Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert

ABSTRACT

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H₂ and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.

IMPORTANCE

The ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment—including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

INTRODUCTION

Endolithic microbial communities (i.e., those that inhabit airspaces within rocks) are self-supporting, marginal ecosystems that are distributed across extreme desert environments and constitute the predominant life-forms in the ice-free areas of Antarctica (1–3). These desert areas are dominated by nutrient-poor (oligotrophic) mineral soil and rocky outcrops (2–5). Due to the extremely harsh conditions of Antarctic deserts—including temperatures below freezing (6), rapid thermal fluctuations, extreme dryness, and high incident ultraviolet and solar radiation—the principal habitat for microorganisms is inside rocks, which provide a refuge for life (1–3, 7). Here, microorganisms are afforded protection, including a thermally buffered and more stable microclimate (1, 2, 8–10), with self-supporting assemblages composed of lichens, free-living algae (Chlorophyta), fungi, and bacteria (1–3, 11–20).

The McMurdo Dry Valleys (Southern Victoria Land), one of the largest ice-free regions of the Antarctic continent (13), are regarded as the coldest and most hyperarid desert on Earth and, therefore, the closest analog to the surface of the planet Mars (21–23). Although the climate of the McMurdo Dry Valleys is characterized by extreme cold and aridity (24), continuous sunshine during the austral summer months can raise the internal temperature of rocks above the freezing point due to the thermal inertia of the substratum (up to 10°C above the ambient temperature) (17). At the same time, porous rocks wetted by snowmelt can retain liquid water internally for several days (8, 9, 13). Together, these hydration events stimulate biological activity, thereby promoting the mobilization of extracellular carbon and increased aerobic respiration by heterotrophic microorganisms (7, 25).

To date, information on biodiversity, structure, and stress responses of endolithic communities comes from both culture-dependent and -independent studies of rocks sampled in the McMurdo Dry Valleys and ice-free mountain peaks on the Transantarctic Mountains of Victoria Land (3, 8, 9, 13–20, 26–33). Recently, the application of genome-resolved metagenomics of 109 endolithically colonized rocks from various Antarctic locations culminated in a catalog of 4,539 metagenome-assembled genomes (MAGs) that represent 2,238 novel candidate species (20). Yet, the functionality of these novel candidate species has been only partially investigated, and to date, the metabolic capacities and adaptations sustaining the success and perpetuation of these microbial assemblages at the edge of life remain still largely unexplored. To address this knowledge gap, in the present study we focus on 17 MAGs that represent four novel classes of the phylum Chloroflexota. The Chloroflexota is among the most abundant phyla in these communities, along with Proteobacteria and Actinomycetota (15, 20). These 17 MAGs, from rocks in McMurdo Dry Valleys and hills and mountains of Northern Victoria Land, are of at least 94% completeness, with 13 being high quality (≥95% completeness and <5% contamination). Based on our interrogation of these MAGs, we endeavored to identify specific metabolic and physiological adaptations that would facilitate the survival of these bacteria in such a harsh and hyperarid environment.

RESULTS AND DISCUSSION

Candidatus classes Spiritibacteria, Martimicrobia, Tarhunnaeia, and Uliximicrobia

All 17 MAGs used in this study (94%–99% completeness and 1.0%–5.5% contamination) were named and assigned to a taxonomic hierarchy according to recommendations for describing novel Candidatus species (34–37) (see Table S1 for supporting metadata). Together these MAGs represent 8 novel genus-level and 17 novel species-level taxa. Based on our phylogenomic analysis (Fig. 1; Fig. S1) and Genome Taxonomy Database (GTDB) taxonomy, these MAGs represent four class-level taxa that are deeply nested within phylum Chloroflexota: Candidatus Spiritibacteria class. nov. (which replaces the placeholder name UBA5177), Candidatus Tarhunnaeia class. nov. (UBA6077), Candidatus Martimicrobia class. nov. (UBA4733), and Candidatus Uliximicrobia class. nov. (UBA2235).

Fig 1.

Maximum-likelihood phylogenomic tree generated from a multiple sequence alignment of 120 bacterial single-copy marker genes, showing Chloroflexota MAGs from this study with proposed Candidatus names (given in bold), and selected neighboring reference GTDB strains labeled with GenBank assembly accession. The source environment of each unnamed reference GTDB strain is listed after the accession. The tree was rooted at Vulcanimicrobiota (not shown), and black circles indicate bootstrap values of 90% or greater. For full tree showing all GTDB reference taxa in the four Chloroflexota classes of interest, see Fig. S1.

The phylum Chloroflexota is prevalent across the 109 endolithic microbiomes, with an occupancy of 89.9% (i.e., the percentage of samples in which they were found) (20). By way of comparison, the two dominant phyla, Actinomycetota and Proteobacteria, had occupancies of 99.1% and 95.4%, respectively. Cyanobacteria, the most abundant bacterial primary producers, had an occupancy of 45.9% (20). Within phylum Chloroflexota, the occupancy values at class level were Chloroflexia (85.3%), Ca. Spiritibacteria (49.5%), Ca. Martimicrobia (26.6%), Ktedonobacteria (22.9%), Ca. Tarhunnaeia (16.0%), Ca. Uliximicrobia (6.4%), and Dehalococcoidia (5.5%) (20). Thus, of the four new classes proposed here, Ca. Spiritibacteria is the most widespread within the Antarctic endolithic samples.

Class Candidatus Spiritibacteria

Eight of the 17 MAGs are assigned to class Ca. Spiritibacteria and represent three genera and eight species (Table 1). The genus Candidatus Spiritibacter gen. nov. (Latin spiritus, the air, in reference to the predicted ability to utilize atmospheric gases + New Latin bacter, bacterium) contains six species: Candidatus Spiritibacter saxicola sp. nov. (type species; Latin saxicola, rock-dweller), Candidatus Spiritibacter pertinax sp. nov. (Latin pertinax, persevering), Candidatus Spiritibacter antarcticus sp. nov. (Latin antarcticus, southern), Candidatus Spiritibacter australis sp. nov. (Latin australis, southern), Candidatus Spiritibacter polaris sp. nov. (Latin polaris, polar), and Candidatus Spiritibacter frigidus sp. nov. (Latin frigidus, cold). Class Ca. Spiritibacteria also includes the genus Candidatus Aglaurobacter gen. nov. (ancient Greek Aglauros, Athenian princess beloved by the god Mars, and turned into stone + New Latin bacter, bacterium), which contains Candidatus Aglaurobacter anningiae sp. nov. (type species; in honor of Mary Anning, English fossil collector and pioneer in the science of paleontology); and genus Candidatus Otrerea gen. nov. (ancient Greek Otrere, consort of the war god Mars), which contains Candidatus Otrerea regina sp. nov. (type species; Latin regina, queen, in reference to Victoria Land).

TABLE 1.

Predicted physiological and metabolic features inferred for MAGs that belong to phylum Chloroflexota classes Candidatus Spiritibacteria (=UBA5177), Candidatus Martimicrobia (=UBA4733), Candidatus Tarhunnaeia (=UBA6077), and Candidatus Uliximicrobia (=UBA2235)^a,b

Spiritibacteria class. nov.

Spiritibacter gen. nov.

Spiritibacter saxicola sp. nov. (type)

Genome size 4.44 Mbp, completeness 98.7%, contamination 2.0%

Atmospheric H2 oxidation (1h, 1l). CO2 fixation using CBB cycle. CO oxidation. Aerobic respiration: cytochrome c oxidase, cytochrome bd ubiquinol oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, kojibiose, glucose, galactose, fructose, mannose, xylose, sorbitol, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, threonine, and phenylalanine], sarcosine, formate, phosphoglycolate, phospholipids, fatty acids, nucleosides. Synthesis of trehalose, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate, molybdate/tungstate. Other transporters: sugars, glycerol, amino acids, glycolate, succinate, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, RHg, Hg, CH2O, methanethiol, Na, Ca, Fl.

Spiritibacter pertinax sp. nov.

Genome size 4.93 Mbp, completeness 98.2%, contamination 2.0%

Atmospheric H2 oxidation (1h). CO2 fixation using CBB cycle. CO oxidation. Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, glucose, galactose, fructose, mannose, sorbitol, glycerol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, threonine, and phenylalanine), glycine betaine, sarcosine, formate, phosphoglycolate, phospholipids, fatty acids, nucleosides. Synthesis of trehalose, glycine betaine, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate. Other transporters: sugars, glycerol, amino acids, glycolate, succinate, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, RHg, Hg, CH2O, methanethiol, Na, Ca, Fl.

Spiritibacter antarcticus sp. nov.

Genome size 4.33 Mbp, completeness 97.7%, contamination 3.0%

Atmospheric H2 oxidation (1h). CO2 fixation using CBB cycle. Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, kojibiose, glucose, galactose, fructose, mannose, sorbitol, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, threonine, and phenylalanine), glycine betaine, sarcosine, formate, phosphoglycolate, fatty acids, nucleosides. Synthesis of trehalose, glycine betaine, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate, molybdate/tungstate. Other transporters: sugars, glycerol, amino acids, glycolate, succinate, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, RHg, Hg, CH2O, Na, Ca, Fl.

Spiritibacter australis sp. nov.

Genome size 5.01 Mbp, completeness 98.7%, contamination 2.0%

Atmospheric H2 oxidation (1h, 1l). CO oxidation. Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, kojibiose, glucose, galactose, fructose, mannose, xylose, sorbitol, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, threonine, and phenylalanine), glycine betaine, sarcosine, formate, phosphoglycolate, phospholipids, fatty acids, nucleosides. Synthesis of trehalose, glycine betaine, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate, molybdate/tungstate. Other transporters: sugars, glycerol, amino acids, glycolate, succinate, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, RHg, Hg, CH2O, Na, Ca, Fl.

Spiritibacter polaris sp. nov.

Genome size 4.36 Mbp, completeness 96.7%, contamination 2.5%

Atmospheric H2 oxidation (1h). CO oxidation. Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, glucose, galactose, fructose, mannose, xylose, sorbitol, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, threonine, and phenylalanine), glycine betaine, sarcosine, formate, phosphoglycolate, phospholipids, fatty acids, nucleosides. Synthesis of trehalose, glycine betaine, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate. Other transporters: sugars, glycerol, amino acids, succinate, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, Hg, CH2O, Na, Ca, Fl.

Spiritibacter frigidus sp. nov.

Genome size 4.38 Mbp, completeness 98.7%, contamination 2.0%

Atmospheric H2 oxidation (1h, 1l). Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, glucose, galactose, fructose, mannose, xylose, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, tryptophan, serine, glycine, and threonine), glycine betaine, sarcosine, formate, phosphoglycolate, phospholipids, fatty acids, nucleosides. Synthesis of trehalose, glycine betaine, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate, molybdate/tungstate. Other transporters: sugars, glycerol, amino acids, glycolate, succinate, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, chromate, As, Hg, CH2O, Na, Ca, Fl.

Aglaurobacter gen. nov.

Aglaurobacter anningiae sp. nov. (type)

Genome size 3.67 Mbp, completeness 96.2%, contamination 1.0%

Atmospheric H2 oxidation (1h). Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides, trehalose, glucose, galactose, fructose, mannose, xylose, ribose, glycerol, peptides, amino acids (BCAA, glutamate, aspartate, methionine, alanine, proline, histidine, serine, and glycine), taurine, sarcosine, formate, phosphoglycolate, phospholipids, nucleosides. Synthesis of polyhydroxyalkanoate, polyphosphate. Bacterial macroglobulin. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, nucleosides, Fe, phosphate, molybdate/tungstate. Other transporters: sugars, glycerol, amino acids, succinate, allantoin/purine, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, Cd, Mn, TeO3, As, Hg, CH2O, Na, Ca, sulfite, Fl.

Otrerea gen. nov.

Otrerea regina sp. nov. (type)

Genome size 6.00 Mbp, completeness 98.0%, contamination 5.5%

Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, Dyp, mycothiol. Rhodopsin-based ion pumping. Organic substrates include oligosaccharides, glucose, galactose, fructose, mannose, xylose, ribose, rhamnose, glycerol, alcohol, peptides, amino acids (BCAA, glutamate, aspartate, proline, serine, glycine, and threonine), sarcosine, formate, lactate, acetate, 2-AEP, phosphoglycolate, phospholipids, fatty acids, nucleosides, AHL. Assimilatory sulfate reduction. Synthesis of trehalose, polyphosphate. Bacterial macroglobulin. ABC transporters: sugars, mannitol, glycerol 3-phosphate, peptides, amino acids, glycine betaine, nucleosides, phosphate, sulfate. Other transporters: sugars, glycerol, ammonia, Co/Mg, Mn, Fe, Ni, K. Efflux/detoxification: Cu, Cd, chromate, As, RHg, Hg, CH2O, Na, Ca, sulfite, Fl.

Martimicrobia class. nov.

Martimicrobium gen. nov.

Martimicrobium tenax sp. nov. (type)

Genome size 5.26 Mbp, completeness 98.0%, contamination 1.0%

Atmospheric H2 oxidation (1h,1l). Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol. Organic substrates include oligosaccharides, glucose, galactose, fructose, mannose, xylose, arabinose, ribose, fucose, rhamnose, sorbitol, mannitol, glycerol, alcohol, galactonate, mannonate, glucuronate, galacturonate, peptides, amino acids (BCAA, glutamate, aspartate, methionine, serine, and glycine), sarcosine, formate, urea, phosphoglycolate, phospholipids, sulfate esters, AHL. Assimilatory sulfate reduction. Synthesis of glycogen, polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: glycerol 3-phosphate, peptides, amino acids, phosphate, phosphonate, molybdate/tungstate. Other transporters: sugars, glycerol, C4-dicarboxylates, acetate, ammonia, Co/Mg, Zn, Mn, Fe, Ni, Mg, K. Efflux/detoxification: Cu, Cd, Pb, Zn, Mn, chromate, As(III), RHg, Hg, CH2O, methanethiol, Na, Ca, sulfite, Fl.

Laranimicrobium gen. nov.

Laranimicrobium antiquum sp. nov. (type)

Genome size 6.19 Mbp, completeness 96.0%, contamination 2.6%

Atmospheric H2 oxidation (1h,1l). Aerobic respiration: cytochrome c oxidase, cytochrome bd ubiquinol oxidase. Oxidative stress response: superoxide dismutase, Dyp, mycothiol. Organic substrates include oligosaccharides, sucrose, glucosylceramides, glucose, galactose, fructose, mannose, ribose, arabinose, rhamnose, sorbitol, glycerol, alcohol, galactonate, mannonate, glucuronate, galacturonate, glucarate, peptides, amino acids (BCAA, glutamate, aspartate, methionine, alanine, serine, and glycine), sarcosine, formate, urea, 2-AEP, phospholipids, sulfate esters. Assimilatory sulfate reduction. Synthesis of polyphosphate. Bacterial macroglobulin. Type IV pili. ABC transporters: glycerol 3-phosphate, peptides, amino acids, phosphate, phosphonate, molybdate/tungstate. Other transporters: sugars, glycerol, monocarboxylates, allantoin/uric acid, ammonia, sulfate, Mn, Fe, Mg, K. Efflux/detoxification: Cu, Cd, Mn, As, CH2O, methanethiol, Na, Ca, sulfite, Fl.

Tarhunnaeia class. nov.

Tarhunnaea gen. nov.

Tarhunnaea remota sp. nov. (type)

Genome size 6.65 Mbp, completeness 97.2%, contamination 1.5%

Atmospheric H2 oxidation (1h, 1l). Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, Dyp, mycothiol, FGD. Organic substrates include oligosaccharides, sucrose, trehalose, glucose, galactose, fructose, xylose, ribose, rhamnose, sorbitol, mannitol, ribitol, glycerol, alcohol, mannonate, glucuronate, galacturonate, glucarate, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, histidine, serine, and glycine), taurine, sarcosine, formate, urea, uric acid, allantoin, phosphonoacetate, phosphoglycolate, phospholipids, fatty acids, lactate, sulfate esters, AHL. Synthesis of trehalose, polyhydroxyalkanoate, polyphosphate. Encapsulin. Metacaspase. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, nucleosides, phosphate, molybdate/tungstate, sulfonate. Other transporters: sugars, glycerol, amino acids, monocarboxylates, glycolate, urea, allantoin/uric acid, Zn, Mn, Mg, K. Efflux/detoxification: Cu, chromate, As, Hg, CH2O, methanethiol, Na, Ca, sulfite, Fl.

Tarhunnaea duricola sp. nov.

Genome size 5.12 Mbp, completeness 95.5%, contamination 2.2%

Atmospheric H2 oxidation (1l). CO2 fixation using CBB cycle. Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides (including starch oligomers), sucrose, trehalose, kojibiose, glucose, galactose, fructose, xylose, ribose, fucose, rhamnose, sorbitol, mannitol, glycerol, alcohol, gluconate, mannonate, glucuronate, galacturonate, glucarate, peptides, amino acids (BCAA, glutamate, aspartate, methionine, proline, serine, glycine, and threonine), sarcosine, formate, uric acid, allantoin, phosphoglycolate, phospholipids, fatty acids. Synthesis of trehalose, polyphosphate. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, nucleosides, phosphate, sulfonate. Other transporters: sugars, glycerol, amino acids, monocarboxylates, glycolate, allantoin/purine, Zn, Mn, Fe, Ni, Mg, K. Efflux/detoxification: Cu, chromate, Hg, CH2O, methanethiol, Na, Ca, Fl.

Sutekhia gen. nov.

Sutekhia aridicola sp. nov. (type)

Genome size 6.62 Mbp, completeness 97.7%, contamination 4.0%

Atmospheric H2 oxidation (1h,1l). Aerobic respiration: cytochrome c oxidase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include starch, oligosaccharides (including cyclo/maltodextrin), sucrose, trehalose, glucose, galactose, fructose, ribose, rhamnose, glycerol, alcohol, gluconate, glucarate, peptides, amino acids (BCAA, glutamate, aspartate, methionine, serine, and glycine), sarcosine, formate, urea, uric acid, allantoin, phosphonoacetate, methylphosphonate, phosphoglycolate, sulfate esters. Synthesis of glycogen, trehalose. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, nucleosides, phosphate, phosphonate, Fe, molybdate/tungstate. Other transporters: sugars, glycerol, monocarboxylates, ammonia, amino acids, Co/Mg, Zn, Mn, Mg, K. Efflux/detoxification: Cu, chromate, Hg, CH2O, Na, sulfite, Fl.

Uliximicrobia class. nov.

Uliximicrobium gen. nov.

Uliximicrobium exili sp. nov. (type)

Genome size 7.56 Mbp, completeness 96.5%, contamination 4.4%

Atmospheric H2 oxidation (1h). Aerobic respiration: cytochrome c oxidase, cytochrome bd ubiquinol oxidase. Anaerobic respiration: fumarate reductase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides, sucrose, glucose, galactose, fructose, ribose, arabinose, rhamnose, sorbitol, mannitol, xylitol, glycerol, galactonate, mannonate, glucuronate, galacturonate, talarate, galactarate, altronate, peptides, amino acids (BCAA, glutamate, aspartate, serine, and glycine), sarcosine, urea, phospholipids, phosphoglycolate, sulfate esters. Assimilatory sulfate reduction. Assimilatory nitrate reduction. Synthesis of trehalose, polyphosphate. Toxin complex (Tc-type). Hemolysin III. Metacaspase. Flagella. Chemotaxis. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, phosphate, Fe, molybdate/tungstate. Other transporters: glycerol, ammonia, amino acids, Co, Mg, K. Efflux/detoxification: Cu, Cd, chromate, Hg, CH2O, Na.

Uliximicrobium arcanum sp. nov.

Genome size 8.35 Mbp, completeness 94.1%, contamination 4.4%

Atmospheric H2 oxidation (1h) Aerobic respiration: cytochrome c oxidase. Anaerobic respiration: fumarate reductase. Oxidative stress response: superoxide dismutase, catalase, Dyp, mycothiol, FGD. Organic substrates include oligosaccharides, sucrose, glucose, galactose, fructose, ribose, arabinose, rhamnose, sorbitol, xylitol, glycerol, galactonate, mannonate, glucuronate, galacturonate, talarate, galactarate, peptides, amino acids (BCAA, glutamate, aspartate, serine, and glycine), sarcosine, urea, phospholipids, phosphoglycolate, sulfate esters. Assimilatory sulfate reduction. Assimilatory nitrate reduction. Synthesis of trehalose, cyclic β-(1,2)-glucan, polyphosphate. Hemolysin III. Metacaspase. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, phosphate, Fe, molybdate/tungstate. Other transporters: glycerol, ammonia, amino acids, Co, Mg, K. Efflux/detoxification: Cu, chromate, CH2O, Na, Fl.

Uliximicrobium absconditum sp. nov.

Genome size 8.16 Mbp, completeness 94.5%, contamination 5.3%

Atmospheric H2 oxidation (1h) Aerobic respiration: cytochrome c oxidase, cytochrome bd ubiquinol oxidase. Anaerobic respiration: fumarate reductase. Oxidative stress response: superoxide dismutase, catalase, Dyp, mycothiol, FGD. Organic substrates include oligosaccharides, sucrose, glucose, galactose, fructose, ribose, arabinose, rhamnose, sorbitol, glycerol, xylitol, galactonate, mannonate, glucuronate, galacturonate, talarate, galactarate, peptides, amino acids (BCAA, glutamate, aspartate, serine, and glycine), urea, methylphosphonate, phospholipids, phosphoglycolate, sulfate esters. Assimilatory sulfate reduction. Assimilatory nitrate reduction. Synthesis of trehalose, cyclic β-(1,2)-glucan, polyphosphate. Hemolysin III. Metacaspase. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, phosphate, phosphonate, Fe, molybdate/tungstate. Other transporters: glycerol, ammonia, amino acids, Co, Mg, K. Efflux/detoxification: Cu, chromate, Hg, CH2O, methanethiol, Na.

Uliximicrobium occultum sp. nov.

Genome size 6.41 Mbp, completeness 95.1%, contamination 5.5%

Atmospheric H2 oxidation (1h). Aerobic respiration: cytochrome c oxidase, cytochrome bd ubiquinol oxidase. Anaerobic respiration: fumarate reductase. Oxidative stress response: superoxide dismutase, catalase, mycothiol, FGD. Organic substrates include oligosaccharides, sucrose, glucose, galactose, fructose, arabinose, ribose, rhamnose, sorbitol, mannitol, glycerol, galactonate, mannonate, glucuronate, galacturonate, talarate, galactarate, peptides, amino acids (BCAA, glutamate, aspartate, serine, and glycine), formate, urea, phospholipids, phosphoglycolate, sulfate esters. Assimilatory sulfate reduction. Synthesis of trehalose, polyphosphate. Hemolysin III. Metacaspase. Type IV pili. ABC transporters: sugars, glycerol 3-phosphate, peptides, amino acids, glycine betaine, phosphate, Fe. Other transporters: glycerol, ammonia, amino acids, allantoin/purine, Co, Mn, Mg, K. Efflux/detoxification: Cu, Cd, CH2O, Na, Ca, Fl.

^a All substrates utilized and all biosynthetic abilities given are based on genomic predictions. All taxa in this table (class, genus, and species) are Candidatus. For full etymologies and metadata for all genera and species, refer to Table S1; for all proteins involved in metabolism and transport, refer to Table S2.

^b 2-AEP, 2-aminoethylphosphonate; ABC, ATP-binding cassette; AHL, acyl-homoserine lactone; BCAA, branched-chain amino acids; CBB cycle, Calvin-Benson-Bassham cycle; CH₂O, formaldehyde; Dyp, dye decolorizing peroxidase; F₄₂₀, 8-hydroxy-5-deazaflavin; FGD, F₄₂₀-dependent glucose dehydrogenase; RHg, organomercurial salts.

Class Ca. Spiritibacteria (UBA5177) includes MAGs that come from the Antarctic endolithic metagenomes, as well as MAGs retrieved from metagenomes from Arctic soil (Axel Heiberg Island) and soil that overlies an ongoing underground coalmine fire (Pennsylvania; Fig. S1; Table S1). Our phylogenomic analysis recovered Ca. Spiritibacteria and Ca. Martimicrobia as sister taxa, forming a clade of terrestrial Chloroflexota that also includes class Ktedonobacteria (Fig. 1).

Class Candidatus Martimicrobia

Two of the MAGs belong to class Ca. Martimicrobia and represent two genera and species (Table 1). The genus Candidatus Martimicrobium gen. nov. (Latin Mars, god of war, in reference to the Mars-like landscape + New Latin microbium, microbe) contains the species Candidatus Martimicrobium tenax sp. nov. (type species; Latin tenax, tenacious); and the genus Candidatus Laranimicrobium gen. nov. [Etruscan Laran, god of war (cognate with the Roman god Mars) + New Latin microbium, microbe] contains the species Candidatus Laranimicrobium antiquum sp. nov. (type species; Latin antiquum, ancient, in reference to the geologically ancient environment). Other MAGs that belong to Ca. Martimicrobia (UBA4733) come from an Arctic soil metagenome (Axel Heiberg Island), as well as acid mine drainage sediment metagenomes (Fig. 1; Table S1), as is also the case for certain MAGs from class Ca. Tarhunnaeia.

Class Candidatus Tarhunnaeia

Three MAGs belong to class Ca. Tarhunnaeia and represent two genera and three species (Table 1). The genus Candidatus Tarhunnaea gen. nov. (Hittite Tarḫunna, god of the weather, in reference to the hostile weather of Antarctica) contains two species: Candidatus Tarhunnaea remota sp. nov. (type species; Latin remota, distant) and Candidatus Tarhunnaea duricola sp. nov. (Latin duricola, dweller in a hard place). The genus Candidatus Sutekhia gen. nov. (ancient Egyptian Sutekh, god of deserts, in reference to the Antarctic desert) contains the species Candidatus Sutekhia aridicola sp. nov. (type species; Latin aridicola, dweller in a dry place).

In addition to the Antarctic endolithic metagenomes, other MAGs that can be assigned to class Ca. Tarhunnaeia (UBA6077) have been retrieved from metagenomes from soil (including rhizosphere samples), groundwater, wastewater (including acid mine drainage sediments), freshwater lake water, woody debris (“hog fuel”), and fossilized dinosaur bone. Those MAGs most closely related to endolithic Ca. Tarhunnaea MAGs were recovered from nutrient-impoverished soils (rhizosphere of Barbacenia macrantha, Brazil) and from temperate grassland soil (Angelo Coast Range; Fig. 1; Table S1). For Ca. Sutekhia aridicola, aside from Antarctic metagenomes (including the Mackay Glacier desert region), the most closely related MAGs come from wastewater of a partial nitritation anammox bioreactor (Table S1).

Class Candidatus Uliximicrobia

The four MAGs of class Ca. Uliximicrobia all belong to a single genus that contains four species (Table 1). The genus Candidatus Uliximicrobium [ancient Greek Ulixes (=Ulysses), warrior and traveller, in reference to inferred toxic and motile abilities + New Latin microbium, microbe] contains four species: Candidatus Uliximicrobium exili sp. nov. (type species) (Latin exili, state of isolation), Candidatus Uliximicrobium arcanum sp. nov. (Latin arcanum, mysterious), Candidatus Uliximicrobium absconditum sp. nov. (Latin absconditum, concealed), and Candidatus Uliximicrobium occultum sp. nov. (Latin occultum, hidden).

MAGs most closely related to Candidatus Uliximicrobium have been retrieved from metagenomes derived from soil biocrust (Negev Desert) and a groundwater planktonic microbiome (Modesto, California) (Fig. S1; Table S1). The majority of MAGs from class Ca. Uliximicrobia (UBA2235) have been retrieved from metagenomes derived from freshwater lakes, estuaries, and marine sponges. Our phylogenomic analysis recovered class Ca. Uliximicrobia as the sister taxon to unnamed Candidatus class UBA11872, retrieved from marine sponge tissue metagenomes (Fig. 1; Fig. S1).

Metabolism and cell envelope

Based on the 17 MAGs from all four Candidatus classes (Spiritibacteria, Martimicrobia, Tarhunnaeia, and Uliximicrobia), these bacteria are inferred to be aerobic heterotrophs, with some additionally inferred to be capable of anaerobic respiration and autotrophy (see “Respiration” and “Trace gas oxidation,” below; Table 2; Fig. 2 to 6). All encode an oxidative tricarboxylic acid (TCA) cycle, an Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, and a pentose phosphate pathway. Aside from oxidative phosphorylation via the EMP pathway, alternative fates for glucose are inferred by the presence in almost all MAGs of a gene for glucose dehydrogenase, as well as other enzymes in certain MAGs for a semiphosphorylative Entner-Doudoroff pathway or for production of 5-ketogluconate (see Supplemental text). Full metabolic pathways and individual proteins (including transporters and enzymes) predicted for the 17 MAGs are presented in Table S2 and summarized in Table 1. For further descriptions regarding the predicted metabolic capacities of these MAGs, including nitrogen, phosphorus, and sulfur metabolism, refer to the Supplemental text.

TABLE 2.

Predicted features inferred for MAGs from classes Candidatus Spiritibacteria, Candidatus Martimicrobia, Candidatus Tarhunnaeia, and Candidatus Uliximicrobia that relate to central carbon metabolism, trace gas oxidation, respiration, light harvesting, osmoadaptation, motility, adherence, and toxic abilities^a

	Spiritibacteria								Martimicrobia		Tarhunnaeia			Uliximicrobia
	Spiritibacter saxicola	Spiritibacter pertinax	Spiritibacter antarcticus	Spiritibacter australis	Spiritibacter polaris	Spiritibacter frigidus	Aglaurobacter anningiae	Otrerea regina	Martimicrobium tenax	Laranimicrobium antiquum	Tarhunnaea remota	Tarhunnaea duricola	Sutekhia aridicola	Uliximicrobium exili	Uliximicrobium arcanum	Uliximicrobium absconditum	Uliximicrobium occultum
tricarboxylic acid cycle
glyoxylate bypass
glycolysis
pentose phosphate pathway
Calvin-Benson-Bassham cycle: form IE RubisCO
H2 oxidation: high-affinity hydrogenase
CO oxidation: carbon monoxide dehydrogenase
aerobic respiration: cytochrome c oxidase complex
aerobic respiration: cytochrome bd ubiquinol oxidase
anaerobic respiration: fumarate reductase
rhodopsin (bacteriorhodopsin-like)
glycine betaine synthesis
trehalose synthesis
glycine betaine/carnitine/choline transporter
α-glucoside/osmoprotectant transporter
maltose/trehalose transporter
bacterial macroglobulin
flagella
type IV pili
toxin complex (Tc-type)

^a All taxa in this table are Candidatus. For full lists of proteins and metabolic pathways, refer to Table S2.

Fig 2.

Predicted metabolic capacities of Candidatus Spiritibacter saxicola gen. et. sp. nov. (Candidatus Spiritibacteria class. nov.). Transporters rendered with stippled lines could not be identified in the MAG but are assumed to be present (also in Fig. 3 to 6). 1h, [NiFe]-hydrogenase group 1h; 1l, [NiFe]-hydrogenase group 1l; 2-PG, 2-phosphoglycolate; BCAA, branched-chain amino acids; bMG, bacterial macroglobulin; CBB cycle, Calvin-Benson-Bassham cycle; Cox, cytochrome c oxidase; Cyd, cytochrome bd ubiquinol oxidase; DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; GH, glycoside hydrolase; Glp, G3P dehydrogenase complex; Kat, catalase; MSH, mycothiol; PAPS, phosphoadenosine phosphosulfate; PPP, pentose phosphate pathway; Sod, superoxide dismutase; Tad, tight adherence; TMA, trimethylarsine.

Fig 6.

Predicted metabolic capacities of Candidatus Uliximicrobium exili gen. et. sp. nov. (Candidatus Uliximicrobia class. nov.). The toxin complex is shown here as extracellular. The identities of the sulfated biopolymers are not known; for simplicity, sulfated oligosaccharides are shown as a substrate. 1h, [NiFe] hydrogenase group 1h; 2-PG, 2-phosphoglycolate; ANR, assimilatory nitrate reduction; ASR, assimilatory sulfate reduction; BCAA, branched-chain amino acids; Cox, cytochrome c oxidase; DHAP, dihydroxyacetone phosphate; Frd, fumarate reductase; G3P, glycerol 3-phosphate; GH, glycoside hydrolase; Kat, catalase; MSH, mycothiol; PPP, pentose phosphate pathway; Sod, superoxide dismutase; Tad, tight adherence; TMA, trimethylarsine.

Clades within the Chloroflexi supergroup have previously been predicted to vary in their capacities to process carbohydrates, with class Chloroflexia relatively enriched in carbohydrate metabolism genes of diverse CAZy (Carbohydrate-Active enZymes) families, whereas Dehalococcoidia and Ca. Dormibacterota encodes relatively few carbohydrate-metabolizing enzymes (38). MAGs of the four candidate Chloroflexota classes examined here encode an array of glycoside hydrolases (GHs) for the degradation of oligosaccharides and simple sugars (Table 3; Supplemental text; Table S2; Table S3).

TABLE 3.

Predicted glycoside hydrolases (GHs) inferred for MAGs from Candidatus Spiritibacteria, Candidatus Martimicrobia, Candidatus Tarhunnaeia, and Candidatus Uliximicrobia, including GH family according to CAZy^a

	Spiritibacteria								Martimicrobia		Tarhunnaea			Uliximicrobia
	Spiritibacter saxicola	Spiritibacter pertinax	Spiritibacter antarcticus	Spiritibacter australis	Spiritibacter polaris	Spiritibacter frigidus	Aglaurobacter anningiae	Otrerea regina	Martimicrobium tenax	Laranimicrobium antiquum	Tarhunnaea remota	Tarhunnaea duricola	Sutekhia aridicola	Uliximicrobium exili	Uliximicrobium arcanum	Uliximicrobium absconditum	Uliximicrobium occultum
α-glucosidase (GH4)
α-glucosidase (GH13)
α-glucosidase (GH31)
β-glucosidase (GH1)
β-glucosidase (GH3)
oligo-1,6-glucosidase (GH13)
6-phospho-β-glucosidase (GH4)
exo-β-1,3-glucanase (GH55)
α-galactosidase (GH4)
α-galactosidase (GH36)
β-galactosidase (GH2)
β-galactosidase (GH35 + GH5)
β-galactosidase (GH42)
β-galactosidase (GH43)
β-fructosidase (GH32)
α-mannosidase (GH38)
α-mannosidase (GH92)
β-mannosidase (GH2)
α-xylosidase (GH31)
β-xylosidase (GH5)
β-xylosidase (GH39)
trehalase (GH15)
levanase (GH32)
invertase (GH100)
cyclomaltodextrinase (GH13)
α-fucosidase (GH29)
α-rhamnosidase (GH78)

^a All taxa in this table are Candidatus. For full lists of GHs, refer to Tables S2 and S3.

Based on the total absence of genes required for the synthesis of lipopolysaccharide (LPS) and the Bam complex, we predict that cells for all 17 MAGs lack LPS and do not possess a typical outer membrane, in common with other members of Chloroflexota (39, 40), as well as the related Candidatus phyla Eulabeiota and Dormibacterota (38, 41). However, cells of certain Chloroflexota (order Chloroflexales) have been observed to have a cell envelope that has a diderm-like architecture despite the absence of genes for both LPS synthesis and Bam complex (40). Therefore, it is possible that the cells of the new Chloroflexota classes possess an outer layer, albeit one lacking LPS. As discussed later, MAGs contain genes for extracellular structures such as pili, flagella, macroglobulins, and toxin complexes (Tables 1 and 2; Fig. 2 to 6).

Respiration

All 17 MAGs encode the cytochrome c oxidase complex for aerobic respiration (Fig. 2). Additionally, six MAGs (representing Ca. Spiritibacteria, Ca. Martimicrobia, and Ca. Uliximicrobia) encode the high O₂ affinity cytochrome bd ubiquinol oxidase (42), suggesting that these bacteria are adapted to microaerobic conditions. We infer the four Ca. Uliximicrobium species to be facultative anaerobes: each MAG encodes a fumarate reductase complex, suggesting the capacity for anaerobic respiration using fumarate as a terminal electron acceptor. The four Ca. Uliximicrobium MAGs encode two distinct stereotypes of citrate synthase: citrate (Si)-synthase, the predominant citrate synthase in bacteria (and encoded in all 17 MAGs); and citrate (Re)-synthase, which unlike citrate (Si)-synthase, is oxygen-sensitive (43–45). Citrate (Re)-synthase is typically found in obligate anaerobes (43, 44), including Dehalococcoides mccartyi (Chloroflexota: Dehalococcoidia), where it is the only citrate synthase (44); and certain members of the class Clostridia (e.g., Clostridium kluyveri, Acetivibrio thermocellus) that encode both citrate (Si)-synthase and citrate (Re)-synthase and express both during the oxidative TCA cycle (43, 46). It has been proposed that expressing dual citrate synthases assists in the initiation of the TCA cycle and in glutamate synthesis (via production of 2-oxoglutarate for ammonia assimilation) (46). Thus, the presence of dual citrate synthases in Ca. Uliximicrobium might be associated with facultatively anaerobic abilities.

Trace gas oxidation

The majority of MAGs (15 out of 17), representing all four classes, encode high-affinity [NiFe] hydrogenases (group 1h or 1l; Fig. S2) that would allow atmospheric H₂ to support aerobic respiration (47–49). Three Ca. Spiritibacter species and one Ca. Tarhunnaea species additionally encode enzymes of the Calvin-Benson-Bassham (CBB) cycle, including ribulose bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase; the RubisCO belongs to form IE, the major form of RubisCO associated with atmospheric chemosynthesis (50). None of these MAGs have any identifiable genes for bacteriochlorophyll-dependent light harvesting. Therefore, the combination of high-affinity [NiFe] hydrogenases and CBB cycle suggests the potential for light-independent H₂-driven carbon fixation (Table 2). The CBB cycle in these four species was the only autotrophic pathway identified in the 17 MAGs. Carbon monoxide dehydrogenase (CODH) is encoded in four Ca. Spiritibacter MAGs, meaning that CO could be oxidized to CO₂ as a source of energy. Thus, of these Ca. Spiritibacter MAGs, two (Ca. Spiritibacter saxicola, Ca. Spiritibacter pertinax) have the potential to utilize H₂, CO₂, and CO for atmospheric chemosynthesis.

Bacterial macroglobulins

MAGs of classes Ca. Spiritibacteria and Ca. Martimicrobia possess genes for bacterial macroglobulins; these are large proteins (≥1,500 aa) that are homologs of the metazoan protease inhibitor α₂-macroglobulin (51). Most bacterial species that have macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts, either as invasive pathogens or colonizing saprophytes, and it has been proposed that these macroglobulins are colonization factors that provide protection to the bacterial cell by inhibiting the action of host proteases (51). The macroglobulin genes in the MAGs examined here have signal peptides, consistent with an extracytoplasmic location (Fig. 2 to 4), as in other bacteria (51, 52). Two types of macroglobulins have been described in bacteria: one type uses a thioester motif (CxEQ) capable of binding and trapping proteases (51, 52); a second type lacks this motif, but still has protease-binding capacity and may function as part of a multiprotein complex (53). The macroglobulins in the two class Ca. Martimicrobia MAGs contain CxEQ, whereas this motif is absent in class Ca. Spiritibacteria MAGs. Ca. Spiritibacteria MAGs also lack any identifiable homologs of a macroglobulin-associated protein complex. Based on gene sequences, the lengths of these macroglobulins (1980–1996 aa for Ca. Spiritibacteria; 1774–1776 aa for Ca. Martimicrobia) are longer than those reported in other bacteria and include at least one (usually two or three) SbsA/Ig-like domain at the N-terminal region. Multiple SbsA/Ig-like domains are found in the cell surface layer protein of Geobacillus stearothermophilus, where this protein constitutes the outer cell envelope (54); thus, these macroglobulins may form part of the cell envelope of Ca. Spiritibacteria and Ca. Martimicrobia cells.

Fig 4.

Predicted metabolic capacities of Candidatus Martimicrobium tenax gen. et. sp. nov. (Candidatus Martimicrobia class. nov.). The identities of the sulfated biopolymers are not known; for simplicity, sulfated glycosaminoglycans (GAGs) are shown as a substrate. 1h, [NiFe]-hydrogenase group 1h; 1l, [NiFe]-hydrogenase group 1l; 2-PG, 2-phosphoglycolate; ASR, assimilatory sulfate reduction; BCAA, branched-chain amino acids; bMG, bacterial macroglobulin; Cox, cytochrome c oxidase; DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; GH, glycoside hydrolase; Kat, catalase; MSH, mycothiol; PPP, pentose phosphate pathway; Sod, superoxide dismutase; Tad, tight adherence.

Potential toxic abilities

There is evidence that certain MAGs encode the ability to lyse or penetrate cells. All four Ca. Uliximicrobium MAGs encode homologs of hemolysin III, a pore-forming cytolytic toxin described in the pathogen Bacillus cereus (55). Furthermore, the Ca. Uliximicrobium exili and Ca. Uliximicrobium arcanum MAGs encode proteins that are homologous to the pore-forming toxin complexes (Tc) of certain pathogenic bacteria that use Tc to inject toxic enzymes into host cells (56, 57) (Fig. 6). In general for Tc, the assembled tripartite complex (holotoxin) comprises TcA, TcB, and TcC subunits (56–60). TcA determines host specificity and facilitates receptor-binding using host cell-surface glycans (60) and subsequently forms a syringe-like translocation channel that perforates the host cell membrane through which the toxic enzyme enters the host cytoplasm (56, 61). TcB and TcC combine to form a hollow “cocoon” that encapsulates the toxic enzyme that is itself part of TcC (56–60). The C-terminal region of TcC is autoproteolytically dissociated within the cocoon (56, 59). In certain Tc-encoding bacteria, TcB and TcC are fused into a single protein (TcBC) (57, 62), which appears to be the case in Ca. Uliximicrobium exili and Ca. Uliximicrobium arcanum. In both Ca. Uliximicrobium MAGs, the two proteins (TcA, TcBC fusion protein) are encoded by adjacent genes (Tables S4 and S5). In Ca. Uliximicrobium exili and Ca. Uliximicrobium arcanum, both proteins are large: 3,333 and 3,075 amino acids (aa), respectively, for TcA; and 2,544 and 2,598 aa, respectively, for TcBC. Between them, the Ca. Uliximicrobium TcA and TcBC proteins include all the features characteristic of the Tc holotoxin: neuraminidase-like region of TcA that is principally responsible for the pH-induced opening of the cocoon, which drives the injection of the TcA channel into the host membrane (56); a TcB-binding domain at the C-terminal of TcA; an N-terminal region of TcB homologous to SpvB toxin; and the rearrangement hotspot (RHS) repeat-associated core domain of TcC that contains the catalytic dyad of the aspartyl protease responsible for cleavage (57, 59). Assuming the Ca. Uliximicrobium TcBC protein is cleaved at the first hydrophobic residue after the boundary between the conserved region and the C-terminal region (57, 59), we estimate that the released C-terminal region polypeptides of Ca. Uliximicrobium exili and Ca. Uliximicrobium arcanum to be 34.74 and 35.73 kDa, respectively; thus, both are around the posited maximum size (~35 kDa) that can be accommodated by the Tc cocoon (57).

In general, among Tc-encoding bacteria, the C-terminal region sequence is highly variable (57, 59) and includes toxic enzymes such as ADP-ribosyltransferase that targets host actin and GTPases (63), and nucleotide deaminase that targets host nucleic acids (57, 64). The C-terminal regions of both Ca. Uliximicrobium exili and Ca. Uliximicrobium arcanum TcBC show no identifiable homology to any known C-terminal region or to each other’s C-terminal region. For both species, the C-terminal region of the TcBC protein lacks any identifiable domains, and we could not infer function(s) based on sequence identities or structure predictions. The C-terminal regions of both Ca. Uliximicrobium proteins are homologous to regions of certain uncharacterized bacterial proteins, such as putative TcC and TcBC proteins of unknown function (see Supplemental text), and we were unable to identify Tc protein homologs in other members of Chloroflexota. Although the C-terminal region polypeptides of the two Ca. Uliximicrobium species have no characterized homologs, the homologies of the other components of the putative Ca. Uliximicrobium TcA-TcBC complexes to the Tc holotoxins of known pathogenic bacteria suggest that the former likewise function as pore-forming toxins that act against eukaryotic cells.

The MAG of Ca. Otrerea regina encodes a large protein (4,592 aa) that is surface-exposed (based on an N-terminal signal peptide; Fig. 3) and potentially involved in adhesion and invasion. The protein sequence includes an N-terminal signal peptide an N-terminal region in the N-terminal half of the protein composed of multiple Big-1 (bacterial immunoglobulin-like domain 1) repeats and an RHS repeat-associated core domain near the C-terminal; Big-1 domains are present in the intimin/invasin family of bacterial adhesins, involved in cell attachment and invasion by pathogenic Escherichia coli and related gammaproteobacterial species (65).

Fig 3.

Predicted metabolic capacities of Candidatus Otrerea regina gen. et. sp. nov. (Candidatus Spiritibacteria class. nov.). 2-AEP, 2-aminoethylphosphonate; 2-PG, 2-phosphoglycolate; ASR, assimilatory sulfate reduction; BCAA, branched-chain amino acids; Big-1, bacterial immunoglobulin-like domain 1; bMG, bacterial macroglobulin; Cox, cytochrome c oxidase; DHAP, dihydroxyacetone phosphate; Dyp, dye decolorizing peroxidase; G3P, glycerol 3-phosphate; GH, glycoside hydrolase; Kat, catalase; MSH, mycothiol; PPP, pentose phosphate pathway; Sod, superoxide dismutase; TMA, trimethylarsine.

Biosynthetic gene clusters

Biosynthetic gene clusters (BGCs) are a locally clustered group of two or more genes that together encode a biosynthetic pathway for secondary metabolite production (66). Previous studies have shown Chloroflexota isolate genomes to contain between 3 and 20 BGC regions (67, 68) and Chloroflexota MAGs to have 0–18 BGC regions (69, 70). Overall, the 17 Chloroflexota MAGs examined here were found to contain one to six BGC regions per MAG (Table S6). Terpenes were the most common BGC type, with all MAGs found to contain at least one terpene BGC associated with carotenoid production. Other BGC types include non-ribosomal peptide synthetases (NRPS) and NRPS-like containing BGCs, found in 10 MAGs, and polyketide synthases (type I or type III), found in 9 MAGs. Several of these BGCs showed the closest similarity to core biosynthesis genes that produce antibiotics active against Gram-positive bacteria, such as gausemycin and icosalide. This antibiotic potential is consistent with previously demonstrated antimicrobial activities in members of Chloroflexota (68).

Metacaspases

Metacaspases are cysteine proteases that are homologous to metazoan caspases, and are found in non-metazoan organisms (71, 72). In metazoans, caspases are involved in cell death, as the major initiators or effectors of apoptosis (73, 74). Metacaspases in plants, fungi, and bacteria are implicated in diverse functions, including cell signaling, stress acclimation, and apoptosis (75, 76). Metacaspases, which have been detected previously in members of the Chloroflexota (72), are encoded in all four Ca. Uliximicrobium MAGs, as well as the Ca. Tarhunnaea remota MAG. In general, it has been hypothesized that metacaspase genes are associated with bacteria that have larger and less streamlined genomes and exhibit complex physiological capacities and enhanced secondary metabolite synthesis (72, 77). This accords with the genome sizes of Ca. Uliximicrobia (estimated 6.4–8.3 Mb) and Ca. Tarhunnaea remota (estimated 6.7 Mb; Table S1) and the relatively high counts of BGCs in these MAGs. The Ca. Uliximicrobium occultum MAG encodes a metacaspase that includes an ATP-dependent helicase domain. Another metacaspase (encoded in Ca. Uliximicrobium arcanum) may have an apoptotic function, given that it also possesses an N-acetylmuramoyl-l-alanine amidase domain, which is a peptidoglycan hydrolase implicated in cellular autolysis (72).

Adaptations to an endolithic and hyperarid environment

Atmospheric chemosynthesis

The energy liberated by atmospheric oxidation of H₂ and CO has been demonstrated to support bacterial persistence during carbon starvation (48, 78, 79). Genes for high-affinity uptake [NiFe]-hydrogenases (group 1h and/or 1l) were identified in 15 out of 17 MAGs, which would allow trace levels of H₂ gas to be used by these bacteria as a reliable energy source in the harsh environment (48, 50, 80). Furthermore, water is the major end-product of the aerobic respiration of atmospheric H₂, and it has been proposed that H₂ oxidation may serve as a mechanism for microorganisms to stay hydrated in the hyperarid deserts of Antarctica (48).

Microaerobic and anaerobic respiration

All 17 MAGs encoded cytochrome c oxidase, but six MAGs additionally encoded the high-affinity cytochrome bd ubiquinol oxidase (Table 2). Ca. Uliximicrobium species MAGs encode both cytochrome bd ubiquinol oxidase for microaerobic respiration and fumarate reductase for anaerobic respiration. The ability to grow under varying oxygen tensions might be beneficial under conditions of oxygen depletion, as occurs in arid environments as a result of rapid respiration by aerobic heterotrophs after hydration events, and may even lead to transient anaerobic microenvironments (7, 25).

Protection against oxidative stress

Aerobic respiration is a potential source of oxidative stress in the form of highly reactive oxygen species (ROS) such as superoxide (O₂^-) and peroxide (H₂O₂) that are formed by the single electron reduction of oxygen (81). Furthermore, a decrease in the environmental temperature increases oxidative stress in bacterial cells (82). All MAGs encode protective enzymes such as catalase and superoxide dismutase, and five MAGs encode Dyp-type peroxidases that may also protect against H₂O₂ (83) (Table S2; Fig. 2 to 6). All 17 MAGs encode the capacity to synthesize carotenoids, which can act against ROS, as well as confer protection against environmental stressors such as solar radiation and freeze-thaw cycles, including by membrane stabilization (84).

All 17 MAGs possess genes for the synthesis of mycothiol. As well as its primary function in maintaining redox homeostasis inside the cell, mycothiol can also serve in detoxification (85); mycothiol is more resistant to autoxidation by heavy metals than glutathione under aerobic conditions (86, 87). The glycolytic intermediate glucose-6-phosphate can serve as an intracellular source of reductant in concert with coenzyme F₄₂₀ (8-hydroxy-5-deazaflavin). Of the 17 MAGs, 14 encode an F₄₂₀-dependent glucose-6-phosphate dehydrogenase, which, in mycobacteria, plays a protective role against oxidative stress (88, 89).

Multiple types of formaldehyde dehydrogenase are encoded across the MAGs for the detoxification of formaldehyde, including mycothiol-, glutathione-, and NAD⁺-dependent formaldehyde dehydrogenase. Another potential route of formaldehyde detoxification involves a spontaneous reaction with tetrahydrofolate (H4F) to form methylene-H4F, with the one-carbon moiety either directed to cellular methylation reactions or further oxidized to formyl-H4F (e.g., for purine synthesis) (90).

Of note is that the Ca. Tarhunnaea remota MAG encodes a type 1 encapsulin shell protein (Enc); Enc forms self-assembling intracellular nanocompartments (Fig. 5) that enclose one or more specific cargo proteins (e.g., ferritin-like proteins, peroxidases) and are important to the cell’s responses to nutrient starvation and/or oxidative stress (91–93). However, we were unable to identify potential cargo protein(s) in the Ca. Tarhunnaea remota MAG based on established criteria (e.g., N- or C-terminal targeting peptides) (92, 93), and so the exact function of this putative Enc nanocompartment is unclear.

Fig 5.

Predicted metabolic capacities of Candidatus Tarhunnaea remota gen. et. sp. nov. (Candidatus Tarhunnaeia class. nov.). 1h, [NiFe] hydrogenase group 1h; 1l, [NiFe] hydrogenase group 1l; 2-PG, 2-phosphoglycolate; BCAA, branched-chain amino acids; Cox, cytochrome c oxidase; DHAP, dihydroxyacetone phosphate; Dyp, dye decolorizing peroxidase; Enc, encapsulin nanocompartment; G3P, glycerol 3-phosphate; GH, glycoside hydrolase; Kat, catalase; MSH, mycothiol; PHA, poly(3-hydroxyalkanoate); PAPS, phosphoadenosine phosphosulfate; PPP, pentose phosphate pathway; Sod, superoxide dismutase; Tad, tight adherence; TMA, trimethylarsine.

Rhodopsin

None of the 17 MAGs showed any genes required for bacteriochlorophyll-dependent light harvesting or photoautotrophy. We infer Ca. Otrerea regina to be photoheterotrophic, based on a gene for bacteriorhodopsin-like rhodopsin (Fig. 3), a multipass membrane protein that uses light energy to generate an ion-motive force for ATP synthesis (94). Rhodopsins have been reported previously in Antarctic Dry Valleys metagenomes (95) and may provide a survival advantage during periods of nutrient deprivation (7).

Osmotic stress

The abilities to synthesize and/or import compatible solutes allow prokaryotes to acclimate to fluctuations in osmolarity and water availability but would be of critical importance to those bacteria that live in hyperarid environments. Water is required for the maintenance of membrane phospholipids in a fluid phase, as well as for the correct folding of many proteins (96). Prokaryotes are capable of survival under limited water availability by osmoadaptation, which consists of intracellular accumulation of compatible solutes that allow for the stabilization of proteins and membranes (97, 98). These compatible solutes are either synthesized de novo or imported from the environment and include glycine betaine, trehalose, and sucrose (97, 99); these abilities are encoded among the MAGs examined here (Table 2; Supplemental text).

Two Ca. Uliximicrobium MAGs encode a homolog of NdvB, a large multidomain enzyme that catalyzes the synthesis of cyclic β-(1, 2)-glucans; in soil-dwelling Rhizobiaceae (class Alphaproteobacteria), these polysaccharides are involved in nodule invasion and virulence (100–102) and have been inferred to function extracytoplasmically in hypoosmotic acclimation, such as by decreasing cytoplasmic membrane turgor pressure (103–105). Unlike Rhizobiaceae that possess NdvB, the Ca. Uliximicrobium MAGs have no identifiable NdvA homolog involved in the export of cyclic β-(1, 2)-glucans.

Pili and flagella

Genes for pilins and the assembly of pili were found in 15 of the 17 MAGs from all four classes (Fig. 2 and Fig. 4–6; Table 2; Table S2), indicating the importance of nonspecific adherence in the colonization of surfaces (106), facilitating the use of rocks as a growth surface (15). Genes for flagellins and proteins for flagella assembly were identified in only one MAG (Ca. Uliximirobium exili) (Fig. 6). Flagellar motility could be beneficial to endolithic bacteria during transient periods when water is present within pores. However, flagella may confer benefits that are not directly related to endolithism, given their pathogenic function in other bacteria (107).

Utilization of sugars

Many of the saccharolytic enzymes encoded across the 17 MAGs can be viewed in the context of the presence of algae, cyanobacteria, and fungi in endolithic communities, especially those that form symbioses as lichens (2, 3, 13). Lichens are symbiotic organisms composed of a fungal partner (mycobiont) and one or more photosynthetic partners (photobiont), which combine to form a symbiotic structure (thallus); this interaction provides the mycobiont with carbon substrates and the photobiont with a habitat that protects them from abiotic and biotic stresses (108). The photobionts and mycobionts of lichens contain sugars in the form of structural cell wall components, extracellular matrices, storage compounds, and other metabolites (109). In lichen thalli, the photosynthetic products glucose and ribitol are transferred from photobiont to mycobiont, where they are converted to mannitol (108). Polyols (especially sorbitol) are synthesized by lichens to cope with both osmotic and oxidative stresses associated with desiccation (110). Sorbitol dehydrogenase, which converts sorbitol to fructose, is encoded in most MAGs from all four classes. The MAGs of both Ca. Tarhunnaea species encode mannitol dehydrogenase, for the conversion of mannitol to fructose, which can be phosphorylated and thereby enter glycolysis. The Ca. Tarhunnaea remota MAG also encodes ribitol dehydrogenase to convert ribitol to the pentose sugar ribulose.

In general, the major monosaccharide components of lichen polysaccharides are glucose, galactose, and mannose, with xylose, arabinose, rhamnose, and fucose present in minor amounts (109, 111, 112). The mycobiont cell wall is composed mainly of the homoglucans lichenin and isolichenin, and galactomannan (112, 113). Some Antarctic lichen photobionts include unicellular green algae (e.g., Trebouxia), which are known to include cellulose in their cell wall, albeit in low amounts, as well as glycoproteins (111, 114). In general, lichen photobionts may produce extracellular polysaccharides that are linked, or loosely attached, to the cell surface, or released into the surrounding environment (114). Microcolonial black fungi, consistently associated with these communities, are reported to produce extracellular matrices, mainly β-glucans, as protectants from hostile environmental conditions (27, 115, 116). Certain endolithic yeasts of the phylum Basidiomycota possess a dense cell wall that is often associated with an exopolysaccharidic capsule, which provides protection against environmental extremes and could also serve as a defensive barrier against lichen toxins (8, 117).

GHs for the release of terminal monosaccharides from polysaccharides or glycoconjugates are represented in the MAGs from across all four classes. β-galactosidase is encoded in all MAGs and could be used for the cleavage of β-linked terminal galactose residues from glycoproteins and glycosaminoglycans. α-galactosidase, for the removal of galactose side-groups from galactomannan, is encoded in 16 MAGs. β-mannosidase, which cleaves the non-reducing end of β-mannosidic oligosaccharides to release mannose, is encoded in eight MAGs. α-mannosidase, for the release of mannose from glycoproteins, is encoded in 10 MAGs. β-xylosidase, which is encoded in 12 MAGs, cleaves xylose from the non-reducing end of oligosaccharides. Exo-β-1,3-glucanase, which successively hydrolyzes the non-reducing ends of β-glucans, is encoded in two MAGs. Enzymes for the catabolism of galactose are encoded in all 17 MAGs, whereas pathways for the catabolism of mannose, xylose, and arabinose are only encoded in certain MAGs (Table S2; Supplemental text). All 17 MAGs encode ATP-binding cassette (ABC) transporters for the uptake of sugars (Fig. 2 to 6). However, whereas most MAGs encode multiple different types of ABC transporters for sugars, the Ca. Uliximicrobium MAGs were exceptional in the paucity of identifiable sugar transporters (Table S2).

Starch granules are known to be present in the lichen photobiont Trebouxia (118, 119). Genes for a starch degradation pathway were identified in one MAG (Ca. Sutekhia aridicola) in the form of a gene cluster that includes cyclomaltodextrin glucanotransferase and cyclomaltodextrinase (both enzymes with signal peptides) for the hydrolysis of cyclic and linear maltodextrins and a maltose/maltodextrin primary uptake system. Oligo-1,6-glucosidase, for hydrolysis of certain oligosaccharides produced from starch by α-amylase, is encoded in nine MAGs (in classes Ca. Spiritibacteria and Ca. Tarhunnaeia).

Sulfated polysaccharides and choline sulfate (both found in lichens; 109, 120) and sulfated glycosaminoglycans (e.g., chondroitin sulfates) are potential sources of sulfate for those MAGs that encode sulfatases and enzymes for assimilatory sulfate reduction (Table S2; Supplemental text). As well as the degradation of sulfated organic compounds to yield sulfate, sulfated polysaccharides can also be “pruned” of sulfate groups to facilitate access to the carbon skeleton for utilization as nutrient sources, as proposed for other bacteria (121, 122).

Utilization of photosynthate

Primary producers, such as lichens, algae, and cyanobacteria, release photosynthates and exudates, including the photorespiratory byproducts glycolate and phosphoglycolate (7, 123). Phosphoglycolate phosphatase, which dephosphorylates 2-phosphoglycolate to glycolate, is encoded in almost all MAGs examined here. This enzyme is also involved in the dissimilation of 2-phosphoglycolate formed intracellularly in bacteria during the DNA repair of 3'-phosphoglycolate ends, a major class of DNA lesions induced by oxidative stress (124). A pathway to convert glycolate (another photorespiratory byproduct) via glyoxylate to malate, and thereby be passed into the TCA cycle (125), was identifiable in 11 of the 17 MAGs (Table S2), although only one MAG (Ca. Otrerea regina) encodes a complete glyoxylate cycle (Table 2).

Utilization of glycerophospholipids

As degradation products of cellular glycerophospholipids, glycerol, and glycerol 3-phosphate (G3P) are ubiquitous in the environment. The ability to utilize glycerol as a carbon source is indicated by genes that are common to all MAGs: glycerol transporter (aquaglyceroporin), G3P ABC transporter, glycerol kinase, and G3P dehydrogenase. MAGs belonging to members of class Ca. Spiritibactera (six Ca. Spiritibacter species and Ca. Aglaurobacter anningiae) encode a heterotrimeric G3P dehydrogenase complex (GlpABC) (126), whereas other MAGs encode the homodimeric G3P dehydrogenase (GlpD). G3P ABC transporters are also capable of importing deacylated phospholipids [glycerophosphoryl diesters (GPDs)] (127). GPDs (e.g., glycerophosphocholine and glycerophosphoglycerol) can be hydrolyzed by the broad-specificity GPD phosphodiesterase to yield G3P, as well as other potential substrates (e.g., choline, glycerol) (128). Most MAGs (all except those of Ca. Spiritibacter) encode cytoplasmic GPD phosphodiesterases, with two Ca. Uliximicrobium MAGs additionally encoding a GPD phosphodiesterase with a signal peptide, which suggests the ability to cleave GPDs extracytoplasmically.

Organic nitrogen sources

Potential nitrogen sources include ammonia excreted or released by diazotrophic cyanobacteria (7), which, as free-living and lichen-associated, are present in endolithic communities (12, 18, 20). Ammonium transporters are encoded in eight MAGs (representing all four candidate classes). The antioxidant glutathione is important for desiccation tolerance in lichens due to the scavenging of free radicals formed during desiccation (129). Peptidases and ABC transporters for oligopeptides (including glutathione) are encoded in all 17 MAGs (Fig. 2 to 6). Allantoin was previously inferred to be directly involved in protection against sun exposure by Antarctic cryptoendolithic lichen-dominated communities, with allantoin and its precursor metabolite uric acid detected using metabolomics (2). This means allantoin and uric acid are potential sources of reduced nitrogen for those bacteria that encode enzymes for their catabolism (e.g., class Ca. Tarhunnaeia MAGs).

Lichen thalli produce urea as a product of arginine catabolism, in both the mycobiont and photobiont (130). Urea can also be generated endogenously by bacteria, such as from purine catabolism. Although individual pinniped carcasses have been reported in McMurdo Dry Valleys (131), a more likely source of urea in this environment is microinvertebrates in meltwater streams that flow for a brief period (~10 weeks) over the austral summer (132). Urease genes were identified in certain MAGs of three classes (Ca. Martimicrobia, Ca. Tarhunnaeia, Ca. Uliximicrobia). Amino acids produced by lichens, including taurine (133), could potentially be used as a source of reduced nitrogen. Enzymes for the catabolism (including transamination and deamination) of certain amino acids are encoded across all MAGs, and two MAGs encode enzymes for the degradation of taurine (Table S2; Supplemental text).

Heavy metal detoxification

An association with lichens would be expected to increase the exposure of bacteria to heavy metals. As components of the Earth’s crust, heavy metals are found naturally in rocks and soil (134, 135). Furthermore, cell wall and extracellular polysaccharides of the lichen thallus accumulate and sequester heavy metals, thereby limiting the entry of these metals into the constituent cells (114). Thus, bacteria in close proximity to lichens and/or utilizing lichen polysaccharides would likely encounter elevated levels of heavy metals. Numerous transporters are encoded in the MAGs for the efflux of heavy metals (e.g., cadmium, iron, cobalt, nickel, zinc, manganese, chromate), as well as enzymes for the detoxification of arsenic (and its derivative ions) and mercury (80, 136, 137) (Fig. 2 to 6; Table S2). However, given the essential roles of metals such as iron, cobalt, nickel, iron, zinc, tungsten, molybdenum, and manganese in metabolism, various uptake systems (including primary transporters) are encoded for cations derived from these elements (Table S2; Fig. 2 to 6). Iron compounds mobilized by endolithic lichens are transported by water within sandstone both downward (when snow melts on the rock surface) and upward (when capillary water rises due to evaporation) (1); the endolithic niche is, therefore, highly enriched with iron (32, 138).

Conclusions

In the Antarctic Dry Valleys desert areas, life is confined to the lithic refugia that provides microorganisms with thermal buffering, moisture, protection from abiotic stresses, a surface for growth, and access to mineral substrates. Based on 17 MAGs retrieved from Antarctic endolithic metagenomes, we propose four new classes within phylum Chloroflexota: Ca. Spiritibacteria, Ca. Martimicrobia, Ca. Tarhunnaeia, and Ca. Uliximicrobia. MAGs from all four classes encode an array of adaptations to hyperarid conditions. These include the capacity for trace gas oxidation, particularly high-affinity uptake hydrogenases, that provide energy and water for survival; in some cases, there is the capacity to couple the energy generated from H₂ and CO oxidation to support carbon fixation (atmospheric chemosynthesis). Also encoded in these Chloroflexota MAGs are transporters and catabolic enzymes that would allow substrates to be sourced from other endolithic organisms such as fungi, lichens, and algae. The adaptations proposed and discussed here—including those that relate to nutrient acquisition, substrate catabolism, energy conservation, and osmoadaptation—are not unique to these endolithic bacteria, but are inferred to play a part in the overall survival strategies that allow these bacteria to persist in a hyperarid desert habitat.

MATERIALS AND METHODS

Endolithic bacteria catalog

In total, the data set included 109 metagenomes, of which 18 (Joint Genome Institute dataset) were generated, sequenced, assembled, and binned into MAGS as described previously (15). A further 91 metagenomes (Fondazione Edmund Mach dataset) were generated, sequenced, assembled, and binned into MAGs as described previously (20). The resulting bins were combined with the 1,660 metagenomic bins obtained previously (15) and analyzed using the metashot/prok-quality (139) v1.2.3 (parameters—gunc_filter --gunc_db gunc_db_2.0.4.dmnd) workflow. Briefly, completeness, redundant, and nonredundant contamination (140) estimates were obtained by CheckM (141) v1.1.2 and GUNC (140). Bins with completeness estimates of <50%, more than 10% contamination and that did not pass the GUNC filter were discarded, resulting in a total of 4,540 filtered prokaryotic MAGs. MAGs were classified into “high-quality draft” (HQ) with >90% completeness and <5% contamination and “medium-quality draft” (MQ) with completeness estimates of ≥50% and less than 10% contamination. Species-level operational taxonomic units (OTUs) were identified by clustering HQ and MQ MAGs at 95% average nucleotide identity (ANI) using dRep (142) v2.6.2, resulting in a total of 2,279 OTUs. For each species-level OTU, the MAG with the highest quality score was chosen as representative. The score was computed using the formula: score = completeness − 5 × contamination + 0.5 × log(N₅₀) (139).

Phylogenomic analysis of Chloroflexota MAGs

Initial classification of MAGs was performed using the classified workflow in GTDB-Tk v2.1.0 with database 207_v2 (143). A total of 233 of the 2,279 OTUs were placed within the Chloroflexota, with 38 MAGs belonging to the undescribed classes UBA2235, UBA4733, UBA5177, and UBA6077. Relationships between these 38 Chloroflexota MAGs and their nearest relatives in the GTDB-Tk reference tree were investigated using CompareM v0.1.2 (https://github.com/dparks1134/CompareM) to calculate average amino acid identity (AAI) and fastANI v1.32 (144) to calculate ANI. The assignment of classes within phylum Chloroflexota was based on GTDB-Tk taxonomic placement results, under which clades at each taxonomic rank are created based on the Relative Evolutionary Divergence values between genomes (145). Species, genera, and family groupings were inferred as previously reported (AAI values of <45% = different family; 45%–65% = same family and different genus; 65%–95% = same genus and different species; 95%–100% = same species) (146–148), with 17 MAGs representing distinct species with high genome completeness (>94%) and low genome contamination (<6%) selected for in-depth metabolic characterization. MAGs were named and assigned to a taxonomic hierarchy according to recommendations for describing novel Candidatus species (34–37).

For the construction of the maximum-likelihood phylogenomic tree, a multiple sequence alignment of 120 bacterial marker genes was obtained from the GTDB-Tk de novo workflow (v2.3.0, database release 214). The alignment was used as input to IQ-tree v2.2.2.5 (149) with automatic selection of the best-fitting model (LG + F + I + G4) and 1,000 ultrafast bootstraps (150), and the resulting tree was visualized using Dendroscope v3.8.5 (151). The tree was rooted in the phylum Vulcanimicrobiota (formerly Candidatus Eremiobacterota) (152).

Genome annotation of Chloroflexota MAGs

The genomic functional potential of MAGs was assessed by considering cellular and metabolic traits based upon manual examination of protein sequences and pathways. This was performed in a similar way to previous assessments of the validity of gene functional assignments (122, 143, 144, 153). In brief, we used automated annotations generated by Prokka (154) and eggNOG (155) as the starting point for a process that included manual vetting of individual protein sequences, which were then used to reconstruct established metabolic pathways. Manual vetting of each protein sequence began with submission to NCBI BLASTP (using the “UniProtKB/Swiss-Prot only” option); proteins needed to show ≥30% sequence identity over the length of the protein and/or an expectation (E) value <10⁻¹⁰ (156) to an experimentally verified protein in the BLASTP database for the functional annotation to be considered valid. If this threshold was not reached, protein sequences were submitted to InterProScan (157) to identify functional domains and potential subcellular locations. All protein annotations in this study are putative. GH families were identified using BLASTP and InterProScan and classified using CAZy (158). Protein sequences that were initially identified as hydrogenases based on catalytic domains were classified further using HydDB (103) and phylogenetic analysis (50). Identification of CODH was dependent upon the presence in these protein sequences of a catalytic cluster (159). The C-terminal regions of toxin complex TcC homologs were investigated using I-TASSER (160).

Phylogenetic analysis of RubisCO and group 1 [NiFe]-hydrogenase

Separate phylogenetic analyses were conducted to identify the form (subtype) of RubisCO large subunit (n = 5) and the type of Group 1 [NiFe]-hydrogenase (n = 22) sequence, using RubisCO large subunit and hydrogenase protein sequences obtained from the 17 MAGs, plus reference sequences obtained from prior phylogenetic analysis (50). Multiple sequence alignment was performed using MAFFT v7.407, employing the L-INS-i iterative refinement method (161, 162). The resulting alignments were then trimmed to remove poorly aligned regions using trimAl v1.4.1, with a gap threshold of 0.5 (163). Sequences with more than 50% gaps after alignment and identical sequences were removed. Maximum likelihood phylogenetic trees were constructed using IQ-Tree v1.6.10 (164), applying 1,000 ultrafast bootstrap iterations, hill-climbing nearest neighbor interchange search, and incorporating additional SH-like approximate likelihood ratio tests (165). ModelFinder was performed to determine the best evolutionary model, which was LG + R4 for the RubisCO tree and LG + R5 for the [NiFe]-hydrogenase tree (164). Sequences that failed the chi2 test during tree building were removed. The final consensus trees were both uploaded to iTOL for visualization (166).

BGC analysis

Nucleotide sequences for the 17 Chloroflexota MAGs were analyzed for BGCs using the antibiotics and secondary metabolite analysis shell (antiSMASH v7.0.0; https://antismash.secondarymetabolites.org) (167–169), with relaxed detection strictness and all extra features enabled [KnownClusterBlast, ClusterBlast, SubClusterBlast, MIBiG cluster comparison, ActiveSiteFinder, RREFinder, Cluster Pfam analysis, Pfam-based GO term annotation, TIGRFam analysis, transcription factor binding site (TFBS) analysis] (167, 169). For each BGC, the core biosynthetic gene sequence was analyzed in NCBI BlastP, and the top hit was recorded. Resulting clusters were verified manually through visual inspection including examination of each BGC for completeness and contiguity. Additionally, for comparison, the complete genome of Chloroflexota NCBI type strain Herpetosiphon aurantiacus DSM 785 (CP000875.1) was also analyzed.

DATA AVAILABILITY

Metagenomes raw data are available under the NCBI accession numbers listed in Table S7. MAGs and annotations for MAGs are available at the zenodo repository (DOI: 10.5281/zenodo.7313591).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.02264-23.

Supplemental text, Figure S1, Figure S2, Table S3, Table S4, Table S5. aem.02264-23-s0001.docx.

Supplemental text: Genus Candidatus Uliximicrobium Tc toxin complexes. Figure S1: Expanded phylogenetic tree. Figure S2: Phylogenetic tree of all [NiFe] hydrogenase genes. Table S3: Glycoside hydrolases. Table S4: Candidatus Uliximicrobium exili gene cluster that contains Tc toxin complex genes, and Candidatus Uliximicrobium absconditum gene cluster that contains genes for homologous proteins. Table S5. Candidatus Uliximicrobium arcanum gene cluster that contains Tc toxin complex genes.

Table S1. aem.02264-23-s0002.xlsx.

MAGs and metadata.

Table S2. aem.02264-23-s0003.xlsx.

Proteins and pathways discussed in the text.

Table S6. aem.02264-23-s0004.xlsx.

Biosynthetic gene clusters.

Table S7. aem.02264-23-s0005.xlsx.

Metagenome raw data links.

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