serum vial sealed with a butyl rubber stopper. The medium was GBS salts medium 50 with 5 mM sodium phosphate pH 7 buffer and a filtered vitamin solution (see below) (1/100 dilution), but with xyloglucan (0.02% mass vol -1 ) as the sole carbon source, reduced with 0.01% sodium sulfide, and with an N 2 headspace. The 100×-concentrated Wolin’s vitamin solution contained per liter: 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine HCl, 5 mg thiamine HCl, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg DL-calcium pantothenate, 0.1 mg vitamin B12, 5 mg p -aminobenzoic acid, and 5 mg lipoic acid 31,32,33 , and was sparged with N 2 for 1 hour prior to sterilization. Unless otherwise noted, the 100×-concentrated Wolin’s vitamin solution was filtered-sterilized (0.2 μm nylon filters; 09-719C, Fisher Scientific, Waltham, MA, USA) and stored at 4 ºC in a foil-covered borosilicate glass bottle the dark until it was added to 1x after the medium was autoclaved. After the importance of folate and its problem with solubility was recognized, the 100×-concentrated Wolin’s vitamin solution was sterilized instead by autoclaving for 30 min at 121 °C. The inoculated medium was brought back to the lab and incubated at 73 °C without shaking within 24 hours of sampling. The enrichment culture was initially transferred to fresh medium (1/1000 dilution) after a one-week incubation for the first two weeks, and then it was transferred every two weeks thereafter. Subsequent experiments replaced xyloglucan with various individual sugars or other substrates. Dilution-to-extinction was performed using fucose (0.02% mass vol -1 ) using 10-fold serial dilutions up to 10 -10 dilution. Isolation was performed using media solidified with Gelrite (0.8% mass vol -1 ) and magnesium chloride hexahydrate (0.4% mass vol -1 ). Plates were prepared in an anaerobic chamber and incubated in anaerobic incubation vessels for 7-10 days with an N 2 headspace 51 . Single colonies were streaked for isolation three times. Well Inyo-BLM 1 and in situ enrichment and isolation of strain SIUC1 T C. inferamans SIUC1 T was isolated from a sterile polyurethane foam plug that was suspended in borehole/well Inyo-BLM 1 (36º 24’04.19 N / 116º 28’06.58 W) for three months. This borehole was completed in 2007 reaching a depth of 883.5 m below land surface (mbls) by The Hydrodynamics Group, LLC (Edmonds, WA, USA) on behalf of the US Department of Energy, Inyo CO, CA, and US National Park Service as part of a suite of holes used to test hydrologic connectivity across the Funeral Mountains adjacent to Death Valley, CA, USA 52 . It transects lithologies ranging from volcanic tuff and valley-fill alluvium to lake sediments, ultimately intersecting Paleozoic dolomite that hosts a low-transmissivity portion 53 of the Lower Carbonate Aquifer of the Death Valley Regional Flow System at 748 mbls 52,54 . The plug was recovered from an inferred fracture zone roughly corresponding to a partially collapsed void 22 immediately below the casing terminus ~750 – 753 mbls. After retrieval, the foam plug was placed into pre-reduced artificial groundwater medium (AGM) as previously described by Hamilton-Brehm et al. 2019 26 . For initial enrichment and isolation attempts, vitamins were supplied as Vitamin Supplement MD-VS™ from the ATCC before autoclaving. An additional sample of the aquifer water without enrichment was obtained from Inyo-BLM 1 at ~752 m via a gas-tight discrete sampler (“bailers,” Comprobe, Inc.; Fort Worth, TX) 22 . 250 ml of the bailed water was filtered using a 0.22 μm PES filter (UX-06730-43, Thermo Scientific; Waltham, MA). 250 μl of Inyo-BLM 1 water was also to the PowerSoil lysis tubes to prevent detection bias against small cells 22 . Lab cultivation was conducted by removing 1 mL of liquid from the stored sponge in AGM and injecting it into a 160 mL serum bottle containing 50 mL (2% vol vol -1 inoculum) of AGM amended with 10 mM xylitol and 0.01% mass vol -1 yeast extract. The final pH of the medium was 8.0 after reduction of the solution. Enrichments were incubated at 60 °C without shaking, and transferred every seven days, three times, using a 0.5% vol vol -1 inoculum into sterile anaerobic AGM media in 160 mL serum bottles. Cell morphology and density was monitored using an Axioskop2 Plus microscope and a Petroff-Hauser counter under phase contrast. Dilution-to-extinction was conducted three times, which successfully enriched the culture to >90% C. inferamans but isolates of C. inferamans were not isolated on the defined AGM medium with vitamins supplied as Vitamin Supplement MD-VS™. A clonal isolate was generated from the third dilution-to-extinction culture by picking a colony and restreaking it two additional times on anaerobic medium solidified with Gelrite (0.8% mass vol -1 ) and magnesium chloride hexahydrate (0.4% mass vol -1 ), with 10 mM xylitol, 0.05% (mass vol -1 ) yeast extract, and 0.05% (mass vol -1 ) casamino acids. 16S rRNA gene qPCR and amplicon analysis of enrichment cultures For GBS enrichments, DNA extraction using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) and 16S rRNA gene amplicon sequencing was done with an Illumina MiSeq (2x250) using 806r reverse primer a modified 515f-mod forward primer (5′ GTGYCAGCMGCCGCGGTAA) to enhance coverage of archaea 46 . Quantitative PCR (qPCR) was done using primers OP9_16S_F (5' AGGAAAGCTGGCCTCTGC) and OP9_16S_R (5' ACCGTCACAGGAAGGAGC) targeting Caldatribacterium with primers at 64 °C annealing temperature or primers 515f-mod and 806r targeting total bacteria and archaea 46 . The standard was a plasmid containing a fragment of the Caldatribacterium 16S rRNA gene (SSW_L1_H02; ref. 55 ). For the Inyo-BLM 1 aquifer sample and in situ enrichment (foam plug), DNA was extracted using the MoBio Powersoil DNA Isolation Kit (Carlsbad, CA, United States). Lab enrichment cultures were extracted using the MoBio UltraClean Microbial DNA Isolation Kit (Carlsbad, CA, United States), both according to the manufacturer’s protocol amended with one freeze/thaw cycle (-80 o C/65 o C) at the beginning of DNA extraction procedure. DNA concentration was determined using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (ThermoScientific, Waltham, MA, USA) with wavelength settings of 260 nm and 280 nm. Extracted DNA was sent to The Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory (Lemont, IL, USA) and sequenced using the Illumina MiSeq platform (2x151 bp). Universal bacterial primers targeting the V4 region of the 16S rRNA gene were used for diversity analysis 56 . Paired-end reads were merged, denoised, and demultiplexed using deML 57 . For both datasets, amplicon sequence variants (ASVs) were processed using the DADA2 pipeline 58 implemented in R using default parameters (https://benjjneb.github.io/dada2/tutorial.html), and taxonomy was assigned using SILVA release v123 (www.arb-silva.de/documentation/release-123/). Stable isotope labeling Stable isotope labeling was performed with xyloglucan mixed cultures and fucose co-cultures derived from GBS. Universally 13 C-labeled substrates (Cambridge Isotope Laboratories) were added to freshly transferred enrichments grown for two days from concentrated, anaerobic stocks to the microcosms at 1 mM final concentration unless otherwise noted: bicarbonate, formate, acetate, glucose, xylose, ribose, or algal amino acids (0.01% mass vol -1 ). Negative controls with no compound added were incubated in parallel. All incubations were done at 73 °C without shaking for 2 hours. After incubation, cells were pelleted by centrifugation for 5 min at 16,100 × g, pooled in 0.5 mL of 1× phosphate-buffered saline (PBS), and 0.25 mL of freshly prepared 3% paraformaldehyde (PFA) was added and mixed. Samples were fixed for 1 hour on ice, cells were pelleted by centrifugation for 5 min at 9,200 ×g, and pellets were washed twice with 1×PBS. Washed cells were resuspended in 200 µL of 50% ethanol, and stored at –20 °C. FISH FISH was performed using protocol v 2.2 59 (www.arb-silva.de/fish-probes/fish-protocols/) using the Caldatribacterium -specific 16S rRNA probe OP9-480 (5’-AGCTRTTCACCCCTYCCCTC-3’) labeled with Cy3 and the Bacteria-specific 16S rRNA probe Bact927 60 (5’-ACCGSTTGTGCGGGCCC-3’) labeled with 6-FAM. Cells from cultures were pelleted by centrifugation (10,000 × g for 5 minutes), washed in 1× PBS, and fixed with 1% paraformaldehyde in 1× PBS on ice for 1 hour. After fixation, the cells were pelleted, washed three times in 1× PBS, resuspended in 50% ethanol and stored at -20 o C. Hybridization was performed on slides at 46 °C with 30% vol vol -1 formamide. Hybridization conditions were optimized for the OP9-480 probe using the Clone-FISH technique 61 via expression of the near-full-length 16S rRNA gene sequence of a Caldatribacterium relative obtained from Mud Hot Springs 55 (SSW_L1_H02) in E. coli strain JM109 (DE3) from the plasmid pGEM-T. After hybridization, cells were counterstained with DAPI (1 µg mL -1 ). Cells were visualized by epifluorescence microscopy using an Eclipse Ti-U inverted microscope (Nikon, Melville, NY, USA) equipped for epifluorescence with Nikon filter sets compatible with Cy3 (96312 G-2E/C), 6-FAM (96343 EN GFP), and DAPI (96310 UV-2E/C), with image capture using a Retiga-SRV camera (QImaging, Surrey, BC, Canada) and Nikon Elements v4.13 software. Isotopically labeled cells were deposited onto ITO-coated slides and visualized with a Leica DM5500B microscope using MetaMorph software with a 100× magnification dry immersion objective. Fluorescence and brightfield images were collected and the X-Y and fiducial locations were noted to enable navigation in the nanoSIMS. NanoSIMS NanoSIMS was conducted on a CAMECA NanoSIMS 50 at Lawrence Livermore National Laboratory. Fiducial locations and locations of FISH-positive cells were found with a charge-coupled device camera using X-Y coordinates and a real-time imaging unit. The primary Cs + ion beam was set to 1.5 pA, corresponding to an approximately 150 nm beam diameter at 16 keV. Rastering was performed over 20 × 20 μm areas with a dwell time of 1 ms pixel –1 for 19-30 scans and generated images containing 256 × 256 pixels, yielding data for 10-114 Caldatribacterium cells per experiment ( Fig. 2 ). Sputtering equilibrium at each area was achieved with an initial beam current of 90 pA to a depth of ~10 nm. After tuning the SIMS for mass resolving power of ~7000, secondary electron images and quantitative secondary ion images were simultaneously collected for 12 C 2 – and 13 C 12 C - on individual electron multipliers in pulse counting mode. NanoSIMS data were initially processed using L’Image (http://limagesoftware.net) to perform deadtime and image shift correction of ion image data before creating 13 C 12 C/ 12 C 2 ratio images, which reflected the level of 13 C incorporation into biomass. Regions of interest for isotopic ratio quantification were drawn manually around each cell. Pure culture experiments Characterization of strain GBS T was performed in 10 mL of GBS salts medium in 20 mL serum vials. Substrates were tested at either 0.05% mass vol -1 for sugars and complex organic substrates, 1 mM for organic acids, 0.1 atm methane, or 0.4/0.1 atm H 2 /CO 2 . Cultures were incubated in the dark without shaking at 73 °C, and growth was assessed by phase-contrast microscopy using a Petroff-Hauser cell counter after seven days of incubation. Growth rate tests for pH (using 5 mM sodium phosphate buffer) and temperature optima were performed using 0.05% fucose as a growth substrate, with samples taken every 12-24 hours after growth was initially observed. Growth was tested at 50-80 o C in 5 o C increments and at 77.5 o C, and at pH values from 4.5-9.5. Strain SIUC1 T cultivation experiments were conducted in 160-mL serum bottles, containing 50 mL volume of AGM and 10 mM xylitol with a 2 atm headspace of 99.9% pure N 2 . Temperature and pH culturing assays were averaged across quadruplicate replicates. Cultivation experiments to define temperature optima were incubated at 55, 60, 65, 70, and 75 o C in the dark without shaking. Cultivation experiments to determine optimal pH utilized alternative buffers, replacing HEPES buffer when appropriate. A final concentration of 10 mM for each buffer was used to achieve the desired pH values ranging from 6.0 - 9.5. The buffers used were: 2-ethanesulfonic acid (MES) for pH 6.0, 1,4-piperazinediethanesulfonic acid (PIPES) for pH 6.75, HEPES for pH 7.5, tris(hydroxymethyl)aminomethane (TRIS) for pH 8.0/8.75 and N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) for pH 9.5. Cell densities for temperature and pH assay were calculated from 24-hour intervals by microscopy using a Petroff-Hausser counting chamber. Growth of strain SIUC1 T on different substrates was determined by quantifying growth in AGM media amended with 10 mM of the selected carbon/energy source at 65 o C as listed in Table 1 , or 2 atm for H 2 /CO 2 (80%:20%) or methane (99.9%). Undefined substrates tested were adjusted to a concentration of 0.1% mass vol -1 , which included casamino acids, peptone, starch, yeast extract, and xylan. Growth was monitored by microscopy using a Petroff-Hausser counting chamber. If a culture reached a cell density of 1 x 10 6 cells mL -1 or greater within 7 days, it was transferred to a new culture. If a culture reached 1 x 10 6 cells mL -1 or greater within 7 days in the 3 rd transfer it was considered a stable culture growing on the specific substrate. Experiments with 10 mM xylitol as the carbon/energy source were combined with sulfur-containing potential electron acceptors at concentrations of 10 mM (sulfate, sulfite, thiosulfate, and elemental sulfur). Sulfide production was assessed qualitatively using lead-acetate strips to indicate reduction of the sulfur compound. For both strains, acetate was quantified by high-performance liquid chromatography (HPLC) using authentic standards and a Shimadzu Prominence-i LC-2030 HPLC equipped with a Resex ROA-Organic Acid H+ 8%, LC-column (300 x 7.8 mm) and Security Guard KJ0-4282 (Phenomenex). 1 mL samples were taken from serum bottles by syringe and centrifuged for 10 minutes at 16,873 x g. Supernatant was transferred to a new tube and acidified by adding 25 µL of a 200 mM sulfuric acid. The acidified sample was then filtered through 0.2 µm filter into an HPLC vial. Samples were run on the HPLC under the following conditions: column temperature: 30 ºC; isocratic; 5 mM sulfuric acid mobile phase at a flow rate of 0.5 mL min -1 ; UV detector set to 254 nm; and run time 30 minutes. Peaks were integrated using LabSolutions LC/GC release 5.87. H 2 was detected qualitatively in headspace samples collected by syringe using a portable hydrogen detector (Forensics Detectors, Model: FD-90A-H2, Rolling Hills Estates, CA, USA). SRB pure cultures were grown in GBS salts medium as prepared for C. saccharofermentans GBS T (5 mM sodium phosphate pH 7 buffer, 0.01% mass vol -1 sodium sulfide) except that vitamins and xyloglucan/sugars were excluded, and instead 1 mM sodium bicarbonate, 1 mM sodium acetate, 1 mM sodium thiosulfate, 5 mM lactate, and 0.66 atm H 2 were added. For experiments with co-cultivation of Caldatribacterium strains and SRBs ( Fig. 3b ), pure cultures for inoculum were diluted 1/100 (0.1 mL in 10 mL total) in medium without added vitamins, and then 0.1 mL of each was used to inoculate 10 mL of Caldatribacterium strain GBS medium with 0.05% mass vol -1 fucose and autoclaved vitamins without added folate; the extra dilution (~10,000-fold total) was used to dilute out folate in the original pure cultures of Caldatribacterium . SRB culture supernatant preparation and analysis of water-soluble vitamins Cultures of SRBs were grown in the absence of added vitamins with 1 mM sodium bicarbonate, 1 mM sodium acetate, 1mM sodium thiosulfate, 5 mM lactate, and 0.66 atm H 2 in 50 mL GBS salts medium in 150 mL serum vials until late exponential phase. Aliquots of culture were centrifuged at 800 × g in 1.5 mL microcentrifuge tubes in an anaerobic chamber, and pooled supernatants were filtered with a 0.2 μm nylon filter into sealed serum vials and autoclaved for 30 min. For Caldatribacterium growth experiments (Fig. 3), 2.5 mL of sterile supernatant was mixed with 7.5 mL of GBS salts medium previously amended with sulfide, and then fucose and autoclaved vitamins without folate were added to yield their typical 0.05% mass vol -1 and 1x concentrations, respectively. Dilution of the Caldatribacterium inocula was performed as above for the co-culture experiments to minimize carryover of folate.. Samples of supernatant for vitamin analysis were frozen at -80 o C and sent on dry ice for analysis at The Metabolomics Innovation Centre (TMIC; Edmonton, AB, Canada) for quantification of water-soluble vitamins using LC−MS/MS and data analysis with Sciex Analyst 1.6.2 62 . Electron microscopy and sample preparation For SEM, late-exponential phase cells were immediately fixed with 2% vol vol -1 glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 hour at 4 o C. Cells were pelleted by centrifugation (10,000 × g for 10 min), washed three times with 0.1 M sodium cacodylate buffer, and resuspended in 1% OsO 4 in 0.1 M sodium cacodylate buffer. Cells were then incubated for 1 hour at 4 ℃ and then rinsed twice with distilled water. Dehydration was performed with 25% increases of ethanol for 15 min for each step from 25 to 95%, then three washes of 100%. Samples were then embedded on a 0.2 μm Millipore filter (13 mm diameter) mounted in a Swinney filter holder. Filters were rinsed with 100% ethanol and kept in ethanol until critical point drying on a Tousimis SAMDRI-790 (Rockville, MD, USA). Sputter coating was carried out using a Denton Vacuum Desk II and the samples were coated with a 600 Å layer of Au-Pd. Imaging was performed using a FEI Quanta FEG 450 SEM, using an acceleration voltage of 20 kV. For cryo-EM, 1 mL of cell culture was centrifuged at 1,000 × g for 2 min to remove insoluble components in the media. The supernatant was centrifuged at 3,000 × g for 5 min to pellet the cells, and the pellet was resuspended in 20 μL of media. Samples were prepared with an automated Leica EM GP plunge freezer set at 21 °C and 95% humidity in the sample chamber. A 3 μL sample of the cell suspension was applied onto glow-discharged copper R2/2 200 grids (Quantifoil), pre-blotted for 60 seconds, blotted for 2 seconds, plunged into liquid ethane, and stored in liquid nitrogen. The samples were imaged on a 120 kV Talos L120C transmission electron microscope at the Netherlands Center for Electron Nanoscopy (NeCEN). Lipid Analysis Cultures of GBS T (0.05% mass vol -1 fucose, 73 o C) and SIUC1 T (0.05% mass vol -1 xylitol, 67.5 o C) were grown to late-exponential phase, and cells were harvested by centrifugation. Cell pellets were stored at -80 o C, and fatty acid methyl ester analysis was performed by Microbial ID (Newark, DE, USA) with the MIDI Sherlock Microbial Identification System and SMOORE6 database. DNA extraction and PCR amplification of the 16S rRNA gene DNA was extracted from cells of strain GBS T grown for seven days and SRB isolates grown for 3 days using the FastDNA Spin Kit for Soil. Strain SIUC1 T cells were grown to a density of 1 × 10 8 cells mL -1 in a cumulative volume of 1 L to 500 mL (10 × 50 mL bottles) and cells were pelleted by centrifugation at 15,000 × g for 30 min in 50 mL conical tubes (Sorvall RC6 Plus centrifuge, rotor F13S14x50cy) at 4°C. The genomic DNA was extracted by cetyltrimethyl ammonium bromide (CTAB) treatment followed by mild homogenization, buffer/chloroform, and finally purified/cleaned with QIAquick PCR (Qiagen) purification columns 63 . The DNA was then assessed for quality and quantity by Qbit and Nanodrop. DNA was aliquoted and stored at -80°C, until needed. For strain GBS T and SRB isolates, the primer 9bF (5’-GRG TTT GAT CCT GGC TCA G-3’) and 1512uR (5' ACGGHTACCTTGTTACGACTT) were used for amplification of 16S rRNA genes 55 , and Sanger sequencing with these primers was performed by Retrogen, Inc. (San Diego, CA, USA). For strain SIUC1 T , universal bacterial primers 27F-YM (5’-AGA GTT TGA TYM TGG CTC AG-3’) and 1492R (5’-TAC CTT GTT ACG ACT T-3’) were used to amplify the 16S rRNA gene, and resulting amplicons were sequenced by MCLAB (South San Francisco, CA, USA). Genome sequencing, assembly, and annotation For strain GBS T ,short read data (2 × 150) was obtained using an Illumina NextSeq 2000 with libraries prepared using the Illumina DNA Prep kit at SeqCenter (Pittsburgh, PA, USA). Long read sequencing was performed with a MinION Mk1B device and FLO-MIN106 flow cell using libraries prepared with the EXP-NBD104 Native Barcoding Expansion Kit (Oxford Nanopore Technologies, Oxford, UK) and the SQK-LSK109 Ligation Sequencing Kit. Short reads were trimmed using Trimmomatic 64 , and hybrid assemblies were performed using Unicycler 65 . Initial annotation was performed using RAST 66 . Genome assembly for strain SIUC1 T was carried out using data from multiple short read runs (Illumina) GeneWiz (San Diego, CA, USA) and UIC Research Resource Center (Chicago, IL, USA) as well as long reads (Oxford Nanopore) using Unicycler 65 . Assemblies were analyzed and compared using QUAST 67 . The Unicycler assembly was selected based on completeness and contiguity for downstream analysis and annotation with JGI annotation pipeline . Phylogenomic and genome distance analysis All genomes from the Genome Taxonomy Database (GTDB) 09-RS220 2 plus those from recent publications 8,21 were used for the construction of a species tree ( Supplementary Data 3 ). The phylogenomic relationships of genomes were inferred based on a concatenation of the bac120 protein marker set identified by GTDB-Tk v. 2.3.2 68 . The concatenated data matrix was analyzed using maximum-likelihood inference in IQ-TREE v.2.2.6 69 , with 1,000 ultrafast bootstraps (UFBoot) and 1,000 SH-like approximate likelihood ratio test (SH-aLRT) replicates (-bb 1000 -alrt 1000), using the best-fit model identified by ModelFinder 70 . Pairwise average nucleotide identity (ANI) and average amino acid identity (AAI) between the MAGs were calculated using FastANI v.1.1 71 and FastAAI v.1 72 , respectively. Annotation of vitamin biosynthesis pathways Tetrahydrofolate biosynthetic potential of Atribacterota species and known or potential syntrophic partners was predicted via manual reconstruction of the Folate Biosynthesis KEGG 73,74,75 reference pathway (map00790) supplemented with COGs 76,77 from the 2021 release of the dataset and PFAM 78,79 version 36 annotations. Genomes were reannotated to a uniform specification. Open reading frames and their resulting protein sequences were predicted using Prodigal 80 version v2.6.3. Functional potential of the protein sequences was then annotated using three methods: (i) kofamscan 81 was used to annotate KEGG orthology (KO), (ii) hmmer 82 version 3.3.1 was used to annotate PFAMs, and (iii) rpsblast from blast+ 83 version 2.9.0+ was used to annotate COGs. COG and PFAM annotations were used to supplement KEGG annotations with putative replacements for ‘missing’ KOs. Domain architectures in the PFAM database indicate that the DHFR-domain (PF00186) -containing proteins often also encode a RibD c-terminus domain (PF01872). However, this feature is not exclusive to DHFR; RibD c-terminus domain is associated with both DHFR and cytidine deaminases. Domain architecture was used to identify possible DHFR genes while distinguishing them from cytidine deaminase: proteins were considered possible unannotated DHFR orthologs if the protein sequence included a RibD c-terminus domain (PF01872), but lacked domains suggestive of deaminase activity (PF14437, PF18785, or PF00383). A similar strategy was employed to identify putative unannotated orthologs of para-aminobenzoate synthase (COG0147) while excluding anthranilate synthase (K01657), and 4-amino-4-deoxychorismate lyase (COG0115) while excluding branched-chain amino acid aminotransferase (K00826). Annotation of secreted and membrane proteins Predicted secreted and transmembrane genome content was analyzed within high-quality (≥90% completeness, <5% contamination) 12 Atribacterota , Thermotogota , Dictyoglomota , Synergistota , and Verrucomicrobiota genomes obtained from the Genome Taxonomy Database 13 release 202 as well as other genomes from bacteria included in the TEMPURA database 84 . Open reading frames were predicted and translated into protein sequences using Prodigal 80 version v2.6.3. Secretion signal cleavage sites were predicted using SignalP-4.1 42 and SignalP-5 43 . Amino acid sequences three residues upstream or two downstream of cleavage sites were extracted and rendered using the R package ggseqlogo 85 version 0.2. Transmembrane helices were predicted using TMHMM 2.0 86 . Hydrophobicity was calculated for each signal peptide sequence by averaging the sum of individual per-residue hydrophobicities as estimated using the Kyte and Doolittle scale 87 . Isoelectric point was estimated for each signal peptide using the isoelectric point calculator web interface 20 . Declarations Acknowledgements We thank Dave and Sandy Jamieson for access to Great Boiling Spring. Inyo-BLM 1 samples were obtained under scientific research permit DEVA-2013-SCI-0069 to D.P.M. from the U.S. National Park Service (NPS). We thank Richard Friese, Josh Hoines, and Dr. Kevin Wilson of the NPS along with Alisa Lembke and the Inyo County, CA Planning Commission for site access and John Bredehoeft and Michael King of The Hydrodynamics Group LLC for hydrogeological context and borehole specifications. We thank John Healey, Brad Lyles, and Chuck Russell of the Desert Research Institute for logistical assistance in obtaining bailed samples and logging data from Inyo-BLM 1. Thanks also to Bill Willborn and the DOE UGTA program for allowing use of their downhole logging system. We thank the NASA Astrobiology Institute node ‘Life Underground’, PI: Dr. Jan Amend at University of Southern California, including Dr. Greg Wanger, Dr. Joshua Sackett, and Dr. Brittany Kruger for permission to use foam plug samples from borehole Inyo-BLM 1. We thank Dr. Bernhard Schink of Universität Konstanz for his help with Greek and Latin grammar. Part of this work was carried out at Lawrence Livermore National Laboratory (LLNL) under Contract DE-AC52-07NA2734 (X.M., P.W., J.P.-R.). Funding was also provided by the U.S. National Science Foundation (DEB 1557042, B.P.H., D.L., C.O.S., J.A.D., X.M., J.P.-R.), NASA (80NNSC17KO548, B.P.H., D.L., C.O.S., J.A.D., X.M., J.P.-R.), NASA Astrobiology Institute (NNA13AA92A, D.P.M., S.H.B.), and Nevada NASA Space Grant (80NSSC20M0043, B.P.H., D.L.). This work was also supported by startup funds from Southern Illinois University Carbondale (S.H.-B.) and the Research-Enriched Academic Challenge (REACH) undergraduate grant (A.M.B.). The scanning electron microscope used in this work was purchased through a grant from National Science Foundation (CHE 0959568). We also acknowledge the National Science Foundation through the grant CHE 0959568 that facilitated the purchase of the FEI Quanta 450 scanning electron microscope. We thank Valerie Jimenez, Sandy Macias, Katelyn Holt, Maidy Ramos, Jorge Torres, Alejandra Moreno, Joseph Mansuri, and Matthew David for assistance with characterization of GBS and SRB strains. Contributions J.A.D. and S.H.B. conceived of the study. B.P.H., J.A.D., and D.P.M., led field campaigns, managed sampling permits, and obtained and managed major grants supporting the work. S.H.B., J.A.D., T.A., J.L., J.J., A.M., A.T., A.V., A.R.M., A.C., J.L., Z.O., A.M.B, E.L, J.T.P, and K.M. conducted culturing enrichment, isolation, and strain characterization. T.A. and J.A.D. performed FISH and 13 C-labeling experiments. 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Wagih O. ggseqlogo: a versatile R package for drawing sequence logos. Bioinformatics 33 , 3645-3647 (2017). Krogh A, Larsson B, Von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305 , 567-580 (2001). Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 157 , 105-132 (1982). Table 1 Table 1 is not available with this version. Additional Declarations There is NO Competing Interest. Supplementary Files AdditionalFile1V29Submit.pdf Supplementary Information AdditionalFile2V29submit.xlsx Supplementary Data 1-11 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6739887","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":462985347,"identity":"eb234139-70df-4491-a065-f32270c8df43","order_by":0,"name":"Brian 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enrichment culture based on 16S rRNA gene amplicons. GBS sediments were the inoculum. \u003cstrong\u003eb,\u003c/strong\u003e Plot of all contigs from a shotgun metagenome derived from the same fucose enrichment culture. \u003cstrong\u003ec,\u003c/strong\u003e Relative abundance of \u003cem\u003eCaldatribacterium\u003c/em\u003e in borehole Inyo-BLM 1; a community enriched in situ on polyurethane foam plug; and a xylitol enrichment culture inoculated with sponge material. All data are based on 16S rRNA gene amplicons. \u003cstrong\u003ed,\u003c/strong\u003e Plot of all contigs from a shotgun metagenome derived from the same xylitol enrichment culture. Color legends for taxa are coordinated for \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb \u003c/strong\u003eand for\u003cstrong\u003e c \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e. Metagenomic binning was performed using MetaWatt. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure1Metagenomeand16StagsV61.png","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/55378467f14700f0092d4324.png"},{"id":95605490,"identity":"31524094-ccaf-4c80-b014-32321bbafebe","added_by":"auto","created_at":"2025-11-11 06:59:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1422537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC incorporation of sugars by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaldatribacterium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in enrichment cultures by FISH-nanoSIMS.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Isotopic enrichments in \u003cem\u003eCaldatribacterium\u003c/em\u003e cells identified by FISH (orange bars) and other cells (grey bars) in a xyloglucan enrichment culture. Each point reflects the percent of \u003csup\u003e13\u003c/sup\u003eC atom fraction excess in a single cell following stable isotope probing with \u003csup\u003e13\u003c/sup\u003eC-labelled substrates. Boxes represent 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles and central mark is the median. Vertical black lines show standard deviations. Red asterisks show significant differences versus unlabeled controls (Wilcoxon rank-sum, *, p \u0026lt; 0.05). Brackets with asterisks show significant differences between \u003cem\u003eCaldatribacterium\u003c/em\u003e cells and other cells (Wilcoxon rank-sum, *, p \u0026lt; 0.05). Inset, FISH showing \u003cem\u003eCaldatribacterium\u003c/em\u003e cells (left, small rod with strong labeling) and nanoSIMS ion ratio images reflecting \u003csup\u003e13\u003c/sup\u003eC assimilation in a \u003cem\u003eCaldatribacterium\u003c/em\u003e cell (right). \u003cstrong\u003eb,\u003c/strong\u003e Parallel experiment with a fucose co-culture containing \u003cem\u003eCaldatribacterium\u003c/em\u003e (orange bars) and \u003cem\u003eT. auxiliatoris\u003c/em\u003e (blue bars). Images shown are representative of ~20 \u003cem\u003eCaldatribacterium\u003c/em\u003e cells imaged after labeling with xylose for 2 hours. Source data for both panels and p-values are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure2FISHNanoSIMSV5WITHinsets.png","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/329c6889296763588671ddf7.png"},{"id":95605499,"identity":"94692d9d-55be-43c6-bff2-79fadcc9775f","added_by":"auto","created_at":"2025-11-11 06:59:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2337810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSemi-syntrophic growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaldatribacterium \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eis due to folate dependency.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Neither \u003cem\u003eCaldatribacterium \u003c/em\u003epure culture grew with filter-sterilized Wolin’s vitamins (Filt. Vit.), but growth could be restored by adding yeast extract (YE, 0.01% mass/vol) or by co-cultivation with a variety of SRBs. All experiments contained filter-sterilized Wolin’s vitamins. \u003cstrong\u003eb\u003c/strong\u003e, \u003cem\u003eCaldatribacterium \u003c/em\u003epure cultures grew with autoclaved Wolin’s vitamins but not filter-sterilized Wolin’s vitamins. Growth could be restored by adding folate (strain GBS\u003csup\u003eT\u003c/sup\u003e and SIUC1\u003csup\u003eT\u003c/sup\u003e) or biotin or riboflavin (strain SIUC1\u003csup\u003eT\u003c/sup\u003e). \u003cstrong\u003ec\u003c/strong\u003e, Growth of \u003cem\u003eCaldatribacterium \u003c/em\u003epure cultures could be restored by adding filtered and autoclaved supernatant (supt.) from SRB cultures to GBS salts medium (1:3 vol vol\u003csup\u003e-1\u003c/sup\u003e) but not SRB medium itself (SRB med.). SRB abbreviations: T.hv, \u003cem\u003eThermodesulfobacterium hveragerdense\u003c/em\u003e DSM 12571\u003csup\u003eT\u003c/sup\u003e; T.com,\u003cem\u003e Thermodesulfobacterium commune\u003c/em\u003e DSM 2178\u003csup\u003eT\u003c/sup\u003e; T. aux,\u003cem\u003e Thermodesulfobacterium auxiliatoris\u003c/em\u003e; and Tvib,\u003cem\u003e Thermodesulfovibrio yellowstonii\u003c/em\u003e. For all panels, bars show the mean and standard deviation. Bars with different letters are significantly different (p \u0026lt;0.05) based on ANOVA and Tukey’s post-hoc tests. N=3. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure3V51.png","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/76e66df1e13eb8c5878641b3.png"},{"id":95657264,"identity":"b64e07d2-c54e-425e-930a-11169d34cee4","added_by":"auto","created_at":"2025-11-11 16:20:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1036916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogeny and genomic relatedness in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaldatribacteriaceae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, A maximum-likelihood phylogeny based on the concatenated, partitioned sequence alignment of 120 conserved bacterial marker sequences (bac120), with appropriate evolutionary models for each partition. Filled circles represent supported nodes based on ≥80% SH-aLRT support and \u0026gt;95% support from ultra-fast bootstrapping (1000 pseudoreplicates). The two new strains are indicated in color and bold. \u003cstrong\u003eb\u003c/strong\u003e, Average nucleotide identity (ANI) and average amino acid identity (AAI) among \u003cem\u003eCaldatribacteriaceae\u003c/em\u003egenomes. The same numbering and color scheme is used. See \u003cstrong\u003eSupplementary Note 4\u003c/strong\u003e for additional discussion taxonomy.\u003c/p\u003e","description":"","filename":"Figure4bac120V31.png","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/cdd9e07dce8c42a2ed54d71b.png"},{"id":95655718,"identity":"392e3c98-015b-43f9-b8ec-5e2d97cc78e9","added_by":"auto","created_at":"2025-11-11 16:16:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2379191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCaldatribacterium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e cell structure. a\u003c/strong\u003e, Cryo-electron micrograph of SIUC1\u003csup\u003eT\u003c/sup\u003e cell with white arrow indicating denser cytosolic region. \u003cstrong\u003eb\u003c/strong\u003e, Closer cryo-electron micrograph view of strain SIUC1\u003csup\u003eT\u003c/sup\u003e with dark arrows indicating 1. outer, 2. middle, and 3. inner lipid membrane-like layers. \u003cstrong\u003ec\u003c/strong\u003e, Epifluorescence micrograph of GBS cells hybridized with Cy3-labeled \u003cem\u003eCaldatribacterium\u003c/em\u003e FISH probe and counterstained with DAPI. The white arrow indicates the cell shown in higher detail in \u003cstrong\u003ed\u003c/strong\u003e, with a profile of Cy3 (rRNA probe, red) and DAPI (DNA, blue) fluorescence along the axis of the cell.\u003c/p\u003e","description":"","filename":"Fig5TEMandFluorlowercase.png","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/0f1c14012ae77211ef27e25e.png"},{"id":95797127,"identity":"8aead8c0-87c8-41d8-86cd-9b030e9a1251","added_by":"auto","created_at":"2025-11-13 08:01:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14684076,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/3d9db781-99d4-4f54-b4ec-8482b01ec75e.pdf"},{"id":95656230,"identity":"c823bc38-d7fd-4c70-aa5e-d8218236d655","added_by":"auto","created_at":"2025-11-11 16:18:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3910468,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u003c/p\u003e","description":"","filename":"AdditionalFile1V29Submit.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/0977f048fdeba27096045df2.pdf"},{"id":95605493,"identity":"949f07bd-488a-4027-8882-555f50576746","added_by":"auto","created_at":"2025-11-11 06:59:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":618597,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 1-11\u003c/p\u003e","description":"","filename":"AdditionalFile2V29submit.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6739887/v1/6f7b92e062e20e0c66e18b69.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCulturability is highly uneven across the prokaryotic tree of life. Despite nearly 150 years of cultivation efforts\u003csup\u003e1\u003c/sup\u003e, most phyla have few or no representatives in pure culture\u003csup\u003e2\u003c/sup\u003e. One such poorly cultivated phylum is the \u003cem\u003eAtribacterota\u003c/em\u003e, formerly OP9/JS1 or “Atribacteria”. \u003cem\u003eAtribacterota\u003c/em\u003e 16S rRNA gene sequences were first obtained from Obsidian Pool, Yellowstone National Park, USA\u003csup\u003e3\u003c/sup\u003e and are now known to be widely distributed in anoxic environments\u003csup\u003e4,5,6,7,8\u003c/sup\u003e. The phylum is divided into two major classes that are physiologically and ecologically distinct. Members of the class \u003cem\u003ePhoenicibacteriia\u003c/em\u003e (JS1) are often abundant in hydrocarbon-containing terrestrial and marine sediments, aquifers, and oil reservoirs, where they may remain active over thousands to millions of years of sedimentation\u003csup\u003e9,10,11\u003c/sup\u003e. One study\u003csup\u003e9\u003c/sup\u003e detected abundant transcripts in deep marine sediments along with corresponding enzyme activities and metabolites and suggested sugar, protein, and allantoin catabolism by \u003cem\u003ePhoenicibacteriia.\u003c/em\u003e Another\u003csup\u003e12\u003c/sup\u003e suggested that some \u003cem\u003ePhoenicibacteriia\u003c/em\u003e were suggested to ferment short-chain alkanes, likely requiring hydrogenotrophic methanogens as a H\u003csub\u003e2\u003c/sub\u003e sink. Jiao et al., (2024)\u003csup\u003e8\u003c/sup\u003e provided experimental evidence of long-chain n-alkane fermentation in an enrichment culture containing \u003cem\u003ePhoenicimicrobium oleiphilum\u003c/em\u003e HX-OS.bin.34\u003csup\u003eTs\u003c/sup\u003e based on the high expression of key genes for alkane fermentation, including genes for fumarate-adding enzymes, hydrogenases, the reverse glycine pathway (RGP), and acetogenesis.\u0026nbsp;These authors\u003csup\u003e8\u003c/sup\u003e also suggested alkane fermentation is widespread in \u003cem\u003ePhoenicibacteriia\u003c/em\u003e based on the broad distribution of these genes. However, to date, no isolates of \u003cem\u003ePhoenicibacteriia\u003c/em\u003e have been described.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe other major class, \u003cem\u003eAtribacteria\u003c/em\u003e (OP9), is more common in terrestrial anoxic environments. Early single-cell genomic and metagenomic studies probed the metabolic capacity of \u003cem\u003eAtribacteria\u003c/em\u003e in hot springs and bioreactors\u003csup\u003e13,14,15\u003c/sup\u003e. In one of these studies, a member of the \u003cem\u003eAtribacteria\u003c/em\u003e, \u003cem\u003eCandidatus\u003c/em\u003e Caldatribacterium saccharofermentans, was enriched on lignocellulose substrates in Great Boiling Spring (GBS), NV, USA\u003csup\u003e16\u003c/sup\u003e and subsequently predicted to be a sugar fermenter based on the presence of genes encoding glycoside hydrolases, hydrogenases, acetogenesis, alcohol dehydrogenase, and a \u003cem\u003eRhodobacter\u003c/em\u003e nitrogen fixation (RNF) complex\u003csup\u003e13,15\u003c/sup\u003e. Other \u003cem\u003eAtribacteria\u003c/em\u003e were also predicted to be sugar\u003csup\u003e9\u003c/sup\u003e or propionate fermenters\u003csup\u003e15\u003c/sup\u003e, and the presence of some genes encoding the Wood-Ljungdahl pathway (WLP) in some \u003cem\u003eAtribacteria\u003c/em\u003e suggested the possibility for syntrophic acetate oxidation or carbon fixation\u003csup\u003e9,17,18\u003c/sup\u003e. Yet, a more recent analysis\u003csup\u003e8\u003c/sup\u003e showed that the carbonyl branch of the WLP is incomplete in \u003cem\u003eAtribacteria\u003c/em\u003e genomes, and most also lack the carbon monoxide dehydrogenase/acetyl-CoA synthetase complex. Instead,\u0026nbsp;they noted the near-universal presence of the RGP in \u003cem\u003eAtribacterota\u003c/em\u003e, indicating that the RGP, rather than WLP, is a conserved core of \u003cem\u003eAtribacterota\u003c/em\u003e metabolism and may enable CO\u003csub\u003e2\u003c/sub\u003e or formate assimilation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe first \u003cem\u003eAtribacteria\u003c/em\u003e pure culture, \u003cem\u003eAtribacter laminatus\u0026nbsp;\u003c/em\u003eRT761\u003csup\u003eT\u003c/sup\u003e, was isolated from a deep, methane-bearing aquifer\u003csup\u003e19\u003c/sup\u003e. \u003cem\u003eA. laminatus\u003c/em\u003e is a moderately thermophilic sugar fermenter that requires yeast extract for growth and produces H\u003csub\u003e2\u003c/sub\u003e, acetate, carbon dioxide, and ethanol. Its slow doubling time (~5 days) and low growth yield were enhanced in co-culture with a hydrogenotrophic methanogen, suggesting a semi-syntrophic interaction involving hydrogen scavenging. \u003cem\u003eA. laminatus\u003c/em\u003e has an unusual cell ultrastructure, described as three lipid membrane-like layers (LMLs), the innermost of which contains the nucleoid and most of the RNA\u003csup\u003e19\u003c/sup\u003e. These were interpreted to be (from outside to in) an outer membrane (outer LML), a cytoplasmic membrane (middle LML), and a separate intracytoplasmic membrane (inner LML). However, alternative interpretations involving a separated cytoplasmic membrane and an outer membrane with a closely associated S-layer or extensive cytoplasmic membrane invaginations have also been suggested\u003csup\u003e19,20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMore recently, Jiao et al., (2024)\u003csup\u003e8\u003c/sup\u003e and Kawamoto et al., (2024)\u003csup\u003e21\u003c/sup\u003e isolated two additional members of \u003cem\u003eAtribacteria\u003c/em\u003e, described as\u003cem\u003e\u0026nbsp;Thermatribacter velox\u003c/em\u003e B11\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eAtrimonas thermophila\u003c/em\u003e M15, from a terrestrial oil well and a terrestrial gas reservoir. However, these two isolates belong to the same species and the name \u003cem\u003eThermatribacter velox\u003c/em\u003e has priority over \u003cem\u003eAtrimonas thermophila\u003c/em\u003e and is used here for both strains, along with the family name \u003cem\u003eThermatribacteriaceae\u003c/em\u003e. Both strains ferment sugars and require yeast extract, producing H\u003csub\u003e2\u003c/sub\u003e and acetate as major products. They also have three LMLs, similar to \u003cem\u003eA. laminatus\u003c/em\u003e. Together,\u0026nbsp;the three isolates\u003cem\u003e\u0026nbsp;\u003c/em\u003ecomprise two of the three known families of \u003cem\u003eAtribacteria\u003c/em\u003e, with \u003cem\u003eCa.\u003c/em\u003e Caldatribacterium representing the third, yet-uncultivated family.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we describe enrichment and isolation of two species of \u003cem\u003eCaldatribacterium\u003c/em\u003e, \u003cem\u003eCaldiatribacterium saccharofermentans\u003c/em\u003e from a hot spring (GBS) and \u003cem\u003eCaldiatribacterium inferamans\u003c/em\u003e from a hot, fractured-rock aquifer in the discharge zone of the Death Valley Regional Flow System. We show that both \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003especies require folate and propose that folate precipitation during standard media preparation protocols is a key problem that limits isolation of all \u003cem\u003eAtribacterota\u003c/em\u003e. We also show that both \u003cem\u003eCaldatribacterium\u003c/em\u003e species contain three LMLs similar to \u003cem\u003eA. laminatus\u003c/em\u003e and \u003cem\u003eT. velox\u003c/em\u003e, but note that ribosomes are partially or fully separated from the nucleoid by the inner LML. The conserved ultrastructure and high percentage of transmembrane proteins support a previously proposed “membrane-centric” biology\u003csup\u003e19\u003c/sup\u003e and an important role for the unusual cell ultrastructure throughout the class.\u003c/p\u003e\n\u003cdiv style=\"bottom: 10px; right: 10px; position: absolute;\"\u003e\u003ca href=\"https://wordtohtml.net/?utm_source=wth_free_link\u0026utm_medium=external\" target=\"_blank\" style=\"font-size:11px; color: #d0d0d0;\"\u003eGenerated using WordToHTML.net - Online Word to HTML Converter\u003c/a\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEnrichment of two \u003cem\u003eCaldatribacterium\u003c/em\u003e strains with distinct SRBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on previous in situ enrichments of \u003cem\u003eCa\u003c/em\u003e. C. saccharofermentans on lignocellulose substrates\u003csup\u003e13,16\u003c/sup\u003e, fresh in situ enrichments containing 1 gram of ammonia fiber expansion (AFEX)-treated corn stover were incubated for five months in the outflow channel of GBS (\u003cstrong\u003eSupplementary Fig. 1\u003c/strong\u003e). Because \u003cem\u003eCa\u003c/em\u003e. C. saccharofermentans was previously suggested to catabolize hemicellulose based on the presence of a gene encoding a member of GH12 family of glycoside hydrolases\u003csup\u003e13\u003c/sup\u003e, an anaerobic medium containing xyloglucan as the sole carbon source was inoculated with material from the in situ enrichment, transported to the lab, and incubated at 73 \u0026deg;C. \u003cem\u003eCaldatribacterium\u003c/em\u003e 16S rRNA gene sequences were detected by PCR after two transfers (1/1000 vol vol\u003csup\u003e-1\u003c/sup\u003e), and the presence of \u003cem\u003eCaldatribacterium\u003c/em\u003e was stable for subsequent transfers, performed every two weeks thereafter. 16S rRNA gene amplicon sequencing of the xyloglucan cultures after one and two years of lab cultivation showed that \u003cem\u003eCaldatribacterium\u003c/em\u003e comprised 8-11% of total reads (2015 XG and 2016 XG, \u003cstrong\u003eFig. 1A\u003c/strong\u003e), and that the community was stable, consisting mainly of fermenters in the phyla \u003cem\u003eDictyoglomota\u0026nbsp;\u003c/em\u003e(two species of \u003cem\u003eDictyoglomus\u003c/em\u003e) and \u003cem\u003eThermotogota\u003c/em\u003e (two species of \u003cem\u003eFervidobacterium\u0026nbsp;\u003c/em\u003eand one of \u003cem\u003ePseudothermotoga\u003c/em\u003e), and the sulfate-reducing bacterium (SRB) \u003cem\u003eThermodesulfobacterium\u003c/em\u003e. These taxa were previously observed in similar\u003cem\u003e\u0026nbsp;\u003c/em\u003ein situ enrichments\u003csup\u003e16\u003c/sup\u003e. Shotgun metagenomics and binning into metagenome-assembled genomes (MAGs) after two years of maintenance yielded similar results (\u003cstrong\u003eSupplementary Fig. 2\u003c/strong\u003e). Fluorescence in situ hybridization (FISH) using an oligonucleotide specific for the 16S rRNA of \u003cem\u003eCaldatribacterium\u003c/em\u003e also confirmed the presence of \u003cem\u003eCaldatribacterium\u003c/em\u003e cells at ~3.5% abundance in xyloglucan enrichments (\u003cstrong\u003eSupplementary Fig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;The xyloglucan culture was used to inoculate media containing a variety of mono- or disaccharides as sole carbon sources to further enrich \u003cem\u003eCa\u003c/em\u003e. C. saccharofermentans. After two transfers, the absolute (1.1 \u0026times;10\u003csup\u003e8\u003c/sup\u003e - 5.5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e 16S rRNA gene copies mL\u003csup\u003e-1\u003c/sup\u003e) and relative (8-53% of compared to total 16S rRNA gene copies mL\u003csup\u003e-1\u003c/sup\u003e) abundance of \u003cem\u003eCaldatribacterium\u003c/em\u003e in these cultures with individual sugars was higher than in xyloglucan cultures (2.5%, 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e copies mL\u003csup\u003e-1\u003c/sup\u003e) (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e). Because the relative enrichment of \u003cem\u003eCaldatribacterium\u003c/em\u003e was highest with fucose as a sole carbon source and because fucose catabolism had been previously predicted from the \u003cem\u003eCa\u003c/em\u003e. C. saccharofermentans MAG\u003csup\u003e13\u003c/sup\u003e, these cultures were used for multiple rounds of dilution-to-extinction. In each dilution series, bottles receiving ~18 cells or more showed growth after 3-week incubations, while those with ~14 cells or less did not (\u003cstrong\u003eSupplementary Fig. 4A\u003c/strong\u003e). After multiple rounds of dilution-to-extinction, the abundance of \u003cem\u003eCaldatribacterium\u003c/em\u003e in the highest dilution showing growth was near or equal to that of total Bacteria and Archaea as assessed by qPCR (\u003cstrong\u003eSupplementary Fig. 4A\u003c/strong\u003e). The dominance of \u003cem\u003eCaldatribacterium\u003c/em\u003e in these dilution cultures, combined with our inability to obtain pure cultures by serial dilution, suggested a metabolic dependency. FISH confirmed enrichment of \u003cem\u003eCaldatribacterium\u003c/em\u003e to ~95% of total cells, but also revealed cells negative for the \u003cem\u003eCaldatribacterium\u003c/em\u003e-specific probe (\u003cstrong\u003eSupplementary Fig. 4B\u003c/strong\u003e). 16S rRNA gene amplicon sequencing and shotgun metagenomics of the fucose culture showed that it was a co-culture of \u003cem\u003eCaldatribacterium\u003c/em\u003e and a \u003cem\u003eThermodesulfobacterium\u003c/em\u003e species; \u0026gt;99.9% of 16S rRNA gene amplicon reads were either \u003cem\u003eCaldatribacterium\u003c/em\u003e or \u003cem\u003eThermodesulfobacterium\u003c/em\u003e (\u003cstrong\u003eFig. 1A\u003c/strong\u003e), and essentially all metagenome contigs were binned as either \u003cem\u003eCaldatribacterium\u003c/em\u003e or \u003cem\u003eThermodesulfobacterium\u003c/em\u003e (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). Additional dilution-to-extinction did not remove \u003cem\u003eThermodesulfobacterium\u003c/em\u003e from the culture, underscoring that the SRB was necessary for growth of \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eenriched from GBS.\u003c/p\u003e\n\u003cp\u003eA distinct enrichment containing \u003cem\u003eCaldatribacterium\u003c/em\u003e was obtained from a borehole, Inyo-BLM 1, which intersects the Lower Carbonate Aquifer near Death Valley National Park, CA\u003csup\u003e22,23,24\u003c/sup\u003e, using an approach inspired by Imachi \u003cem\u003eet al\u003c/em\u003e., 2011\u003csup\u003e25\u003c/sup\u003e. Sterile polyurethane foam plugs were suspended at regular intervals in an uncased section (depth of ~751 m) of Inyo-BLM 1 for three months (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e). The plugs were then removed from the borehole and transferred to a bottle containing reduced artificial groundwater medium (AGM)\u003csup\u003e26\u003c/sup\u003e, transported to the lab, and stored at 4 \u0026deg;C. In the lab, liquid from the plug/AGM sample was inoculated into serum bottles containing AGM amended with xylitol (10 mM) and yeast extract (0.01% mass vol\u003csup\u003e-1\u003c/sup\u003e), based on previous observations of sugar alcohol catabolism by some subsurface microorganisms\u003csup\u003e26\u003c/sup\u003e. Bottles were incubated at 60 \u0026deg;C and transferred (1/50 vol vol\u003csup\u003e-1\u003c/sup\u003e) to fresh medium every week. Although \u003cem\u003eCaldatribacterium\u003c/em\u003e was not detected in situ, 16S rRNA gene amplicon sequencing showed that the plug enriched for \u003cem\u003eCaldatribacterium\u003c/em\u003e (19.1% of reads), and the xylitol enrichment led to high relative abundance of \u003cem\u003eCaldatribacterium\u003c/em\u003e after multiple transfers (97.6% of reads) (\u003cstrong\u003eFig. 1C\u003c/strong\u003e). The most abundant non-\u003cem\u003eCaldatribacterium\u003c/em\u003e 16S rRNA gene amplicon in the xylitol enrichment was assigned to the SRB \u003cem\u003eThermodesulfovibrio\u003c/em\u003e (1.1% of reads), and \u003cem\u003eThermodesulfovibrio\u003c/em\u003e remained in low abundance after multiple rounds of dilution, suggesting the Inyo-BLM 1 \u003cem\u003eCaldatribacterium\u003c/em\u003e strain was also dependent on a SRB, albeit one belonging to a distinct phylum (\u003cem\u003eNitrospirota\u003c/em\u003e) from the SRB that was co-enriched with \u003cem\u003eCaldatribacterium\u003c/em\u003e from GBS (genus \u003cem\u003eThermodesulfobacterium\u003c/em\u003e and phylum \u003cem\u003eDesulfobacterota\u003c/em\u003e). A shotgun metagenome of the xylitol enrichment culture confirmed the high abundance of a \u003cem\u003eCaldatribacterium\u003c/em\u003e strain and \u003cem\u003eThermodesulfovibrio\u003c/em\u003e (\u003cstrong\u003eFig. 1D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAttempts to isolate either \u003cem\u003eCaldatribacterium\u003c/em\u003e strain on solid media with fucose (GBS enrichment) or xylitol (Inyo-BLM 1 enrichment) as sole carbon sources were unsuccessful, yet pure cultures of the respective SRBs were readily obtained. The \u003cem\u003eThermodesulfobacterium\u003c/em\u003e strain in the GBS fucose enrichment was isolated on GBS salts medium plates solidified with 1% mass vol\u003csup\u003e-1\u003c/sup\u003e Gelrite and 0.4% mass vol\u003csup\u003e-1\u003c/sup\u003e magnesium chloride hexahydrate containing 1 mM each of sodium bicarbonate, sodium thiosulfate, and sodium acetate, with 0.66 atm H\u003csub\u003e2\u003c/sub\u003e in the headspace (\u003cstrong\u003eSupplementary Note 1\u003c/strong\u003e). The \u003cem\u003eThermodesulfovibrio\u003c/em\u003e strain in the subsurface xylitol enrichments was isolated under similar conditions but with the addition of 5 mM lactate. For the \u003cem\u003eThermodesulfobacterium\u003c/em\u003e strain, autotrophic growth with H\u003csub\u003e2\u003c/sub\u003e as the electron donor was stimulated by acetate, and the isolate could also grow using lactate (\u003cstrong\u003eSupplementary Data 1\u003c/strong\u003e). It represents a new species, with 75.8-80.1% average nucleotide identity (ANI) to other members of the genus \u003cem\u003eThermodesulfobacterium\u003c/em\u003e, herein proposed as \u003cem\u003eThermodesulfobacterium auxiliatoris\u003c/em\u003e for its ability to help \u003cem\u003eCaldatribacterium\u003c/em\u003e grow (see below). The MAG of the \u003cem\u003eThermodesulfovibrio\u003c/em\u003e strain from the Inyo-BLM 1 xylitol enrichment had a genomic ANI of 98.55% with \u003cem\u003eThermodesulfovibrio yellowstonii\u0026nbsp;\u003c/em\u003eDSM 11347\u003csup\u003eT\u003c/sup\u003e and the 16S rRNA gene of the isolate was identical to that strain; it could grow using lactate or a combination of H\u003csub\u003e2\u003c/sub\u003e and acetate, similar to other members of this species\u003csup\u003e27\u003c/sup\u003e (\u003cstrong\u003eSupplementary Note 2\u003c/strong\u003e). Neither of these SRBs were able to grow on their own under the conditions of the \u003cem\u003eCaldatribacterium\u003c/em\u003e enrichment cultures they were isolated from (i.e., fucose or xylitol as sole carbon sources) or with other sugars as sole carbon sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCaldatribacterium\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in GBS enrichments assimilates sugars and amino acids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the absence of pure cultures, we used nanometer-scale secondary-ion mass spectrometry (nanoSIMS) coupled with FISH targeting \u003cem\u003eCaldatribacterium\u003c/em\u003e to assess incorporation of \u003csup\u003e13\u003c/sup\u003eC-labeled substrates in the GBS xyloglucan and fucose dilution-to-extinction cultures (\u003cstrong\u003eFig. 2\u003c/strong\u003e). In the xyloglucan enrichments, most cells assimilated \u003csup\u003e13\u003c/sup\u003eC atoms from labeled glucose, xylose, ribose, and amino acids, yet only \u003csup\u003e13\u003c/sup\u003eC incorporation from xylose was higher in \u003cem\u003eCaldatribacterium\u003c/em\u003e cells than other cells, suggesting direct uptake (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). In contrast,\u003cem\u003e\u0026nbsp;\u003c/em\u003ealthough both \u003cem\u003eCaldatribacterium\u003c/em\u003e cells and other cells were labeled after incubation with \u003csup\u003e13\u003c/sup\u003eC-bicarbonate, formate, or acetate, labeling was low (\u003csup\u003e13\u003c/sup\u003eC atom percent enrichment (\u003csup\u003e13\u003c/sup\u003eC APE) \u0026lt;0.5%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn fucose enrichments, uptake of glucose, xylose, and ribose was higher in \u003cem\u003eCaldatribacterium\u003c/em\u003e cells compared to \u003cem\u003eThermodesulfobacterium\u0026nbsp;\u003c/em\u003ecells, again suggesting direct uptake, yet acetate and amino acid assimilation was higher in \u003cem\u003eThermodesulfobacterium\u0026nbsp;\u003c/em\u003ecells (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). Little or no uptake of \u003csup\u003e13\u003c/sup\u003eC-bicarbonate, formate, or acetate was detected in \u003cem\u003eCaldatribacterium\u003c/em\u003e cells (APE \u0026lt;0.5%). These taxon-specific carbon uptake patterns suggest distinct roles for \u003cem\u003eCaldatribacterium\u003c/em\u003e and \u003cem\u003eThermodesulfobacterium\u003c/em\u003e in the GBS enrichment cultures and suggest potential metabolic interactions between \u003cem\u003eCaldatribacterium\u003c/em\u003e and the co-enriched SRBs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of \u003cem\u003eCaldatribacterium\u003c/em\u003e strains and semi-syntrophic growth with SRBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParallel co-enrichment of the two different\u003cem\u003e\u0026nbsp;Caldatribacterium\u003c/em\u003e cultures with unrelated hydrogenotrophic SRBs initially suggested a semi-syntrophic interaction based on interspecies electron transfer via H\u003csub\u003e2\u003c/sub\u003e or other fermentation products, as observed between \u003cem\u003eA. laminatus\u003c/em\u003e and \u003cem\u003eMethanothermobacter thermoautotrophicus\u003c/em\u003e\u003csup\u003e19\u003c/sup\u003e. Consortial fermentation of sugars was supported by the nanoSIMS results (\u003cstrong\u003eFigure 2\u003c/strong\u003e). For example, the rapid uptake of acetate but not sugars by \u003cem\u003eThermodesulfobacterium\u003c/em\u003e suggested that \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eand \u003cem\u003eThermodesulfobacterium\u003c/em\u003e work together to mineralize sugars, with acetate as a waste product of \u003cem\u003eCaldatribacterium\u003c/em\u003e sugar fermentation, as previously predicted\u003csup\u003e13,15\u003c/sup\u003e, and confirmed for both \u003cem\u003eA. laminatus\u003c/em\u003e\u003csup\u003e19\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;T. velox\u003c/em\u003e\u003csup\u003e8,21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, an alternative hypothesis is that \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003estrains\u003cem\u003e\u0026nbsp;\u003c/em\u003erely on SRBs for resources during growth on sugars as sole carbon sources. To address this alternative hypothesis and try to obtain pure cultures, dilution-to-extinction was repeated with the GBS fucose cultures, and the lowest dilution that did not exhibit visible growth was used to inoculate media in quintuplicate with fucose alone or with fucose plus yeast extract and casamino acids (0.1% mass vol\u003csup\u003e-1\u003c/sup\u003eeach). While cultures with fucose alone showed no visible growth after two weeks, all replicate cultures containing yeast extract and casamino acids (0.1% mass vol\u003csup\u003e-1\u003c/sup\u003e each) showed robust growth (up to 10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e) after four days of incubation. Because it appeared that complex organics, in addition to a carbohydrate fermentation substrate, enabled growth of \u003cem\u003eCaldatribacterium\u003c/em\u003e on its own, solid media with yeast extract and casamino acids (0.1% mass vol\u003csup\u003e-1\u003c/sup\u003eeach) in addition to fucose (for GBS enrichments) or fucose and xylitol (for Inyo-BLM 1 enrichments) was used to isolate \u003cem\u003eCaldatribacterium\u003c/em\u003e strains from the dilution-to-extinction cultures. Under these conditions, colonies were obtained and streak-purified multiple times to obtain axenic cultures. Once obtained, both \u003cem\u003eCaldatribacterium\u003c/em\u003e pure cultures could grow in liquid medium supplemented with yeast extract, casamino acids, and either fucose or xylitol. Culture purity was confirmed by FISH (\u003cstrong\u003eSupplementary Fig. 6\u003c/strong\u003e) in combination with total cell counts, genome sequencing (see below), and viable plate counts for both strains. Subsequent experiments determined that yeast extract alone, and not casamino acids, was necessary and sufficient for growth of the \u003cem\u003eCaldatribacterium\u003c/em\u003e pure cultures in the presence of fucose or xylitol.\u003c/p\u003e\n\u003cp\u003eThe isolated \u003cem\u003eCaldatribacterium\u003c/em\u003e strains, herein proposed as \u003cem\u003eCaldatribacterium saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eCaldatribacterium inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e under the rules of the International Code of Nomenclature of Prokaryotes (protologues below), were used to confirm that various SRBs could enable growth on defined media and to further probe the nature of semi-syntrophic growth. In the absence of yeast extract, growth of both \u003cem\u003eCaldatribacterium\u003c/em\u003e strains with fucose was supported by the addition of pure cultures of the \u003cem\u003eThermodesulfobacterium auxiliatoris\u003c/em\u003e and \u003cem\u003eThermodesulfovibrio yellowstonii\u0026nbsp;\u003c/em\u003estrains isolated in this study, as well as \u003cem\u003eThermodesulfobacterium commune\u003c/em\u003e DSM 2178\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eThermodesulfobacterium hveragerdense\u003c/em\u003e DSM 12571\u003csup\u003eT\u003c/sup\u003e (\u003cstrong\u003eFig. 3A\u003c/strong\u003e); under these conditions, \u003cem\u003eCaldatribacterium\u003c/em\u003e constituted the vast majority of cells as determined by FISH (\u003cstrong\u003eSupplementary Fig. 7\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCharacterization of the isolated \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003estrains showed that they could ferment a variety of sugars, with \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e having a somewhat wider range of fermentation substrates (\u003cstrong\u003eSupplementary Data 2)\u003c/strong\u003e. However, \u003cem\u003eC. saccharofermentans\u0026nbsp;\u003c/em\u003ecould not grow with xylose as a sole carbon and energy source even though xylose uptake was observed by nanoSIMS. Both strains grew best at circumneutral pH and had similar doubling times (7.6-8.3 hr), with \u003cem\u003eC. saccharofermentans\u003c/em\u003e having a slightly higher optimal growth temperature (70 \u0026deg;C) than \u003cem\u003eC. inferamans\u003c/em\u003e (60-70 \u0026deg;C), consistent with the environments from which they were isolated (\u003cstrong\u003eSupplementary Data 2, Supplementary Fig. 8)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo determine whether \u003cem\u003eCaldatribacterium\u003c/em\u003e growth could be stimulated by the removal of fermentation products by the SRBs, we added potential fermentation products to \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e pure cultures in fucose- and yeast-extract-amended media (\u003cstrong\u003eSupplementary Fig. 9\u003c/strong\u003e). Acetate and formate (1 mM) had no significant effect on growth and lactate up to 10 mM had only a mild inhibitory effect, with higher concentrations yielding a longer lag phase and slower growth. H\u003csub\u003e2\u003c/sub\u003e at up to 0.08 atm in the headspace slowed growth, while higher levels were inhibitory. Since \u003cem\u003eT. velox\u003c/em\u003e M15 was not stimulated by co-cultivation with a hydrogenotrophic methanogen\u003csup\u003e21\u003c/sup\u003e, and \u003cem\u003eCaldatribacterium inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e (\u003cstrong\u003eFig. 3\u003c/strong\u003e) and \u003cem\u003eT. velox\u003c/em\u003e B11\u003csup\u003eT\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e grow to high cell densities without a hydrogenotrophic partner, we infer they are also not strongly inhibited by their fermentation products. Thus, the \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eand \u003cem\u003eThermatribacter\u003c/em\u003e strains do not depend on syntrophic depletion of a fermentation product, in contrast to the much stronger stimulation of \u003cem\u003eA. laminatus\u003c/em\u003e growth following removal of H\u003csub\u003e2\u003c/sub\u003e from the headspace or in co-culture with a hydrogenotrophic methanogen\u003csup\u003e19\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistinct hydrogenase complexes may alter H\u003csub\u003e2\u003c/sub\u003e tolerance in different \u003cem\u003eAtribacteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; To probe the mechanism behind the differences in H\u003csub\u003e2\u003c/sub\u003e sensitivity between the \u003cem\u003eCaldatribacterium\u003c/em\u003e strains and \u003cem\u003eT. velox\u0026nbsp;\u003c/em\u003eB11\u003csup\u003eT\u003c/sup\u003e versus \u003cem\u003eA. laminatus\u003c/em\u003e, and to uncover other genomic features, we sequenced the genomes of both \u003cem\u003eCaldatribacterium\u003c/em\u003e strains with a combination of Illumina and Oxford Nanopore technologies and then assembled the data into single circular contigs that were 2,255,665 bp for \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and 1,967,957 bp for \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e. A maximum-likelihood phylogeny based on the bac120 marker protein set\u003csup\u003e28\u003c/sup\u003e confirmed that the two new isolates form a monophyletic clade with the previously published \u003cem\u003eCa\u003c/em\u003e. C. saccharofermentans MAG and the \u003cem\u003eCa\u003c/em\u003e. C. californiense SAG\u003csup\u003e13\u003c/sup\u003e, along with MAGs from the Shengli Oilfield in China and hot springs in Tengchong, China, and Tibet (taxa 1-8) (\u003cstrong\u003eFig. 4A\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Supplementary Data 3\u003c/strong\u003e). Based on relative evolutionary divergence and average amino acid identity, this clade is a distinct family of \u003cem\u003eAtribacterota\u003c/em\u003e, herein proposed as \u003cem\u003eCaldatribacteriaceae\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eSupplementary Note 3\u003c/strong\u003e). The \u003cem\u003eCaldatribacterium\u003c/em\u003e isolate genomes shared 92% ANI and 85% AAI (\u003cstrong\u003eFig. 4\u003c/strong\u003e), confirming they represent distinct species of a single genus.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e genomes each encoded at least two putative [FeFe] hydrogenases belonging to groups A1 (HydABC) and A3 (HndABCD) and an [FeFe] group C1 hydrogenase (\u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e), as previously noted for the \u003cem\u003eCa.\u003c/em\u003e C. saccharofermentans MAG and \u003cem\u003eCa.\u003c/em\u003e C. californiense SAG\u003csup\u003e8,13,15\u003c/sup\u003e. Group A3 hydrogenases are electron-bifurcating/confurcating enzymes that enable fermentative H\u003csub\u003e2\u003c/sub\u003e production with simultaneous oxidation of ferredoxin and NADH, and this enzyme was originally predicted to confer insensitivity to high H\u003csub\u003e2\u003c/sub\u003e concentrations in \u003cem\u003eCaldatribacterium\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e based on the insensitivity of the characterized homolog in \u003cem\u003eThermotoga maritima\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e; however, all of these [FeFe] hydrogenases have close orthologs in the genomes of \u003cem\u003eT. velox\u003c/em\u003e B11\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eT. velox\u003c/em\u003e M15, and \u003cem\u003eA. laminatus\u003c/em\u003e RT761\u003csup\u003eT\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e (\u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e). These cultivated \u003cem\u003eAtribacteria\u003c/em\u003e do however differ in that all except for \u003cem\u003eA. laminatus\u003c/em\u003e RT761\u003csup\u003eT\u003c/sup\u003e encode a [NiFe] group 3b hydrogenase. Characterized members of this group are bidirectional sulfhydrogenases that can oxidize NADH, with protons or sulfur as electron acceptors and H\u003csub\u003e2\u003c/sub\u003e or sulfide as products\u003csup\u003e30\u003c/sup\u003e. This [NiFe] group 3b hydrogenase was also speculated to enable \u003cem\u003eT. velox\u003c/em\u003e B11\u003csup\u003eT\u003c/sup\u003e and other \u003cem\u003eAtribacteria\u003c/em\u003e to participate in syntrophic anaerobic acetate oxidation by providing reducing power for the RGP\u003csup\u003e8\u003c/sup\u003e. We propose that it has a role in the relative H\u003csub\u003e2\u003c/sub\u003e-insensitivity of \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Thermatribacter\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFolate permits \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003egrowth in defined media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince semi-syntrophic growth of the \u003cem\u003eCaldatribacterium\u003c/em\u003e strains with SRBs was not due to the consumption of fermentation products, we hypothesized that one or more vitamin may be required for growth and could be provided either by the SRBs or by yeast extract. We initially carried out all cultivation experiments with Wolin\u0026rsquo;s vitamin solution\u003csup\u003e31,32\u003c/sup\u003e from a concentrated stock that was filter-sterilized, as recommended for media preparation\u003csup\u003e33\u003c/sup\u003e and commonly used in our labs, or with Vitamin Supplement MD-VS\u0026trade; from the American Type Culture Collection (ATCC); however, these solutions did not enable \u003cem\u003eCaldatribacterium\u003c/em\u003e growth in the experiments described above, such as the dilution-to-extinction cultures. We noted a precipitate in the concentrated Wolin\u0026rsquo;s vitamin solution that was removed during filter-sterilization, so we reasoned that one or more vitamins necessary for \u003cem\u003eCaldatribacterium\u003c/em\u003e growth may be removed during this step. To test this, we added unfiltered Wolin\u0026rsquo;s vitamin solution that was sterilized by autoclaving rather than filtration, and showed that it restores growth to levels equal to cultures with yeast extract (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). Knowing that autoclaving did not destroy the necessary vitamins, we tested single autoclaved vitamins to determine whether they could support \u003cem\u003eCaldatribacterium\u003c/em\u003e growth when added with the filtered Wolin\u0026rsquo;s vitamin solution. An initial analysis of \u003cem\u003eCaldatribacterium\u003c/em\u003e genomes suggested auxotrophy for folate, riboflavin, and biotin\u003csup\u003e13\u003c/sup\u003e (\u003cstrong\u003eSupplementary Data 5\u003c/strong\u003e). Although addition of autoclaved biotin or riboflavin to media with filtered vitamins failed to enable growth of \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and enabled only poor growth of \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e, folate completely restored growth in \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and substantially improved growth of \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). Subsequent experiments focusing on \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e showed that folate, riboflavin, and panthothenate were necessary and sufficient for growth on fucose in the absence of yeast extract, and these three vitamins plus biotin and cobalamin allowed for growth comparable to that with the full vitamin mixture and/or yeast extract (\u003cstrong\u003eSupplementary Fig. 10\u003c/strong\u003e). Additional experiments determined that a minimum of 0.5 ng mL\u003csup\u003e-1\u003c/sup\u003e of folate was necessary to support growth of strain GBS, although folate concentrations lower than 20 ng mL\u003csup\u003e-1\u003c/sup\u003e resulted in slower growth (\u003cstrong\u003eSupplementary Fig. S11\u003c/strong\u003e). We note that folate is nearly insoluble at neutral pH and insoluble below pH ~6\u003csup\u003e34,35\u003c/sup\u003e, and that recipes for concentrated Wolin\u0026rsquo;s vitamin solutions are unbuffered\u003csup\u003e31,33\u003c/sup\u003e. We also note that Wolin\u0026rsquo;s vitamin solution is sometimes referred to as Wolfe\u0026rsquo;s vitamin solution or by other names. For example, the ATCC sells these vitamins under the name Vitamin Supplement MD-VS\u0026trade; and describes them as a modification of \u0026ldquo;Wolfe\u0026rsquo;s Vitamin Solution\u0026rdquo; without citation. MD-VS\u0026trade; is identical to Wolin\u0026rsquo;s vitamin solution but is phosphate-buffered (900 mg L\u003csup\u003e-1\u003c/sup\u003e). We measured a pH of 6.0 in Vitamin Supplement MD-VS\u0026trade; and reiterate that AGW media prepared with MD-VS\u0026trade; successfully co-enriched \u003cem\u003eC. inferamans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eThermodesulfovibrio\u003c/em\u003e but did not enable isolation of \u003cem\u003eC. inferamans\u003c/em\u003e, presumably due to folate removal during filter-sterilization of Vitamin Supplement MD-VS\u0026trade; at the ATCC.\u003c/p\u003e\n\u003cp\u003eTo explore the possibility that the SRBs that enabled growth in co-culture could produce and secrete folate required by \u003cem\u003eCaldatribacterium\u003c/em\u003e, filter-sterilized supernatants from the SRB cultures or uninoculated SRB medium were added to the \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003emedium in the presence of filtered vitamins. All four SRB supernatants, but not the uninoculated SRB medium, supported growth of both \u003cem\u003eCaldatribacteirum\u003c/em\u003e strains (\u003cstrong\u003eFig. 3C\u003c/strong\u003e). Although these results suggested that folate was present in the SRB supernatants, a targeted metabolomics analysis did not detect folate monoglutamate with a minimal level of detection of ~0.25 ng mL\u003csup\u003e-1\u003c/sup\u003e (\u003cstrong\u003eSupplementary Data 6\u003c/strong\u003e). This could be because the supernatants contained a form of folate that was not detected by the metabolomics assay or that they contained another factor that could substitute for folate. Folate can take on a variety of forms, including polyglutamylated and N5- and N10-substituted forms that would have evaded detection in our metabolomics analysis\u003csup\u003e36,37\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFolate auxotrophy is conserved in \u003cem\u003eAtribacterota\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; To further assess folate metabolism in \u003cem\u003eCaldatribacterium\u003c/em\u003e and examine the broader significance of folate in \u003cem\u003eAtribacterota\u003c/em\u003e, we examined folate-dependent enzymes, biosynthesis, and transport pathways in the two complete \u003cem\u003eCaldatribacterium\u003c/em\u003e genomes and other \u003cem\u003eAtribacterota\u003c/em\u003e genomes (\u003cstrong\u003eSupplementary Data 4 and 5\u003c/strong\u003e). Folates are near-universal one-carbon donors and acceptors that can be synthesized de novo from GTP, chorismate, and glutamate by many bacteria via a pathway consisting of a pterin branch, a para-aminobenzoic acid (pABA) branch, and glutamylation reactions\u003csup\u003e38\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig. 12\u003c/strong\u003e). Both \u003cem\u003eCaldatribacterium\u003c/em\u003e isolates and most \u003cem\u003eAtribacterota\u003c/em\u003e MAGs and SAGs encode methionyl-tRNA formyltransferase (Fmt) and the formate-dependent form of thymidylate kinase (ThyA), indicating folate dependency for both translation and purine metabolism. Yet, \u003cem\u003eAtribacterota\u003c/em\u003e genomes lacked most known folate biosynthesis genes, including the key gene of the pterin branch hydroxymethyldihydropterin pyrophosphokinase (HPPK), the key genes of the pABA branch aminodeoxychorismate (ADC) synthase and ADC lyase, and dihydropteroate synthase (DHPS), which joins the pterin and pABA branches by condensing 6-hydroxymethyl-7,8-dihydropterin pyrophosphate and pABA. The absence of DHPS also suggests that pterin and pABA salvage would not satisfy folate needs in \u003cem\u003eAtribacterota\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eSupplementary Fig. 12\u003c/strong\u003e). Intact folate salvage has been partially characterized in some bacteria\u003csup\u003e39\u003c/sup\u003e and is the basis of its inclusion in media vitamin solutions. We found that energy-coupling factor (ECF) transporters with substrate-binding domains for folate were conserved across the \u003cem\u003eAtribacterota\u003c/em\u003e, as originally noted for \u003cem\u003eCa.\u003c/em\u003e Caldatribacterium genomes\u003csup\u003e13\u003c/sup\u003e (\u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e). Consistent with their use of added folate, \u003cem\u003eAtribacterota\u0026nbsp;\u003c/em\u003egenomes also encode folylpolyglutamyl synthase (FPGS) necessary for glutamylation reactions that would be necessary for use of transported folate. In contrast to the \u003cem\u003eAtribacterota\u003c/em\u003e genomes, the genomes of the SRBs that enabled \u003cem\u003eCaldatribacterium\u003c/em\u003e growth as defined co-cultures encoded complete de novo folate synthesis pathways (\u003cstrong\u003eSupplementary Fig. 13;\u003c/strong\u003e \u003cstrong\u003eSupplementary Data 5\u003c/strong\u003e). We also noted the absence of biosynthetic pathways for riboflavin and biotin across the \u003cem\u003eAtribacterota\u003c/em\u003e along with ECF substrate-binding domains for both riboflavin and biotin, although neither riboflavin nor biotin fully rescued growth of the \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003estrains in pure culture (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). Riboflavin was detected in spent medium from all four SRB pure cultures, as was niacinamide, with niacin and pantothenate detected in medium from some, but not all SRBs (\u003cstrong\u003eSupplementary Data 6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; To directly test the hypothesis that folate was removed during media preparation by filter-sterilization of the concentrated Wolin\u0026rsquo;s vitamin solution, we prepared Wolin\u0026rsquo;s vitamin solution as described\u003csup\u003e33\u003c/sup\u003e, added it to GBS salts medium, and quantified folate and other vitamins by targeted metabolomics. This experiment showed that folate, but not other vitamins, was removed by filter sterilization of the Wolin\u0026rsquo;s vitamin solution. In contrast, folate added as part of the unfiltered Wolin\u0026rsquo;s vitamin solution added prior to autoclaving was measurable in the final medium (\u003cstrong\u003eSupplementary Data 6\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eshares unusual cell structure and secreted proteins with other \u003cem\u003eAtribacterota\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the unusual cell ultrastructure of existing \u003cem\u003eAtribacterota\u003c/em\u003e isolates\u003csup\u003e8,19,21\u003c/sup\u003e and the predicted bacterial microcompartments (BMCs) in \u003cem\u003eCaldatribacterium\u003c/em\u003e and other \u003cem\u003eAtribacterota\u003c/em\u003e\u003csup\u003e15,40\u003c/sup\u003e, we examined their cell structure. Strains GBS\u003csup\u003eT\u003c/sup\u003e and SIUC1\u003csup\u003eT\u003c/sup\u003e were both rod-shaped or ovoid cells with somewhat pointed ends (\u003cstrong\u003eFig. 5 A,B; Supplementary Fig. S14\u003c/strong\u003e). The ultrastructure of strains GBS\u003csup\u003eT\u003c/sup\u003e and SIUC1\u003csup\u003eT\u003c/sup\u003e was observed by cryo-electron microscopy \u003cstrong\u003e(Fig. 5 A,B; Supplementary Fig. S14; Supplementary Data 7; Supplementary Data 8)\u003c/strong\u003e. Similar to \u003cem\u003eA. laminatus\u003c/em\u003e\u003csup\u003e19\u003c/sup\u003e, \u003cem\u003eT. velox\u003c/em\u003e B11\u003csup\u003eT\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e, and \u003cem\u003eT. velox\u003c/em\u003e M15\u003csup\u003e21\u003c/sup\u003e, dense cytosolic regions were observed for both strains, as well as three LMLs comprised of an internal membrane and a double layer at the cell boundary that could represent two membranes or a membrane and another structure such as an S-layer. We noted previously\u003csup\u003e13\u003c/sup\u003e that draft \u003cem\u003eCaldatribacterium\u003c/em\u003e genomes encode genes for synthesis and export of lipid A (\u003cem\u003elpxABCD\u003c/em\u003e; \u003cem\u003emsbA\u003c/em\u003e), but lacked genes required for production and attachment of keto-deoxyoctulosonate and the liposaccharide inner core. Analysis of the GBS\u003csup\u003eT\u003c/sup\u003e and SIUC1\u003csup\u003eT\u003c/sup\u003e genomes, along with all other \u003cem\u003eAtribacterota\u003c/em\u003e genomes, confirmed these observations (\u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e), consistent with an outer membrane with unusual lipopolysaccharide throughout the phylum.\u003c/p\u003e\n\u003cp\u003eEpifluorescence microscopy using the DNA stain DAPI and FISH with \u003cem\u003eCaldatribacterium\u003c/em\u003e 16S rRNA-targeted probes showed that rRNA and DNA were not colocalized within the cell. DAPI fluorescence was visible only within the compartmentalized nucleoids (more condensed in strain \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e) typically at or near the center of cells, yet the rRNA probe signal was distinct and concentrated near cell poles (\u003cstrong\u003eFig. 5 C,D; Supplementary Figs. 15 and 16)\u003csub\u003e.\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003eThis contrasts with similar analyses of \u003cem\u003eA. laminatus\u003c/em\u003e, which showed co-localization of DNA and total RNA within the central part of the cell, ribosomes in both the inner LML-bounded space and the middle LML-bounded space, and signal from rRNA-targeted FISH probes distributed throughout the middle of the cell but not at cell poles\u003csup\u003e19\u003c/sup\u003e. The localization of rRNA outside of the compartmentalized nucleoids is inconsistent with the interpretation that the three LMLs in \u003cem\u003eCaldatribacterium\u003c/em\u003e are an S-layer, an outer membrane, and a separated cytoplasmic membrane\u003csup\u003e19,20\u003c/sup\u003e, because that model would place rRNA in the periplasm. Thus, our data favor models where the nucleoids of \u003cem\u003eAtribacterota\u003c/em\u003e are either bound by a third membrane (inner LML) or by invaginations of the cytoplasmic membrane, as observed in some \u003cem\u003ePlanctomycetota\u003c/em\u003e\u003csup\u003e41\u003c/sup\u003e. However, invaginations of the second LML are not apparent in any images of any cultivated \u003cem\u003eAtribacterota\u003c/em\u003e\u003csup\u003e8,21\u003c/sup\u003e, including cryo-electron tomograms of \u003cem\u003eA.\u0026nbsp;\u003c/em\u003elaminatus\u003csup\u003e19\u003c/sup\u003e. BMCs were not observed in either strain, but their expression could depend on growth conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the unusual ultrastructure of \u003cem\u003eCaldatribacterium\u003c/em\u003e and since a \u0026ldquo;membrane-centric\u0026rdquo; metabolism was suggested previously for \u003cem\u003eA. laminatus\u003c/em\u003e based on a high percentage and expression of transmembrane proteins, proteins with unique N-terminal extensions, and a unique secretome that was poorly annotated by SignalP-4.1\u003csup\u003e19,42\u003c/sup\u003e, we examined the proteomes of the \u003cem\u003eCaldatribacterium\u003c/em\u003e strains and other \u003cem\u003eAtribacterota\u003c/em\u003e. We observed a similarly high proportion of proteins with transmembrane helices encoded by GBS\u003csup\u003eT\u003c/sup\u003e (29.7% of all proteins) and SIUC1\u003csup\u003eT\u003c/sup\u003e (30.2% of all proteins) and an abundance of unusual signal sequences that were recognized by the recurrent neural network implemented in SignalP-5.0\u003csup\u003e43\u003c/sup\u003e but not by Hidden-Markov Models (HMMs) implemented by SignalP-4.1 (\u003cstrong\u003eSupplementary Fig. 18\u003c/strong\u003e). In contrast to a previous report\u003csup\u003e19\u003c/sup\u003e, we note that \u003cem\u003eAtribacterota\u003c/em\u003e signal peptides recognized by SignalP-5.0 are less hydrophobic, have a lower isoelectric point, and are longer than other signal peptides in \u003cem\u003eAtribacterota\u003c/em\u003e and other diderms with unusual cell structures (\u003cstrong\u003eSupplementary Fig. 19; Supplementary Fig. 20\u003c/strong\u003e; \u003cstrong\u003eSupplementary Fig. 21\u003c/strong\u003e). Analysis of the signal peptidase recognition sites predicted by SignalP-5.0 and SignalP-4.1 revealed an enrichment of phenylalanine at the second position in the A-X-A consensus in \u003cem\u003eAtribacterota\u003c/em\u003e, \u003cem\u003eDicytoglomota\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Thermotogota\u003c/em\u003e (\u003cstrong\u003eSupplementary Fig. 22\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough \u003cem\u003eAtribacterota\u003c/em\u003e was designated as a candidate phylum 27 years ago (as OP9), it has continued to resist lab cultivation, with isolates representing two families being described only recently. Here, we used metagenomic data derived from a corn stover enrichment in GBS over a decade ago\u003csup\u003e13,15\u003c/sup\u003e to target members of a third family of\u003cem\u003e\u0026nbsp;Atribacterota\u003c/em\u003e. The annotation of pathways for sugar fermentation in the original \u003cem\u003eCa.\u003c/em\u003e C. saccharofermentans MAG and \u003cem\u003eCa.\u003c/em\u003e C. californiense SAG led us to provide sugars as sole carbon and energy substrates without terminal electron acceptors, and this approach allowed us to enrich and eventually isolate \u003cem\u003eC. saccharofermentans\u003c/em\u003e and \u003cem\u003eC. inferamans\u003c/em\u003e. Both species were indeed shown to be strictly anaerobic sugar and sugar alcohol fermenters that produce H\u003csub\u003e2\u003c/sub\u003e and acetate (\u003cstrong\u003eSupplementary Data 9\u003c/strong\u003e). This core metabolism is conserved in the three previously described isolates representing the two other families of \u003cem\u003eAtribacteria\u003c/em\u003e. Genes for this metabolism are conserved and ancestral in the class\u003csup\u003e8\u003c/sup\u003e, suggesting a common role for all \u003cem\u003eAtribacteria\u003c/em\u003e in consortial sugar fermentation. Basic phenotypic comparisons of all five \u003cem\u003eAtribacterota\u003c/em\u003e isolates, including membrane fatty acids, are presented in \u003cstrong\u003eSupplementary Data 2\u003c/strong\u003e and \u003cstrong\u003eSupplementary Data 10\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSugar fermentation to H\u003csub\u003e2\u003c/sub\u003e and acetate is highly exergonic\u003csup\u003e44\u003c/sup\u003e, and the isolation of primary sugar fermenters producing H\u003csub\u003e2\u003c/sub\u003e and acetate as the major fermentation products was described at least 83 years ago\u003csup\u003e45\u003c/sup\u003e. So why have \u003cem\u003eAtribacterota\u003c/em\u003e resisted lab cultivation? \u003cem\u003eA. laminatus\u003c/em\u003e was reported to be sensitive to H\u003csub\u003e2\u003c/sub\u003e accumulation and stimulated by the hydrogenotrophic methanogen \u003cem\u003eMethanothermobacter thermoautotrophicus\u003c/em\u003e through interspecies electron transfer. The \u003cem\u003eCaldatribacterium\u003c/em\u003e species described here cohabitate with hydrogenotrophs and probably also engage in interspecies electron transfer. \u003cem\u003eC. saccharofermentans\u003c/em\u003e was enriched in situ along with a newly discovered hydrogenotrophic methanogen in the \u003cem\u003eArchaeoglobaceae\u003c/em\u003e\u003csup\u003e16,46\u003c/sup\u003e and with the hydrogenotrophic SRB \u003cem\u003eThermodesulfobacterium auxiliatoris\u003c/em\u003e. In contrast, neither methanogens nor methanogenesis activity were evident in the Inyo-BLM 1 borehole from which \u003cem\u003eC. inferamans\u003c/em\u003e was obtained, yet hydrogenotrophic SRBs were abundant and active in situ\u003csup\u003e47\u003c/sup\u003e. The co-enrichment of \u003cem\u003eCaldatribacterium\u003c/em\u003e species in GBS and Inyo-BLM 1 with hydrogenotrophic SRBs also supports this model of consortial sugar fermentation, yet we showed that both \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003especies are relatively insensitive to H\u003csub\u003e2\u003c/sub\u003e accumulation. However, since the \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eisolates grew well in pure culture when supplemented with yeast extract or in defined co-culture with a variety of SRBs, we were forced to consider other mechanisms of dependency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter reexamining our genomic data, we designed experiments to test the hypothesis that vitamin auxotrophy is the basis of \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003eenrichment with SRBs and for semi-syntrophic growth with other anaerobes – a hypothesis that was supported by all of our experiments. This finding was surprising because most researchers seeking to cultivate new lineages - including ourselves - use Wolin’s vitamin solution\u003csup\u003e31,32\u003c/sup\u003e (also called Wolfe’s vitamin solution), which contains ten different vitamins and was developed 62 years ago for this purpose. For example, MediaDive\u003csup\u003e33\u003c/sup\u003e lists 362 different media using Wolin’s vitamin solution and all use filter sterilization rather than autoclaving to sterilize the stock vitamin solution. We show that filter sterilization removes precipitated folate from the stock vitamin solution and that folate and all seven other vitamins that were assayed were detectable after autoclaving. Since all \u003cem\u003eAtribacterota\u003c/em\u003e lack pathways for folate biosynthesis, we suggest that the unintentional removal of folate during media preparation\u003cem\u003e\u0026nbsp;\u003c/em\u003emay be a key problem limiting our ability to cultivate\u003cem\u003e\u0026nbsp;Atribacterota\u003c/em\u003e for nearly 150 years of microbiology research focused on pure cultures\u003csup\u003e1\u003c/sup\u003e. We note that neither Wolin\u003csup\u003e31,32\u003c/sup\u003e nor Balch\u003csup\u003e31,32\u003c/sup\u003e described how this vitamin solution should be sterilized, but we strongly suggest amendment of all media preparation instructions to avoid filter-sterilization of folate. The stability and solubility of folate solutions is also affected by other vitamins, with degradation catalyzed by riboflavin, ascorbic acid, and degradation products of thiamine, and solubility promoted by nicotinamide\u003csup\u003e48\u003c/sup\u003e. Other factors affecting folate solubility and stability have been reviewed\u003csup\u003e48\u003c/sup\u003e. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eCaldatribacterium\u003c/em\u003e isolates described here were similar in their cell ultrastructure to other isolates in the \u003cem\u003eAtribacteria\u003c/em\u003e, with what appears to be an intracellular membrane containing a nucleoid. One difference between the \u003cem\u003eCaldatribacterium\u003c/em\u003e isolates and the other\u003cem\u003e\u0026nbsp;Atribacterota\u0026nbsp;\u003c/em\u003eisolates is the profile of DNA and rRNA in the cell. \u003cem\u003eCaldatribacterium\u0026nbsp;\u003c/em\u003ehas a distinct distribution of DNA in the cell center and rRNA at the cell ends, apparently separated by the inner LML. The \u003cem\u003eAtribacterota\u003c/em\u003e are also unique, along with other thermophiles with unusual membrane architectures - \u003cem\u003eDicytoglomota\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Thermotogota\u0026nbsp;\u003c/em\u003e-\u003cem\u003e\u0026nbsp;\u003c/em\u003eby possessing a high percentage of unusual signal peptides and a high percentage of transmembrane proteins, congruent with a membrane-centric metabolism\u003cem\u003e.\u003c/em\u003e We suggest that the unique cell ultrastructure of these phyla should be a focus of future studies to better understand their origins and roles in ecophysiology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtologues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCaldiatribacterium\u003c/em\u003e gen. nov.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eCaldiatribacterium\u003c/em\u003e gen. nov. (L. adj. kal -di, hot; L. adj. ater -tra -trum, black; L. neut. n. bacterium, rod or staff. N.L. neut. n., \u003cem\u003eCaldiatribacterium\u003c/em\u003e, ‘black’ or ‘dark’ references both ‘microbial dark matter,’ referring to the poor culturability of the genus and phylum, and the dark, anoxic environments where it is found.\u003c/p\u003e\n\u003cp\u003eCells are rod-shaped or ovoid cells with pointed ends. Cells stain Gram-variable, but genomic data and cryo-EM reveal three lipid membrane-like structures including an outer membrane and a nucleoid that appears to be membrane-bound. No endospore formation is observed. Thermophilic. Obligately anaerobic chemoorganoheterotroph. Fermentation end products are acetate and H\u003csub\u003e2\u003c/sub\u003e. The major fatty acids (\u0026gt;10%) were C\u003csub\u003e13:0 iso 3OH\u003c/sub\u003e, C\u003csub\u003e15:0 iso\u003c/sub\u003e, C\u003csub\u003e16:0\u003c/sub\u003e, and C\u003csub\u003e18:0\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe type species is \u003cem\u003eCaldiatribacterium saccharofermentans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCaldiatribacterium saccharofermentans\u003c/em\u003e sp. nov.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eCaldiatribacterium saccharofermentans\u003c/em\u003e sp. nov.\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003esac.cha.ro.fer.men′tans. Gr. n. \u003cem\u003esakchâr\u003c/em\u003e sugar; L. part. adj. \u003cem\u003efermentans\u003c/em\u003e fermenting; N.L. neut. n. \u003cem\u003eSaccharofermentans\u003c/em\u003e sugar-fermenting). \u003cem\u003eCaldiatribacterium saccharofermentans\u003c/em\u003e, “The mysterious heat-loving sugar-fermenting rod.”\u003c/p\u003e\n\u003cp\u003eCells are rod-shaped or ovoid cells with somewhat pointed ends 1.5-3.0 µm long and 0.5-0.9 µm wide, occurring singly or in pairs. Flagellar motility rarely observed, but flagella observed by cryo-EM. Flagellar biosynthesis gene clusters complete. Optimal growth temperature is 70 \u003csup\u003eo\u003c/sup\u003eC, with a maximum of 77.5 \u003csup\u003eo\u003c/sup\u003eC and minimum of 60 \u003csup\u003eo\u003c/sup\u003eC. Optimal pH is 7.0 with a range of 5.5–9.0. Obligately anaerobic chemoorganoheterotroph that ferments sugars and sugar alcohols as sole carbon sources and fermentation substrates: adonitol, cellobiose, fructose, fucose, galactose, gluconate, glucose, inositol, lyxose, maltose, mannose, N-acetylglucosamine, raffinose, rhamnose, ribose, sorbitol, sucrose, tagatose, and xylitol. Fermentation end products are acetate and H\u003csub\u003e2\u003c/sub\u003e. The major fatty acids (\u0026gt;10%) were C\u003csub\u003e13:0 iso 3OH\u003c/sub\u003e, C\u003csub\u003e16:0\u003c/sub\u003e, and C\u003csub\u003e18:0\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe type strain is GBS\u003csup\u003eT\u003c/sup\u003e (DSM 110181\u003csup\u003eT\u003c/sup\u003e and JCM C190371\u003csup\u003eT\u003c/sup\u003e), which was isolated from Great Boiling Spring, NV., USA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCaldiatribacterium inferamans\u003c/em\u003e sp. nov.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eCaldiatribacterium inferamans\u003c/em\u003e sp. nov.\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eL. pl. n. inferi, the underworld; L. pres. part. amans, loving; N.L. part. adj. \u003cem\u003einferamans\u003c/em\u003e, loving the underworld referring to the origin of the strain). \u003cem\u003eCaldiatribacterium inferamans\u003c/em\u003e, “The mysterious heat-lover from the underworld” or “The mysterious heat-loving rod that loves the hot underworld.”\u003c/p\u003e\n\u003cp\u003eCells are rod or ovoid cells with somewhat pointed ends 1.0-2.6 µm long and 0.5-0.8 µm wide, typically occurring singly. Flagellar motility rarely observed, but flagella observed by cryo-EM. Flagellar biosynthesis gene clusters complete. Optimal growth temperature is 60-70 \u003csup\u003eo\u003c/sup\u003eC, with a maximum of 75 \u003csup\u003eo\u003c/sup\u003eC and minimum of 55 \u003csup\u003eo\u003c/sup\u003eC. Optimal pH is 7.4-8.7 with a range of 5.9–9.5. Obligately anaerobic chemoorganoheterotroph that ferments sugars, sugar alcohols, and proteins as a sole carbon source and electron donor/acceptor for growth: arabinose, cellobiose, fructose, fucose, fumarate, galactose, gluconate, glucose, lactose, maltose, mannitol, mannose, peptone, raffinose, sorbitol, starch, succinate, sucrose, trehalose, xylose, xylitol, xylan, and yeast extract. Fermentation end product is acetate and H\u003csub\u003e2\u003c/sub\u003e. The estimated genome size of SIUC1\u003csup\u003eT\u003c/sup\u003e is 1,967,957 bp, with 56.5 mol% DNA G + C content. The major fatty acids (\u0026gt;10%) were C\u003csub\u003e13:0 iso 3OH\u003c/sub\u003e, C\u003csub\u003e15:0 iso\u003c/sub\u003e, C\u003csub\u003e16:0\u003c/sub\u003e, and C\u003csub\u003e18:0\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe type strain is SIUC1\u003csup\u003eT\u003c/sup\u003e (DSM 110249\u003csup\u003eT\u003c/sup\u003e and JCM 39079\u003csup\u003eT\u003c/sup\u003e), which was isolated from the Deep Carbonate Aquifer located in the southern hydrographic Great Basin, NV, USA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCaldatribacteriaceae\u003c/em\u003e fam nov.\u003c/strong\u003e \u003cem\u003eCaldiatribacteriaceae\u003c/em\u003e (L. adj. kal -di, hot; L. adj. ater -tra -trum, black; L. neut. n. bacterium, rod or staff. N.L. masc. n. \u003cem\u003eCaldatribacterium\u003c/em\u003e type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. \u003cem\u003eCaldiatribacteriaceae\u003c/em\u003e the family of the genus \u003cem\u003eCaldatribacterium\u003c/em\u003e). Belongs to the order \u003cem\u003eAtribacterales\u003c/em\u003e, class \u003cem\u003eAtribacteria\u003c/em\u003e, and phylum \u003cem\u003eAtribacterota\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eThermodesulfobacterium auxiliatoris\u0026nbsp;\u003c/em\u003esp. nov.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eThermodesulfobacterium auxiliatoris\u003c/em\u003e sp. nov (L. gen. masc. n. auxiliatoris, one who gives aid). \u003cem\u003eThermodesulfobacterium auxiliatoris\u003c/em\u003e, \"The thermophilic sulfate reducing rod who provides aid,\" based on its ability to facilitate growth of \u003cem\u003eCaldatribacterium\u003c/em\u003e in co-culture.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cells are regular rods averaging 1.5 μm long and 0.5 μm wide, usually occurring singly. Diderm cell envelope structure. Optimal growth temperature is 70-75 ºC with a maximum of 80 ºC and a minimum of 50 ºC. Optimal pH is 7.0 with a range of 5.5-8.0. Facultative chemolithoautotroph capable of using H\u003csub\u003e2\u003c/sub\u003e as the electron donor and CO\u003csub\u003e2\u003c/sub\u003e and the carbon source, with sulfate as the terminal electron acceptor. Acetate stimulates growth under autotrophic conditions, and formate and lactate can serve as electron donors and carbon sourcesfor heterotrophic growth (\u003cstrong\u003eSupplementary Data 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe type strain is TA1\u003csup\u003eT\u003c/sup\u003e (DSM 107971\u003csup\u003eT\u003c/sup\u003e and JCM 32932\u003csup\u003eT\u003c/sup\u003e), isolated from Great Boiling Spring, NV, USA.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInclusion and ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research included local researchers as full authors, when possible, to recognize both logistical and intellectual contributions. No potential or listed authors were discriminated against on the basis of gender, race, ethnicity or any other factors not related to scientific contributions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e genomes and are available in GenBank under accession numbers CP187957 and CP189974. Raw reads for all metagenomes and 16S rRNA gene amplicons are available from the Sequence Read Archive (SRA) within BioProjects PRJNA1250235 and BioProject PRJNA956542 at the accessions in \u003cstrong\u003eSupplementary Data 11\u003c/strong\u003e. \u0026nbsp;GenBank accession numbers for GBS\u003csup\u003eT\u003c/sup\u003e, SIUC1\u003csup\u003eT\u003c/sup\u003e, and \u003cem\u003eT. auxiliatoris\u003c/em\u003e 16S rRNA genes are MN114535, MT023787, and MH513620. All accessions are tabulated in \u003cstrong\u003eSupplementary Data 11\u003c/strong\u003e. Source data are provided with this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Scripts used to account for vitamin biosynthetic pathways and compare signal peptides are available on GitHub: https://github.com/Hedlund-Lab/atribacterota-2025.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eGBS in situ enrichment and isolation of strain GBS\u003csup\u003eT\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e was obtained from GBS, a circumneutral pH, Na–Cl spring in northwestern Nevada (40°39’41.3”N, 119°21’58.1”W)\u003csup\u003e49\u003c/sup\u003e. In situ enrichments consisting of 1 g of AFEX-treated corn stover sealed in 100 µm nylon mesh bags were performed essentially as described in ref\u003csup\u003e16\u003c/sup\u003e, because previous in situ\u003cem\u003e \u003c/em\u003eenrichments contained populations of \u003cem\u003eCa.\u003c/em\u003e Caldatribacterium saccharofermentans\u003csup\u003e13\u003c/sup\u003e. The bags were incubated at the water/sediment interface at the \"C site\" in the outflow of GBS from 26 October 2013 to 28 March 2014. Immediately after removal, the corn stover was transferred to a 25 mL serum tube containing 10 mL of sterile, anaerobic GBS spring water (previously collected, sparged with N\u003csub\u003e2\u003c/sub\u003e, autoclaved, and reduced by addition of cysteine to 0.05%) under a stream of N\u003csub\u003e2\u003c/sub\u003e. The tube was sealed and shaken, and then 0.1 mL of the contents was transferred using an N\u003csub\u003e2\u003c/sub\u003e-flushed, sterile needle and syringe to 50 mL of anaerobic medium in a 150 mL serum vial sealed with a butyl rubber stopper. The medium was GBS salts medium\u003csup\u003e50\u003c/sup\u003e with 5 mM sodium phosphate pH 7 buffer and a filtered vitamin solution (see below) (1/100 dilution), but with xyloglucan (0.02% mass vol\u003csup\u003e-1\u003c/sup\u003e) as the sole carbon source, reduced with 0.01% sodium sulfide, and with an N\u003csub\u003e2\u003c/sub\u003e headspace. The 100×-concentrated Wolin’s vitamin solution contained per liter: 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine HCl, 5 mg thiamine HCl, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg DL-calcium pantothenate, 0.1 mg vitamin B12, 5 mg \u003cem\u003ep\u003c/em\u003e-aminobenzoic acid, and 5 mg lipoic acid\u003csup\u003e31,32,33\u003c/sup\u003e, and was sparged with N\u003csub\u003e2\u003c/sub\u003e for 1 hour prior to sterilization. Unless otherwise noted, the 100×-concentrated Wolin’s vitamin solution was filtered-sterilized (0.2 μm nylon filters; 09-719C, Fisher Scientific, Waltham, MA, USA) and stored at 4 ºC in a foil-covered borosilicate glass bottle the dark until it was added to 1x after the medium was autoclaved. After the importance of folate and its problem with solubility was recognized, the 100×-concentrated Wolin’s vitamin solution was sterilized instead by autoclaving for 30 min at 121 °C.\u003c/p\u003e\n\u003cp\u003eThe inoculated medium was brought back to the lab and incubated at 73 °C without shaking within 24 hours of sampling. The enrichment culture was initially transferred to fresh medium (1/1000 dilution) after a one-week incubation for the first two weeks, and then it was transferred every two weeks thereafter. Subsequent experiments replaced xyloglucan with various individual sugars or other substrates. Dilution-to-extinction was performed using fucose (0.02% mass vol\u003csup\u003e-1\u003c/sup\u003e) using 10-fold serial dilutions up to 10\u003csup\u003e-10\u003c/sup\u003e dilution. Isolation was performed using media solidified with Gelrite (0.8% mass vol\u003csup\u003e-1\u003c/sup\u003e) and magnesium chloride hexahydrate (0.4% mass vol\u003csup\u003e-1\u003c/sup\u003e). Plates were prepared in an anaerobic chamber and incubated in anaerobic incubation vessels for 7-10 days with an N\u003csub\u003e2\u003c/sub\u003e headspace\u003csup\u003e51\u003c/sup\u003e. Single colonies were streaked for isolation three times. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWell Inyo-BLM 1 and in situ enrichment and isolation of strain SIUC1\u003csup\u003eT\u003c/sup\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. inferamans\u003c/em\u003e SIUC1\u003csup\u003eT\u003c/sup\u003e was isolated from a sterile polyurethane foam plug that was suspended in borehole/well Inyo-BLM 1 (36º 24’04.19 N / 116º 28’06.58 W) for three months. This borehole was completed in 2007 reaching a depth of 883.5 m below land surface (mbls) by The Hydrodynamics Group, LLC (Edmonds, WA, USA) on behalf of the US Department of Energy, Inyo CO, CA, and US National Park Service as part of a suite of holes used to test hydrologic connectivity across the Funeral Mountains adjacent to Death Valley, CA, USA\u003csup\u003e52\u003c/sup\u003e. It transects lithologies ranging from volcanic tuff and valley-fill alluvium to lake sediments, ultimately intersecting Paleozoic dolomite that hosts a low-transmissivity portion\u003csup\u003e53\u003c/sup\u003e of the Lower Carbonate Aquifer of the Death Valley Regional Flow System at 748 mbls\u003csup\u003e52,54\u003c/sup\u003e. The plug was recovered from an inferred fracture zone roughly corresponding to a partially collapsed void\u003csup\u003e22\u003c/sup\u003e immediately below the casing terminus ~750 – 753 mbls. After retrieval, the foam plug was placed into pre-reduced artificial groundwater medium (AGM) as previously described by Hamilton-Brehm et al. 2019\u003csup\u003e26\u003c/sup\u003e. For initial enrichment and isolation attempts, vitamins were supplied as Vitamin Supplement MD-VS™ from the ATCC before autoclaving. An additional sample of the aquifer water without enrichment was obtained from Inyo-BLM 1 at ~752 m via a gas-tight discrete sampler (“bailers,” Comprobe, Inc.; Fort Worth, TX)\u003csup\u003e22\u003c/sup\u003e. 250 ml of the bailed water was filtered using a 0.22 μm PES filter (UX-06730-43, Thermo Scientific; Waltham, MA). 250 μl of Inyo-BLM 1 water was also to the PowerSoil lysis tubes to prevent detection bias against small cells\u003csup\u003e22\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eLab cultivation was conducted by removing 1 mL of liquid from the stored sponge in AGM and injecting it into a 160 mL serum bottle containing 50 mL (2% vol vol\u003csup\u003e-1\u003c/sup\u003e inoculum) of AGM amended with 10 mM xylitol and 0.01% mass vol\u003csup\u003e-1\u003c/sup\u003e yeast extract. The final pH of the medium was 8.0 after reduction of the solution. Enrichments were incubated at 60 °C without shaking, and transferred every seven days, three times, using a 0.5% vol vol\u003csup\u003e-1\u003c/sup\u003e inoculum into sterile anaerobic AGM media in 160 mL serum bottles. Cell morphology and density was monitored using an Axioskop2 Plus microscope and a Petroff-Hauser counter under phase contrast. Dilution-to-extinction was conducted three times, which successfully enriched the culture to \u0026gt;90% \u003cem\u003eC. inferamans\u003c/em\u003e but isolates of \u003cem\u003eC. inferamans\u003c/em\u003e were not isolated on the defined AGM medium with vitamins supplied as Vitamin Supplement MD-VS™. A clonal isolate was generated from the third dilution-to-extinction culture by picking a colony and restreaking it two additional times on anaerobic medium solidified with Gelrite (0.8% mass vol\u003csup\u003e-1\u003c/sup\u003e) and magnesium chloride hexahydrate (0.4% mass vol\u003csup\u003e-1\u003c/sup\u003e), with 10 mM xylitol, 0.05% (mass vol\u003csup\u003e-1\u003c/sup\u003e) yeast extract, and 0.05% (mass vol\u003csup\u003e-1\u003c/sup\u003e) casamino acids. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16S rRNA gene qPCR and amplicon analysis of enrichment cultures \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor GBS enrichments, DNA extraction using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) and 16S rRNA gene amplicon sequencing was done with an Illumina MiSeq (2x250) using 806r reverse primer a modified 515f-mod forward primer (5′ GTGYCAGCMGCCGCGGTAA) to enhance coverage of archaea\u003csup\u003e46\u003c/sup\u003e. Quantitative PCR (qPCR) was done using primers OP9_16S_F (5' AGGAAAGCTGGCCTCTGC) and OP9_16S_R (5' ACCGTCACAGGAAGGAGC) targeting \u003cem\u003eCaldatribacterium\u003c/em\u003e with primers at 64 °C annealing temperature or primers 515f-mod and 806r targeting total bacteria and archaea\u003csup\u003e46\u003c/sup\u003e. The standard was a plasmid containing a fragment of the \u003cem\u003eCaldatribacterium\u003c/em\u003e 16S rRNA gene (SSW_L1_H02; ref.\u003csup\u003e55\u003c/sup\u003e). \u003c/p\u003e\n\u003cp\u003eFor the Inyo-BLM 1 aquifer sample and in situ enrichment (foam plug), DNA was extracted using the MoBio Powersoil DNA Isolation Kit (Carlsbad, CA, United States). Lab enrichment cultures were extracted using the MoBio UltraClean\u003csup\u003e \u003c/sup\u003eMicrobial DNA Isolation Kit (Carlsbad, CA, United States), both according to the manufacturer’s protocol amended with one freeze/thaw cycle (-80 \u003csup\u003eo\u003c/sup\u003eC/65 \u003csup\u003eo\u003c/sup\u003eC) at the beginning of DNA extraction procedure. DNA concentration was determined using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (ThermoScientific, Waltham, MA, USA) with wavelength settings of 260 nm and 280 nm. Extracted DNA was sent to The Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory (Lemont, IL, USA) and sequenced using the Illumina MiSeq platform (2x151 bp). Universal bacterial primers targeting the V4 region of the 16S rRNA gene were used for diversity analysis\u003csup\u003e56\u003c/sup\u003e. Paired-end reads were merged, denoised, and demultiplexed using deML\u003csup\u003e57\u003c/sup\u003e. For both datasets, amplicon sequence variants (ASVs) were processed using the DADA2 pipeline\u003csup\u003e58\u003c/sup\u003e implemented in R using default parameters (https://benjjneb.github.io/dada2/tutorial.html), and taxonomy was assigned using SILVA release v123 (www.arb-silva.de/documentation/release-123/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStable isotope labeling \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable isotope labeling was performed with xyloglucan mixed cultures and fucose co-cultures derived from GBS. Universally \u003csup\u003e13\u003c/sup\u003eC-labeled substrates (Cambridge Isotope Laboratories) were added to freshly transferred enrichments grown for two days from concentrated, anaerobic stocks to the microcosms at 1 mM final concentration unless otherwise noted: bicarbonate, formate, acetate, glucose, xylose, ribose, or algal amino acids (0.01% mass vol\u003csup\u003e-1\u003c/sup\u003e). Negative controls with no compound added were incubated in parallel. All incubations were done at 73 °C without shaking for 2 hours. After incubation, cells were pelleted by centrifugation for 5 min at 16,100 × g, pooled in 0.5 mL of 1× phosphate-buffered saline (PBS), and 0.25 mL of freshly prepared 3% paraformaldehyde (PFA) was added and mixed. Samples were fixed for 1 hour on ice, cells were pelleted by centrifugation for 5 min at 9,200 ×g, and pellets were washed twice with 1×PBS. Washed cells were resuspended in 200 µL of 50% ethanol, and stored at –20 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFISH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFISH was performed using protocol v 2.2\u003csup\u003e59\u003c/sup\u003e (www.arb-silva.de/fish-probes/fish-protocols/) using the \u003cem\u003eCaldatribacterium\u003c/em\u003e-specific 16S rRNA probe OP9-480 (5’-AGCTRTTCACCCCTYCCCTC-3’) labeled with Cy3 and the Bacteria-specific 16S rRNA probe Bact927\u003csup\u003e60\u003c/sup\u003e (5’-ACCGSTTGTGCGGGCCC-3’) labeled with 6-FAM. Cells from cultures were pelleted by centrifugation (10,000 × g for 5 minutes), washed in 1× PBS, and fixed with 1% paraformaldehyde in 1× PBS on ice for 1 hour. After fixation, the cells were pelleted, washed three times in 1× PBS, resuspended in 50% ethanol and stored at -20 \u003csup\u003eo\u003c/sup\u003eC. Hybridization was performed on slides at 46 °C with 30% vol vol\u003csup\u003e-1\u003c/sup\u003e formamide. Hybridization conditions were optimized for the OP9-480 probe using the Clone-FISH technique\u003csup\u003e61\u003c/sup\u003e via expression of the near-full-length 16S rRNA gene sequence of a \u003cem\u003eCaldatribacterium\u003c/em\u003e relative obtained from Mud Hot Springs\u003csup\u003e55\u003c/sup\u003e (SSW_L1_H02) in \u003cem\u003eE. coli\u003c/em\u003e strain JM109 (DE3) from the plasmid pGEM-T. After hybridization, cells were counterstained with DAPI (1 µg mL\u003csup\u003e-1\u003c/sup\u003e). Cells were visualized by epifluorescence microscopy using an Eclipse Ti-U inverted microscope (Nikon, Melville, NY, USA) equipped for epifluorescence with Nikon filter sets compatible with Cy3 (96312 G-2E/C), 6-FAM (96343 EN GFP), and DAPI (96310 UV-2E/C), with image capture using a Retiga-SRV camera (QImaging, Surrey, BC, Canada) and Nikon Elements v4.13 software. Isotopically labeled cells were deposited onto ITO-coated slides and visualized with a Leica DM5500B microscope using MetaMorph software with a 100× magnification dry immersion objective. Fluorescence and brightfield images were collected and the X-Y and fiducial locations were noted to enable navigation in the nanoSIMS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanoSIMS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanoSIMS was conducted on a CAMECA NanoSIMS 50 at Lawrence Livermore National Laboratory. Fiducial locations and locations of FISH-positive cells were found with a charge-coupled device camera using X-Y coordinates and a real-time imaging unit. The primary Cs\u003csup\u003e+\u003c/sup\u003e ion beam was set to 1.5 pA, corresponding to an approximately 150 nm beam diameter at 16 keV. Rastering was performed over 20 × 20 μm areas with a dwell time of 1 ms pixel\u003csup\u003e–1\u003c/sup\u003e for 19-30 scans and generated images containing 256 × 256 pixels, yielding data for 10-114 \u003cem\u003eCaldatribacterium\u003c/em\u003e cells per experiment (\u003cstrong\u003eFig. 2\u003c/strong\u003e). Sputtering equilibrium at each area was achieved with an initial beam current of 90 pA to a depth of ~10 nm. After tuning the SIMS for mass resolving power of ~7000, secondary electron images and quantitative secondary ion images were simultaneously collected for \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e–\u003c/sup\u003e and \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e-\u003c/sup\u003e on individual electron multipliers in pulse counting mode. NanoSIMS data were initially processed using L’Image (http://limagesoftware.net) to perform deadtime and image shift correction of ion image data before creating \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e12\u003c/sup\u003eC/\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e ratio images, which reflected the level of \u003csup\u003e13\u003c/sup\u003eC incorporation into biomass. Regions of interest for isotopic ratio quantification were drawn manually around each cell. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePure culture experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of strain GBS\u003csup\u003eT\u003c/sup\u003e was performed in 10 mL of GBS salts medium in 20 mL serum vials. Substrates were tested at either 0.05% mass vol\u003csup\u003e-1\u003c/sup\u003e for sugars and complex organic substrates, 1 mM for organic acids, 0.1 atm methane, or 0.4/0.1 atm H\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e. Cultures were incubated in the dark without shaking at 73 °C, and growth was assessed by phase-contrast microscopy using a Petroff-Hauser cell counter after seven days of incubation. Growth rate tests for pH (using 5 mM sodium phosphate buffer) and temperature optima were performed using 0.05% fucose as a growth substrate, with samples taken every 12-24 hours after growth was initially observed. Growth was tested at 50-80 \u003csup\u003eo\u003c/sup\u003eC in 5 \u003csup\u003eo\u003c/sup\u003eC increments and at 77.5 \u003csup\u003eo\u003c/sup\u003eC, and at pH values from 4.5-9.5. \u003c/p\u003e\n\u003cp\u003eStrain SIUC1\u003csup\u003eT \u003c/sup\u003ecultivation experiments were conducted in 160-mL serum bottles, containing 50 mL volume of AGM and 10 mM xylitol with a 2 atm headspace of 99.9% pure N\u003csub\u003e2\u003c/sub\u003e. Temperature and pH culturing assays were averaged across quadruplicate replicates. Cultivation experiments to define temperature optima were incubated at 55, 60, 65, 70, and 75 \u003csup\u003eo\u003c/sup\u003eC in the dark without shaking. Cultivation experiments to determine optimal pH utilized alternative buffers, replacing HEPES buffer when appropriate. A final concentration of 10 mM for each buffer was used to achieve the desired pH values ranging from 6.0 - 9.5. The buffers used were: 2-ethanesulfonic acid (MES) for pH 6.0, 1,4-piperazinediethanesulfonic acid (PIPES) for pH 6.75, HEPES for pH 7.5, tris(hydroxymethyl)aminomethane (TRIS) for pH 8.0/8.75 and N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) for pH 9.5. Cell densities for temperature and pH assay were calculated from 24-hour intervals by microscopy using a Petroff-Hausser counting chamber.\u003c/p\u003e\n\u003cp\u003eGrowth of strain SIUC1\u003csup\u003eT\u003c/sup\u003e on different substrates was determined by quantifying growth in AGM media amended with 10 mM of the selected carbon/energy source at 65 \u003csup\u003eo\u003c/sup\u003eC as listed in \u003cstrong\u003eTable 1\u003c/strong\u003e, or 2 atm for H\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e (80%:20%) or methane (99.9%). Undefined substrates tested were adjusted to a concentration of 0.1% mass vol\u003csup\u003e-1\u003c/sup\u003e, which included casamino acids, peptone, starch, yeast extract, and xylan. Growth was monitored by microscopy using a Petroff-Hausser counting chamber. If a culture reached a cell density of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e or greater within 7 days, it was transferred to a new culture. If a culture reached 1 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e or greater within 7 days in the 3\u003csup\u003erd\u003c/sup\u003e transfer it was considered a stable culture growing on the specific substrate. Experiments with 10 mM xylitol as the carbon/energy source were combined with sulfur-containing potential electron acceptors at concentrations of 10 mM (sulfate, sulfite, thiosulfate, and elemental sulfur). Sulfide production was assessed qualitatively using lead-acetate strips to indicate reduction of the sulfur compound.\u003c/p\u003e\n\u003cp\u003eFor both strains, acetate was quantified by high-performance liquid chromatography (HPLC) using authentic standards and a Shimadzu Prominence-i LC-2030 HPLC equipped with a Resex ROA-Organic Acid H+ 8%, LC-column (300 x 7.8 mm) and Security Guard KJ0-4282 (Phenomenex). 1 mL samples were taken from serum bottles by syringe and centrifuged for 10 minutes at 16,873 x g. Supernatant was transferred to a new tube and acidified by adding 25 µL of a 200 mM sulfuric acid. The acidified sample was then filtered through 0.2 µm filter into an HPLC vial. Samples were run on the HPLC under the following conditions: column temperature: 30 ºC; isocratic; 5 mM sulfuric acid mobile phase at a flow rate of 0.5 mL min\u003csup\u003e-1\u003c/sup\u003e; UV detector set to 254 nm; and run time 30 minutes. Peaks were integrated using LabSolutions LC/GC release 5.87. H\u003csub\u003e2\u003c/sub\u003e was detected qualitatively in headspace samples collected by syringe using a portable hydrogen detector (Forensics Detectors, Model: FD-90A-H2, Rolling Hills Estates, CA, USA).\u003c/p\u003e\n\u003cp\u003eSRB pure cultures were grown in GBS salts medium as prepared for \u003cem\u003eC. saccharofermentans\u003c/em\u003e GBS\u003csup\u003eT\u003c/sup\u003e (5 mM sodium phosphate pH 7 buffer, 0.01% mass vol\u003csup\u003e-1\u003c/sup\u003e sodium sulfide) except that vitamins and xyloglucan/sugars were excluded, and instead 1 mM sodium bicarbonate, 1 mM sodium acetate, 1 mM sodium thiosulfate, 5 mM lactate, and 0.66 atm H\u003csub\u003e2\u003c/sub\u003e were added. For experiments with co-cultivation of \u003cem\u003eCaldatribacterium\u003c/em\u003e strains and SRBs (\u003cstrong\u003eFig. 3b\u003c/strong\u003e), pure cultures for inoculum were diluted 1/100 (0.1 mL in 10 mL total) in medium without added vitamins, and then 0.1 mL of each was used to inoculate 10 mL of \u003cem\u003eCaldatribacterium\u003c/em\u003e strain GBS medium with 0.05% mass vol\u003csup\u003e-1\u003c/sup\u003e fucose and autoclaved vitamins without added folate; the extra dilution (~10,000-fold total) was used to dilute out folate in the original pure cultures of \u003cem\u003eCaldatribacterium\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSRB culture supernatant preparation and analysis of water-soluble vitamins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultures of SRBs were grown in the absence of added vitamins with 1 mM sodium bicarbonate, 1 mM sodium acetate, 1mM sodium thiosulfate, 5 mM lactate, and 0.66 atm H\u003csub\u003e2\u003c/sub\u003e in 50 mL GBS salts medium in 150 mL serum vials until late exponential phase. Aliquots of culture were centrifuged at 800 × g in 1.5 mL microcentrifuge tubes in an anaerobic chamber, and pooled supernatants were filtered with a 0.2 μm nylon filter into sealed serum vials and autoclaved for 30 min. For \u003cem\u003eCaldatribacterium\u003c/em\u003e growth experiments (Fig. 3), 2.5 mL of sterile supernatant was mixed with 7.5 mL of GBS salts medium previously amended with sulfide, and then fucose and autoclaved vitamins without folate were added to yield their typical 0.05% mass vol\u003csup\u003e-1\u003c/sup\u003e and 1x concentrations, respectively. Dilution of the \u003cem\u003eCaldatribacterium\u003c/em\u003e inocula was performed as above for the co-culture experiments to minimize carryover of folate.. Samples of supernatant for vitamin analysis were frozen at -80 \u003csup\u003eo\u003c/sup\u003eC and sent on dry ice for analysis at The Metabolomics Innovation Centre (TMIC; Edmonton, AB, Canada) for quantification of water-soluble vitamins using LC−MS/MS and data analysis with Sciex Analyst 1.6.2\u003csup\u003e62\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectron microscopy and sample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor SEM, late-exponential phase cells were immediately fixed with 2% vol vol\u003csup\u003e-1\u003c/sup\u003e glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 hour at 4 \u003csup\u003eo\u003c/sup\u003eC. Cells were pelleted by centrifugation (10,000 × g for 10 min), washed three times with 0.1 M sodium cacodylate buffer, and resuspended in 1% OsO\u003csub\u003e4\u003c/sub\u003e in 0.1 M sodium cacodylate buffer. Cells were then incubated for 1 hour at 4 ℃ and then rinsed twice with distilled water. Dehydration was performed with 25% increases of ethanol for 15 min for each step from 25 to 95%, then three washes of 100%. Samples were then embedded on a 0.2 μm Millipore filter (13 mm diameter) mounted in a Swinney filter holder. Filters were rinsed with 100% ethanol and kept in ethanol until critical point drying on a Tousimis SAMDRI-790 (Rockville, MD, USA). Sputter coating was carried out using a Denton Vacuum Desk II and the samples were coated with a 600 Å layer of Au-Pd. Imaging was performed using a FEI Quanta FEG 450 SEM, using an acceleration voltage of 20 kV.\u003c/p\u003e\n\u003cp\u003eFor cryo-EM, 1 mL of cell culture was centrifuged at 1,000 × g for 2 min to remove insoluble components in the media. The supernatant was centrifuged at 3,000 × g for 5 min to pellet the cells, and the pellet was resuspended in 20 μL of media. Samples were prepared with an automated Leica EM GP plunge freezer set at 21 °C and 95% humidity in the sample chamber. A 3 μL sample of the cell suspension was applied onto glow-discharged copper R2/2 200 grids (Quantifoil), pre-blotted for 60 seconds, blotted for 2 seconds, plunged into liquid ethane, and stored in liquid nitrogen. The samples were imaged on a 120 kV Talos L120C transmission electron microscope at the Netherlands Center for Electron Nanoscopy (NeCEN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultures of GBS\u003csup\u003eT\u003c/sup\u003e (0.05% mass vol\u003csup\u003e-1\u003c/sup\u003e fucose, 73 \u003csup\u003eo\u003c/sup\u003eC) and SIUC1\u003csup\u003eT\u003c/sup\u003e (0.05% mass vol\u003csup\u003e-1\u003c/sup\u003e xylitol, 67.5 \u003csup\u003eo\u003c/sup\u003eC) were grown to late-exponential phase, and cells were harvested by centrifugation. Cell pellets were stored at -80 \u003csup\u003eo\u003c/sup\u003eC, and fatty acid methyl ester analysis was performed by Microbial ID (Newark, DE, USA) with the MIDI Sherlock Microbial Identification System and SMOORE6 database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and PCR amplification of the 16S rRNA gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from cells of strain GBS\u003csup\u003eT\u003c/sup\u003e grown for seven days and SRB isolates grown for 3 days using the FastDNA Spin Kit for Soil. Strain SIUC1\u003csup\u003eT\u003c/sup\u003e cells were grown to a density of 1 × 10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e in a cumulative volume of 1 L to 500 mL (10 × 50 mL bottles) and cells were pelleted by centrifugation at 15,000 × g for 30 min in 50 mL conical tubes (Sorvall RC6 Plus centrifuge, rotor F13S14x50cy) at 4°C. The genomic DNA was extracted by cetyltrimethyl ammonium bromide (CTAB) treatment followed by mild homogenization, buffer/chloroform, and finally purified/cleaned with QIAquick PCR (Qiagen) purification columns\u003csup\u003e63\u003c/sup\u003e. The DNA was then assessed for quality and quantity by Qbit and Nanodrop. DNA was aliquoted and stored at -80°C, until needed.\u003c/p\u003e\n\u003cp\u003eFor strain GBS\u003csup\u003eT\u003c/sup\u003e and SRB isolates, the primer 9bF (5’-GRG TTT GAT CCT GGC TCA G-3’) and 1512uR (5' ACGGHTACCTTGTTACGACTT) were used for amplification of 16S rRNA genes\u003csup\u003e55\u003c/sup\u003e, and Sanger sequencing with these primers was performed by Retrogen, Inc. (San Diego, CA, USA). For strain SIUC1\u003csup\u003eT\u003c/sup\u003e, universal bacterial primers 27F-YM (5’-AGA GTT TGA TYM TGG CTC AG-3’) and 1492R (5’-TAC CTT GTT ACG ACT T-3’) were used to amplify the 16S rRNA gene, and resulting amplicons were sequenced by MCLAB (South San Francisco, CA, USA). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing, assembly, and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor strain GBS\u003csup\u003eT\u003c/sup\u003e,short read data (2 × 150) was obtained using an Illumina NextSeq 2000 with libraries prepared using the Illumina DNA Prep kit at SeqCenter (Pittsburgh, PA, USA). Long read sequencing was performed with a MinION Mk1B device and FLO-MIN106 flow cell using libraries prepared with the EXP-NBD104 Native Barcoding Expansion Kit (Oxford Nanopore Technologies, Oxford, UK) and the SQK-LSK109 Ligation Sequencing Kit. Short reads were trimmed using Trimmomatic\u003csup\u003e64\u003c/sup\u003e, and hybrid assemblies were performed using Unicycler\u003csup\u003e65\u003c/sup\u003e. Initial annotation was performed using RAST\u003csup\u003e66\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGenome assembly for strain SIUC1\u003csup\u003eT\u003c/sup\u003e was carried out using data from multiple short read runs (Illumina) GeneWiz (San Diego, CA, USA) and UIC Research Resource Center (Chicago, IL, USA) as well as long reads (Oxford Nanopore) using Unicycler\u003csup\u003e65\u003c/sup\u003e. Assemblies were analyzed and compared using QUAST\u003csup\u003e67\u003c/sup\u003e. The Unicycler assembly was selected based on completeness and contiguity for downstream analysis and annotation with JGI annotation pipeline\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenomic and genome distance analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll genomes from the Genome Taxonomy Database (GTDB) 09-RS220\u003csup\u003e2\u003c/sup\u003e plus those from recent publications\u003csup\u003e8,21\u003c/sup\u003e were used for the construction of a species tree (\u003cstrong\u003eSupplementary Data 3\u003c/strong\u003e). The phylogenomic relationships of genomes were inferred based on a concatenation of the bac120 protein marker set identified by GTDB-Tk v. 2.3.2\u003csup\u003e68\u003c/sup\u003e. The concatenated data matrix was analyzed using maximum-likelihood inference in IQ-TREE v.2.2.6\u003csup\u003e69\u003c/sup\u003e, with 1,000 ultrafast bootstraps (UFBoot) and 1,000 SH-like approximate likelihood ratio test (SH-aLRT) replicates (-bb 1000 -alrt 1000), using the best-fit model identified by ModelFinder\u003csup\u003e70\u003c/sup\u003e. Pairwise average nucleotide identity (ANI) and average amino acid identity (AAI) between the MAGs were calculated using FastANI v.1.1\u003csup\u003e71\u003c/sup\u003e and FastAAI v.1\u003csup\u003e72\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnotation of vitamin biosynthesis pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTetrahydrofolate biosynthetic potential of \u003cem\u003eAtribacterota\u003c/em\u003e species and known or potential syntrophic partners was predicted via manual reconstruction of the Folate Biosynthesis KEGG\u003csup\u003e73,74,75\u003c/sup\u003e reference pathway (map00790) supplemented with COGs\u003csup\u003e76,77\u003c/sup\u003e from the 2021 release of the dataset and PFAM\u003csup\u003e78,79\u003c/sup\u003e version 36 annotations. Genomes were reannotated to a uniform specification. Open reading frames and their resulting protein sequences were predicted using Prodigal\u003csup\u003e80\u003c/sup\u003e version v2.6.3. Functional potential of the protein sequences was then annotated using three methods: (i) kofamscan\u003csup\u003e81\u003c/sup\u003e was used to annotate KEGG orthology (KO), (ii) hmmer\u003csup\u003e82\u003c/sup\u003e version 3.3.1 was used to annotate PFAMs, and (iii) rpsblast from blast+\u003csup\u003e83\u003c/sup\u003e version 2.9.0+ was used to annotate COGs.\u003c/p\u003e\n\u003cp\u003eCOG and PFAM annotations were used to supplement KEGG annotations with putative replacements for ‘missing’ KOs. Domain architectures in the PFAM database indicate that the DHFR-domain (PF00186) -containing proteins often also encode a RibD c-terminus domain (PF01872). However, this feature is not exclusive to DHFR; RibD c-terminus domain is associated with both DHFR and cytidine deaminases. Domain architecture was used to identify possible DHFR genes while distinguishing them from cytidine deaminase: proteins were considered possible unannotated DHFR orthologs if the protein sequence included a RibD c-terminus domain (PF01872), but lacked domains suggestive of deaminase activity (PF14437, PF18785, or PF00383). A similar strategy was employed to identify putative unannotated orthologs of para-aminobenzoate synthase (COG0147) while excluding anthranilate synthase (K01657), and 4-amino-4-deoxychorismate lyase (COG0115) while excluding branched-chain amino acid aminotransferase (K00826).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnotation of secreted and membrane proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePredicted secreted and transmembrane genome content was analyzed within high-quality (≥90% completeness, \u0026lt;5% contamination)\u003csup\u003e12\u003c/sup\u003e \u003cem\u003eAtribacterota\u003c/em\u003e, \u003cem\u003eThermotogota\u003c/em\u003e, \u003cem\u003eDictyoglomota\u003c/em\u003e, \u003cem\u003eSynergistota\u003c/em\u003e, and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e genomes obtained from the Genome Taxonomy Database\u003csup\u003e13\u003c/sup\u003e release 202 as well as other genomes from bacteria included in the TEMPURA database\u003csup\u003e84\u003c/sup\u003e. Open reading frames were predicted and translated into protein sequences using Prodigal\u003csup\u003e80\u003c/sup\u003e version v2.6.3. Secretion signal cleavage sites were predicted using SignalP-4.1\u003csup\u003e42\u003c/sup\u003e and SignalP-5\u003csup\u003e43\u003c/sup\u003e. Amino acid sequences three residues upstream or two downstream of cleavage sites were extracted and rendered using the R package ggseqlogo\u003csup\u003e85\u003c/sup\u003e version 0.2. Transmembrane helices were predicted using TMHMM 2.0\u003csup\u003e86\u003c/sup\u003e. Hydrophobicity was calculated for each signal peptide sequence by averaging the sum of individual per-residue hydrophobicities as estimated using the Kyte and Doolittle scale\u003csup\u003e87\u003c/sup\u003e. Isoelectric point was estimated for each signal peptide using the isoelectric point calculator web interface\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dave and Sandy Jamieson for access to Great Boiling Spring. Inyo-BLM 1 samples were obtained under scientific research permit DEVA-2013-SCI-0069 to D.P.M. from the U.S. National Park Service (NPS). We thank Richard Friese, Josh Hoines, and Dr. Kevin Wilson of the NPS along with Alisa Lembke and the Inyo County, CA Planning Commission for site access and John Bredehoeft and Michael King of The Hydrodynamics Group LLC for hydrogeological context and borehole specifications. We thank John Healey, Brad Lyles, and Chuck Russell of the Desert Research Institute for logistical assistance in obtaining bailed samples and logging data from Inyo-BLM 1. Thanks also to Bill Willborn and the DOE UGTA program for allowing use of their downhole logging system. We thank the NASA Astrobiology Institute node \u0026lsquo;Life Underground\u0026rsquo;, PI: Dr. Jan Amend at University of Southern California, including Dr. Greg Wanger, Dr. Joshua Sackett, and Dr. Brittany Kruger for permission to use foam plug samples from borehole Inyo-BLM 1. We thank Dr. Bernhard Schink of Universit\u0026auml;t Konstanz for his help with Greek and Latin grammar.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePart of this work was carried out at Lawrence Livermore National Laboratory (LLNL) under Contract DE-AC52-07NA2734 (X.M., P.W., J.P.-R.). Funding was also provided by the U.S. National Science Foundation (DEB 1557042, B.P.H., D.L., C.O.S., J.A.D., X.M., J.P.-R.), NASA (80NNSC17KO548, B.P.H., D.L., C.O.S., J.A.D., X.M., J.P.-R.), NASA Astrobiology Institute (NNA13AA92A, D.P.M., S.H.B.), and Nevada NASA Space Grant (80NSSC20M0043, B.P.H., D.L.). This work was also supported by startup funds from Southern Illinois University Carbondale (S.H.-B.) and the Research-Enriched Academic Challenge (REACH) undergraduate grant (A.M.B.). The scanning electron microscope used in this work was purchased through a grant from National Science Foundation (CHE 0959568). We also acknowledge the National Science Foundation through the grant CHE 0959568 that facilitated the purchase of the FEI Quanta 450 scanning electron microscope. We thank Valerie Jimenez, Sandy Macias, Katelyn Holt, Maidy Ramos, Jorge Torres, Alejandra Moreno, Joseph Mansuri, and Matthew David for assistance with characterization of GBS and SRB strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.A.D. and S.H.B. conceived of the study. B.P.H., J.A.D., and D.P.M., led field campaigns, managed sampling permits, and obtained and managed major grants supporting the work. S.H.B., J.A.D., T.A., J.L., J.J., A.M., A.T., A.V., A.R.M., A.C., J.L., Z.O., A.M.B, E.L, J.T.P, and K.M. conducted culturing enrichment, isolation, and strain characterization. T.A. and J.A.D. performed FISH and \u003csup\u003e13\u003c/sup\u003eC-labeling experiments. X.M., J.J., P.K.W., J.P.-R., and D.L. analyzed samples with nanoSIMS and nanoSIMS data analysis/interpretation. T.S. prepared cells for SEM imaging. A.R.M. and A.B. conducted cryo-EM and led structural interpretations. T.R.M., R.X., and J.A.D. conducted genome sequencing and assembly. D.L. led on bioinformatic analysis related to taxonomy and B.P.H. led on interpretations of the taxonomy. C.O.S. led on bioinformatic analysis focused on vitamin biosynthetic pathways and N-terminal signal sequences. 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A simple method for displaying the hydropathic character of a protein. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e157\u003c/strong\u003e, 105-132 (1982).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Geothermal, subsurface, Atribacterota, Atribacteria, OP9, sulfate-reducing bacteria, folate, Wolin’s vitamins, Wolfe’s vitamins, NanoSIMS","lastPublishedDoi":"10.21203/rs.3.rs-6739887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6739887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The phylum \u003ci\u003eAtribacterota\u003c/i\u003e is ubiquitous in anoxic environments where it plays important roles in syntrophic carbon and hydrogen metabolism; however, only two species have been isolated. Here, we report the isolation of two additional species representing a new family, \u003ci\u003eCaldatribacterium saccharofermentans\u003c/i\u003e from a hot spring and \u003ci\u003eCaldatribacterium inferamans\u003c/i\u003e from a deep fractured-rock aquifer. Both were co-enriched on carbohydrates with sulfate-reducing bacteria (SRB). Despite unsuccessful attempts to isolate them on the defined enrichment medium, we finally isolated both species by adding yeast extract to the medium. We show that folate was the key factor provided by the SRBs and the yeast extract and that folate provided in vitamin solutions was inadvertently removed by filter sterilization according to standard media preparation protocols. We further confirm the absence of folate biosynthesis pathways across the phylum and suggest that folate precipitation during media preparation limits cultivation of \u003ci\u003eAtribacterota\u003c/i\u003e in toto. The two \u003ci\u003eCaldatribacterium\u003c/i\u003e species share unusual features with other \u003ci\u003eAtribacterota\u003c/i\u003e, including three lipid membrane-like layers (LMLs), with the inner LML that appears to surround a nucleoid, and a high percentage of transmembrane proteins. This study expands the culturability of \u003ci\u003eAtribacterota\u003c/i\u003e and identifies conserved features, including sugar fermentation, unusual cell ultrastructure, and vitamin dependencies necessary for their cultivation.","manuscriptTitle":"Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 06:59:14","doi":"10.21203/rs.3.rs-6739887/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b94add1-d6a8-42a6-b1ea-6a3ea1104224","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49160345,"name":"Biological sciences/Microbiology/Environmental microbiology"},{"id":49160346,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"}],"tags":[],"updatedAt":"2026-03-24T00:25:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 06:59:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6739887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6739887","identity":"rs-6739887","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}