# Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Yulin Song, Vincent Hervé, Renate Radek, Fabienne Pfeiffer, Hao Zheng, Andreas Brune

► To cite this version:

Yulin Song, Vincent Hervé, Renate Radek, Fabienne Pfeiffer, Hao Zheng, et al.. Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches. Environmental Microbiology, 2021, 23 (8), pp.4228-4245. 10.1111/1462-2920.15600. hal-03701894

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# Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Yulin Song, Vincent Hervé, Renate Radek, Fabienne Pfeiffer, Hao Zheng, Andreas Brune

<figure><img src="image_1.png" /></figure>

To cite this version:

Yulin Song, Vincent Hervé, Renate Radek, Fabienne Pfeiffer, Hao Zheng, et al.. Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches. Environmental Microbiology, 2021, 23 (8), pp.4228-4245. 10.1111/1462-2920.15600. hal-03701894

HAL Id: hal-03701894

https://hal.science/hal-03701894v1

Submitted on 10 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Characterization and phylogenomic analysis of *Breznakiella homolactica* gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Yulin Song<sup>1</sup>,<sup>†</sup> Vincent Hervé Radek<sup>1</sup>,<sup>†</sup> Renate Radek<sup>1</sup>,<sup>‡</sup> Fabienne Pfeiffer,<sup>1</sup> Hao Zheng<sup>1</sup> and Andreas Brune<sup>1*</sup>

<sup>1</sup>Research Group Insect Gut Microbiology and Symbiosis, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, Marburg, 35043, Germany.

<sup>2</sup>Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Str. 1-3, Berlin, 14195, Germany.

## Summary

Spirochetes of the genus *Treponema* are surprisingly abundant in termite guts, where they play an important role in reductive acetogenesis. Although they occur in all termites investigated, their evolutionary origin is obscure. Here, we isolated the first representative of ‘termite gut treponemes’ from cockroaches, the closest relatives of termites. Phylogenomic analysis revealed that *Breznakiella homolactica* gen. nov. sp. nov. represents the most basal lineage of the highly diverse ‘termite cluster I’, a deep-branching sister group of *Treponemataceae* (fam. ‘Termitinemataceae’) that was present already in the cockroach ancestor of termites and subsequently coevolved with its host. *Breznakiella homolactica* is obligately anaerobic and catalyses the homolactic fermentation of both hexoses and pentoses. Resting cells produced acetate in the presence of oxygen. Genome analysis revealed the presence of pyruvate oxidase and catalase, and a cryptic potential for the formation of acetate, ethanol, formate, CO₂ and H₂ - the fermentation products of termite gut isolates. Genes encoding key enzymes of reductive acetogenesis, however, are absent, confirming the hypothesis that the ancestral metabolism of the cluster was fermentative, and that the capacity for acetogenesis from H₂ plus CO₂ - the most intriguing

property among termite gut treponemes - was acquired by lateral gene transfer.

## Introduction

Spirochetes (phylum *Spirochaetes*) are morphologically distinct bacteria with highly motile, helical cells composed of a protoplasmic cylinder and periplasmic flagella that are enclosed in an outer sheath (Paster, 2015). They are widespread in a variety of environments but typically occur only in low numbers. A notable exception is the hindgut of termites, where spirochetes predominate the bacterial microbiota, particularly in the wood-feeding taxa (Brune, 2014; Mikaelyan et al., 2015a). Here, they occur either free-living or associated with cellulolytic protists and can make up more than one-half of the bacterial population (Breznak, 2002; Breznak and Leadbetter, 2006; Ohkuma and Brune, 2011).

The high abundance of spirochetes in the hindgut of termites was first described by Joseph Leidy ('Vibrio termitis'; Leidy, 1881) and has puzzled biologists ever since. For a long time, termite gut spirochetes remained uncultured and (if at all) were classified only on a morphological basis; none of these names has been validly published (Breznak, 2002; Paster, 2018). However, with the advent of 16S rRNA-gene-based phylogenies, it became clear that almost all termite gut spirochetes fall into the radiation of the genus *Treponema*, where they form two distinct clades (Berchtold et al., 1994; Ohkuma and Kudo, 1996; Paster et al., 1996). The larger of the two clades, referred to as 'termite cluster' (Lilburn et al., 1999) or 'termite *Treponema* cluster I' (Ohkuma et al., 1999), hereafter 'termite cluster I', is extremely diverse and seems to occur in all termite species (Mikaelyan et al., 2015b). It represents a separate line of descent in the *Treponema* complex that comprises the free-living *Treponema caldarium* and *Treponema stenostreptum* (Lilburn et al., 1999; Ohkuma et al., 1999; Abt et al., 2013). The other clade, which has been referred to as 'termite *Treponema* cluster II' (Ohkuma et al., 1999; Noda et al., 2003), hereafter 'termite cluster

Received 18 March, 2021; revised 12 May, 2021; accepted 13 May, 2021. *For correspondence. E-mail brune@mpi-marburg.mpg.de; Tel. +49-6421-178-101; Fax +49-6421-178-999.
# Characterization and phylogenomic analysis of *Breznakiella homolactica* gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Yulin Song<sup>1</sup>,<sup>†</sup> Vincent Hervé<sup>1</sup>,<sup>†</sup> Renate Radek<sup>1,2</sup>
Fabienne Pfeiffer,<sup>1</sup> Hao Zheng<sup>1</sup> and Andreas Brune<sup>1*</sup>

## Summary

Spirochetes of the genus *Treponema* are surprisingly abundant in termite guts, where they play an important role in reductive acetogenesis. Although they occur in all termites investigated, their evolutionary origin is obscure. Here, we isolated the first representative of ‘termite gut treponemes’ from cockroaches, the closest relatives of termites. Phylogenomic analysis revealed that *Breznakiella homolactica* gen. nov. sp. nov. represents the most basal lineage of the highly diverse ‘termite cluster I’, a deep-branching sister group of *Treponemataceae* (fam. ‘Termitinemataceae’) that was present already in the cockroach ancestor of termites and subsequently coevolved with its host. *Breznakiella homolactica* is obligately anaerobic and catalyses the homolactic fermentation of both hexoses and pentoses. Resting cells produced acetate in the presence of oxygen. Genome analysis revealed the presence of pyruvate oxidase and catalase, and a cryptic potential for the formation of acetate, ethanol, formate, CO₂ and H₂ - the fermentation products of termite gut isolates. Genes encoding key enzymes of reductive acetogenesis, however, are absent, confirming the hypothesis that the ancestral metabolism of the cluster was fermentative, and that the capacity for acetogenesis from H₂ plus CO₂ - the most intriguing

property among termite gut treponemes - was acquired by lateral gene transfer.

## Introduction

Spirochetes (phylum *Spirochaetes*) are morphologically distinct bacteria with highly motile, helical cells composed of a protoplasmic cylinder and periplasmic flagella that are enclosed in an outer sheath (Paster, 2015). They are widespread in a variety of environments but typically occur only in low numbers. A notable exception is the hindgut of termites, where spirochetes predominate the bacterial microbiota, particularly in the wood-feeding taxa (Brune, 2014; Mikaelyan et al., 2015a). Here, they occur either free-living or associated with cellulolytic protists and can make up more than one-half of the bacterial population (Breznak, 2002; Breznak and Leadbetter, 2006; Ohkuma and Brune, 2011).

The high abundance of spirochetes in the hindgut of termites was first described by Joseph Leidy ('Vibrio termitis'; Leidy, 1881) and has puzzled biologists ever since. For a long time, termite gut spirochetes remained uncultured and (if at all) were classified only on a morphological basis; none of these names has been validly published (Breznak, 2002; Paster, 2018). However, with the advent of 16S rRNA-gene-based phylogenies, it became clear that almost all termite gut spirochetes fall into the radiation of the genus *Treponema*, where they form two distinct clades (Berchtold et al., 1994; Ohkuma and Kudo, 1996; Paster et al., 1996). The larger of the two clades, referred to as 'termite cluster' (Lilburn et al., 1999) or 'termite *Treponema* cluster I' (Ohkuma et al., 1999), hereafter 'termite cluster I', is extremely diverse and seems to occur in all termite species (Mikaelyan et al., 2015b). It represents a separate line of descent in the *Treponema* complex that comprises the free-living *Treponema caldarium* and *Treponema stenostreptum* (Lilburn et al., 1999; Ohkuma et al., 1999; Abt et al., 2013). The other clade, which has been referred to as 'termite *Treponema* cluster II' (Ohkuma et al., 1999; Noda et al., 2003), hereafter 'termite cluster

<footnote n="1"><sup>1</sup>Research Group Insect Gut Microbiology and Symbiosis, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, Marburg, 35043, Germany.</footnote>

<footnote n="2"><sup>2</sup>Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Str. 1-3, Berlin, 14195, Germany.</footnote>
II', is much less diverse and more closely related to the *Treponema* species associated with other animals. Its members occur only in a few evolutionary lower termites, where they are associated with certain gut flagellates but are absent from all higher termites (fam. Termitidae) (lida et al., 2000; Noda et al., 2018).

The only termite gut treponemes cultured to date are representatives of termite cluster I and have been isolated from lower termites. The first isolate, *Treponema primitia*, is a homoacetogen capable of reductive acetogenesis from H₂ + CO₂ via the Wood–Ljungdahl pathway (Leadbetter et al., 1999; Graber and Breznak, 2004). Gene inventories of key enzymes of the bacterial Wood–Ljungdahl pathway, namely, formyltetrahydrofolate synthetase (FTHFS; Salmassi and Leadbetter, 2003; Pester and Brune, 2006; Warnecke et al., 2007; Ottesen and Leadbetter, 2010, 2011), the formate dehydrogenase subunit (FDH_H) of the hydrogen-dependent carbon dioxide reductase (HDCR; Zhang et al., 2011; Zhang and Leadbetter, 2012), and the betasubunit (CooS) of the CO dehydrogenase/acetyl-CoA synthase complex (Warnecke et al., 2007; Matson et al., 2011), showed that homologues from termite gut treponemes are abundantly represented in various wood-feeding lower or higher termites (including the wood-feeding cockroach *Cryptocercus punctulatus*, the closest relative of termites), but not in omnivorous cockroaches.

Although these findings suggest that termite gut treponemes play an important role in reductive acetogenesis, which is the most important hydrogen sink in termite guts (Brune, 2014), the distribution of the capacity for reductive acetogenesis among the mostly uncultured members of termite cluster I is entirely unclear. Unlike *T. primitia*, the other species in termite cluster I, *Treponema azotonutricium* (Lilburn et al., 2001; Graber et al., 2004) and *Treponema isoptericolens* (Dröge et al., 2008), are not acetogens but rather classical sugar fermenters. Moreover, the gene inventories are potentially misleading because elements of the Wood–Ljungdahl pathway occur also in non-acetogenic organisms, where they are involved in the assimilation and interconversion of C₁ metabolites (Drake, 1994).

Importantly, a complete Wood–Ljungdahl pathway occurs also in *Candidatus* Treponema intracellularis, the only identified member of termite cluster II (Ohkuma et al., 2015), and in *Candidatus* Adiutrix intracellularis, the first representative of a deep-branching clade of *Deltaproteobacteria* (Ikeda-Ohtsubo et al., 2016); these two uncultured endosymbionts of termite gut flagellates contribute substantially to the high rates of reductive acetogenesis from H₂ + CO₂ in the hindgut of their respective termite host (Rosenthal et al., 2013; Ohkuma et al., 2015). Although these bacteria represent lines of descent that are different from *T. primitia*, all possess key

genes of the Wood–Ljungdahl pathway that are closely related to homologues from *Firmicutes*, which indicates that the capacity for reductive acetogenesis has been independently acquired by lateral gene transfer (Salmassi and Leadbetter, 2003; Matson et al., 2011; Ikeda-Ohtsubo et al., 2016). Consequently, it remains unsettled whether the putative ancestor of termite cluster I, which was probably present already in the common ancestor of *Cryptocercus* and termites, was already acetogenic, or whether this trait was acquired - possibly even more than once - only in individual lineages.

Almost nothing is known about the presence of treponemes in cockroaches. Members of termite clusters I and II were detected, albeit in low abundance, in short-read amplicon libraries of the bacterial gut microbiota of various cockroaches, including the wood-feeding *Cryptocercus punctulatus* (Dietrich et al., 2014; Schauer et al., 2014; Tai et al., 2015; Berlanga et al., 2016; Lampert et al., 2019). However, none of these phylotypes were represented in any of the 16S-rRNA-based clone libraries of the bacterial gut microbiota of cockroaches (Schauer et al., 2012; Bauer et al., 2015; Mikaelyan et al., 2015b), and their phylogenetic relationship to termite gut treponemes remains unclear.

Here, we report the isolation of the first representative of termite cluster I from the gut of an omnivorous Madeira cockroach (*Rhyparobia maderae*), using a combination of membrane filtration and deep-agar dilution series. We conducted a detailed ultrastructural and physiological characterization of strain RmG30, including its response to oxygen, and investigated its relationship to members of the termite cluster I and other members of the family *Treponemataceae* by phylogenomic analysis.

## Results

### Morphological characterization

Strain RmG30 showed visible growth in anoxic deep-agar tubes after 2 weeks of incubation. Initially, the colonies (<0.1 mm in diameter) had an irregular shape, but after 3 weeks, they became spherical with a well-defined edge (~0.2 mm in diameter). After 2–3 months, the colonies were white and cotton-ball-like with blurred edges and had a diameter of approximately 1–2 mm.

Phase-contrast microscopy of liquid cultures showed spiral-shaped cells with a length of 10–25 (some 3–90) µm (Fig. 1A). Cells were highly motile. Spherical bodies with a diameter of 2–4 µm were present in all cultures and increased in abundance during the stationary phase (Fig. 1B). Scanning electron microscopy revealed a cell width of 0.20 ± 0.01 µm and a wavelength of 1.0 ± 0.1 (some 0.8–1.6) µm (Fig. 1C). Transmission electron microscopy of ultrathin sections rarely showed two,
Il', is much less diverse and more closely related to the *Treponema* species associated with other animals. Its members occur only in a few evolutionary lower termites, where they are associated with certain gut flagellates but are absent from all higher termites (fam. Termitidae) (lida et al., 2000; Noda et al., 2018).

The only termite gut treponemes cultured to date are representatives of termite cluster I and have been isolated from lower termites. The first isolate, *Treponema primitia*, is a homoacetogen capable of reductive acetogenesis from H₂ + CO₂ via the Wood–Ljungdahl pathway (Leadbetter et al., 1999; Graber and Breznak, 2004). Gene inventories of key enzymes of the bacterial Wood–Ljungdahl pathway, namely, formyltetrahydrofolate synthetase (FTHFS; Salmassi and Leadbetter, 2003; Pester and Brune, 2006; Warnecke et al., 2007; Ottesen and Leadbetter, 2010, 2011), the formate dehydrogenase subunit (FDH_H) of the hydrogen-dependent carbon dioxide reductase (HDCR; Zhang et al., 2011; Zhang and Leadbetter, 2012), and the betasubunit (CooS) of the CO dehydrogenase/acetyl-CoA synthase complex (Warnecke et al., 2007; Matson et al., 2011), showed that homologues from termite gut treponemes are abundantly represented in various wood-feeding lower or higher termites (including the wood-feeding cockroach *Cryptocercus punctulatus*, the closest relative of termites), but not in omnivorous cockroaches.

Although these findings suggest that termite gut treponemes play an important role in reductive acetogenesis, which is the most important hydrogen sink in termite guts (Brune, 2014), the distribution of the capacity for reductive acetogenesis among the mostly uncultured members of termite cluster I is entirely unclear. Unlike *T. primitia*, the other species in termite cluster I, *Treponema azotonutricium* (Lilburn et al., 2001; Graber et al., 2004) and *Treponema isoptericolens* (Dröge et al., 2008), are not acetogens but rather classical sugar fermenters. Moreover, the gene inventories are potentially misleading because elements of the Wood–Ljungdahl pathway occur also in non-acetogenic organisms, where they are involved in the assimilation and interconversion of C₁ metabolites (Drake, 1994).

Importantly, a complete Wood–Ljungdahl pathway occurs also in *Candidatus* Treponema intracellularis, the only identified member of termite cluster II (Ohkuma et al., 2015), and in *Candidatus* Adiutrix intracellularis, the first representative of a deep-branching clade of *Deltaproteobacteria* (Ikeda-Ohtsubo et al., 2016); these two uncultured endosymbionts of termite gut flagellates contribute substantially to the high rates of reductive acetogenesis from H₂ + CO₂ in the hindgut of their respective termite host (Rosenthal et al., 2013; Ohkuma et al., 2015). Although these bacteria represent lines of descent that are different from *T. primitia*, all possess key

genes of the Wood–Ljungdahl pathway that are closely related to homologues from *Firmicutes*, which indicates that the capacity for reductive acetogenesis has been independently acquired by lateral gene transfer (Salmassi and Leadbetter, 2003; Matson et al., 2011; Ikeda-Ohtsubo et al., 2016). Consequently, it remains unsettled whether the putative ancestor of termite cluster I, which was probably present already in the common ancestor of *Cryptocercus* and termites, was already acetogenic, or whether this trait was acquired - possibly even more than once - only in individual lineages.

Almost nothing is known about the presence of treponemes in cockroaches. Members of termite clusters I and II were detected, albeit in low abundance, in short-read amplicon libraries of the bacterial gut microbiota of various cockroaches, including the wood-feeding *Cryptocercus punctulatus* (Dietrich et al., 2014; Schauer et al., 2014; Tai et al., 2015; Berlanga et al., 2016; Lampert et al., 2019). However, none of these phylotypes were represented in any of the 16S-rRNA-based clone libraries of the bacterial gut microbiota of cockroaches (Schauer et al., 2012; Bauer et al., 2015; Mikaelyan et al., 2015b), and their phylogenetic relationship to termite gut treponemes remains unclear.

Here, we report the isolation of the first representative of termite cluster I from the gut of an omnivorous Madeira cockroach (*Rhyparobia maderae*), using a combination of membrane filtration and deep-agar dilution series. We conducted a detailed ultrastructural and physiological characterization of strain RmG30, including its response to oxygen, and investigated its relationship to members of the termite cluster I and other members of the family *Treponemataceae* by phylogenomic analysis.

## Results

### Morphological characterization

Strain RmG30 showed visible growth in anoxic deep-agar tubes after 2 weeks of incubation. Initially, the colonies (<0.1 mm in diameter) had an irregular shape, but after 3 weeks, they became spherical with a well-defined edge (~0.2 mm in diameter). After 2–3 months, the colonies were white and cotton-ball-like with blurred edges and had a diameter of approximately 1–2 mm.

Phase-contrast microscopy of liquid cultures showed spiral-shaped cells with a length of 10–25 (some 3–90) µm (Fig. 1A). Cells were highly motile. Spherical bodies with a diameter of 2–4 µm were present in all cultures and increased in abundance during the stationary phase (Fig. 1B). Scanning electron microscopy revealed a cell width of 0.20 ± 0.01 µm and a wavelength of 1.0 ± 0.1 (some 0.8–1.6) µm (Fig. 1C). Transmission electron microscopy of ultrathin sections rarely showed two,
<figure><img src="image_2.png" /><figcaption>**Fig 1.** Morphology and ultrastructure of strain RmG30. Phase-contrast micrographs of cells in a growing culture (A) and spherical bodies formed in the stationary phase (B); scanning electron micrograph of cells in a growing culture (C); transmission electron micrograph of the cross-section of a cell (D); the arrows indicate the periplasmic flagella. Scale bars are 10 µm (A), 5 µm (B, C) and 0.2 µm (D).</figcaption></figure>

typically one, and sometimes no periplasmic flagella, indicating that the cells possess single periplasmic flagella at each end that do not always overlap at mid-cell (Fig. 1D).

## Phylogenetic analyses

16S rRNA gene sequence analysis indicated that strain RmG30 falls into the radiation of the genus *Treponema* (Fig. 2). It represents a novel lineage that is the most basal member of termite cluster I. Together with *T. caldarium* and *T. stenostreptum*, they form a well-supported family-level clade that occupies a sister position to all other members of the genus *Treponema* (family *Treponemataceae*).

A phylogenomic analysis of all members of the order *Spirochaetales* with sequenced genomes confirmed the basal position of strain RmG30 in termite cluster I (Fig. 3 and Supplementary Fig. S1). As in the 16S rRNA-based analysis, the strain represents a line of descent that is separate from those of *T. primitia* and *T. azotonutricium* (the genome of *T. isoptericolens* has not been sequenced), and from numerous MAGs from higher termites (Hervé *et al.*, 2020). The sister position of the ectosymbiotic spirochete of *Oxymonas* sp. from *Neotermes koshunensis* (NkOx-clu11) to *T. primitia* agrees with the original report (Utami *et al.*, 2019). Notably, a MAG from the fungus-cultivating termite *Macrotermes natalensis* does not cluster with other MAGs from higher termites but represents the same lineage as strain RmG30.

The sequence similarities between the 16S rRNA genes of RmG30 and the other species of the clade range between 90.6% and 93.2% (Fig. 4), which is below the threshold typically observed among members of the same genus (Yarza

*et al.*, 2014). Likewise, the low average nucleotide identities between the genomes of strain RmG30 and the corresponding type strains (Fig. 4) justifies to consider each species a separate genus-level taxon. This agrees with the results obtained with the GTDB toolkit (Chaumeil *et al.*, 2020), which classified *T. caldarium*, strain RmG30, and the termite gut isolates (*T. primitia* and *T. azotonutricium*) as separate genera in the family ‘Treponemataceae_B’ and elevated the family *Treponemataceae* to an order-level lineage (‘Treponematales’) separate from the other families in the *Spirochaetales* (Fig. 3; Supplementary Table S1). Also, members of the genus *Rectinema* (which are currently assigned to *Treponemataceae*; Hördt *et al.*, 2020) were identified as a separate, family-level lineage (f__UBA8932) in the order ‘Treponematales’.

## Growth and physiology

Strain RmG30 grew at temperatures between 20 and 37°C, with the highest growth rate at 35°C; growth yields showed a broad optimum between 25 and 35°C (Fig. 5A). The pH range of growth was between 6.5 and 7.8, with a broad optimum above pH 7.3 (Fig. 5B). Growth yields decreased drastically as soon as the pH fell below 7.3. Doubling times were similar above pH 7 (16–18 h) but increased at lower pH. Acid production stopped at a final pH of 6.0. No growth occurred at pH 8.5 (not shown).

Strain RmG30 grew fermentatively on D-glucose and a variety of other carbohydrates, including D-mannose, D-galactose, D-fructose, D-xylose, L-arabinose, D-ribose, D-trehalose and N-acetyl-glucosamine. Liquid culture on glucose showed no lag phase and reached maximal
<figure><img src="image_3.png" /><figcaption>**Fig 1.** Morphology and ultrastructure of strain RmG30. Phase-contrast micrographs of cells in a growing culture (A) and spherical bodies formed in the stationary phase (B); scanning electron micrograph of cells in a growing culture (C); transmission electron micrograph of the cross-section of a cell (D); the arrows indicate the periplasmic flagella. Scale bars are 10 µm (A), 5 µm (B, C) and 0.2 µm (D).</figcaption></figure>

typically one, and sometimes no periplasmic flagella, indicating that the cells possess single periplasmic flagella at each end that do not always overlap at mid-cell (Fig. 1D).

## Phylogenetic analyses

16S rRNA gene sequence analysis indicated that strain RmG30 falls into the radiation of the genus *Treponema* (Fig. 2). It represents a novel lineage that is the most basal member of termite cluster I. Together with *T. caldarium* and *T. stenostreptum*, they form a well-supported family-level clade that occupies a sister position to all other members of the genus *Treponema* (family *Treponemataceae*).

A phylogenomic analysis of all members of the order *Spirochaetales* with sequenced genomes confirmed the basal position of strain RmG30 in termite cluster I (Fig. 3 and Supplementary Fig. S1). As in the 16S rRNA-based analysis, the strain represents a line of descent that is separate from those of *T. primitia* and *T. azotonutricium* (the genome of *T. isoptericolens* has not been sequenced), and from numerous MAGs from higher termites (Hervé *et al.*, 2020). The sister position of the ectosymbiotic spirochete of *Oxymonas* sp. from *Neotermes koshunensis* (NkOx-clu11) to *T. primitia* agrees with the original report (Utami *et al.*, 2019). Notably, a MAG from the fungus-cultivating termite *Macrotermes natalensis* does not cluster with other MAGs from higher termites but represents the same lineage as strain RmG30.

The sequence similarities between the 16S rRNA genes of RmG30 and the other species of the clade range between 90.6% and 93.2% (Fig. 4), which is below the threshold typically observed among members of the same genus (Yarza

*et al.*, 2014). Likewise, the low average nucleotide identities between the genomes of strain RmG30 and the corresponding type strains (Fig. 4) justifies to consider each species a separate genus-level taxon. This agrees with the results obtained with the GTDB toolkit (Chaumeil *et al.*, 2020), which classified *T. caldarium*, strain RmG30, and the termite gut isolates (*T. primitia* and *T. azotonutricium*) as separate genera in the family ‘Treponemataceae_B’ and elevated the family *Treponemataceae* to an order-level lineage (‘Treponematales’) separate from the other families in the *Spirochaetales* (Fig. 3; Supplementary Table S1). Also, members of the genus *Rectinema* (which are currently assigned to *Treponemataceae*; Hördt *et al.*, 2020) were identified as a separate, family-level lineage (f__UBA8932) in the order ‘Treponematales’.

## Growth and physiology

Strain RmG30 grew at temperatures between 20 and 37°C, with the highest growth rate at 35°C; growth yields showed a broad optimum between 25 and 35°C (Fig. 5A). The pH range of growth was between 6.5 and 7.8, with a broad optimum above pH 7.3 (Fig. 5B). Growth yields decreased drastically as soon as the pH fell below 7.3. Doubling times were similar above pH 7 (16–18 h) but increased at lower pH. Acid production stopped at a final pH of 6.0. No growth occurred at pH 8.5 (not shown).

Strain RmG30 grew fermentatively on D-glucose and a variety of other carbohydrates, including D-mannose, D-galactose, D-fructose, D-xylose, L-arabinose, D-ribose, D-trehalose and N-acetyl-glucosamine. Liquid culture on glucose showed no lag phase and reached maximal
<figure><img src="image_3.png" /><figcaption>**Fig 2.** 16S rRNA gene tree illustrating the relationship of strain RmG30 (red) to other lineages of termite cluster I and other members of the family *Treponemataceae*. Type strains that represent genus-level taxa in the newly proposed family *Termitinemataceae* are shown in bold. Members of *Spirochaetaceae* were used as outgroup. Bullets indicate node support (● ≥ 95%; ◦ ≥ 70%). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

turbidity within 10 days. Cells precultivated on glucose showed a lag phase of 7–14 days when transferred on D-mannose, D-ribose and D-trehalose, and a lag phase of 14–28 days on D-galactose, D-fructose, L-arabinose and N-acetyl-glucosamine, indicating that the corresponding pathways require induction. No growth was observed on L-rhamnose, D-mannitol, D-gluconic acid, D-glucuronic acid, D-cellobiose, D-maltose, D-sucrose, D-lactose, starch, cellulose, xylan, pyruvate, L-lactate, formate and H₂ + CO₂.

Lactate was the only product detected from all sugars tested. Acetate was formed only from N-acetyl-glucosamine and in stoichiometric amounts, indicating that it stems exclusively from deacetylation (Table 1). Ethanol, formate, or H₂ were not detected, and carbon and electron recoveries were complete on all substrates.

The lactate-to-substrate stoichiometries on glucose (1.96), N-acetyl-glucosamine (2.08) and xylose (1.73) matched those expected from homolactic fermentation of hexoses (2.00) and pentoses (1.67). Growth rates on xylose and N-acetyl-glucosamine were close to those on glucose, but growth yields were higher on N-acetyl-glucosamine and lower on xylose (Table 1).

Fumarate was not reduced to succinate in the presence of glucose, formate, or H₂. The addition of H₂ to the headspace did not affect growth and product formation on glucose. Cells did not grow in mineral medium without yeast extract and Casamino acids. Higher concentrations of yeast extract (0.2%) slightly stimulated the growth.

Strain RmG30 grew in non-reduced anoxic medium under N₂/CO₂, but no growth occurred if 0.5% O₂ was added to the headspace. Catalase and oxidase activity
<figure><img src="image_4.png" /><figcaption>**Fig 2.** 16S rRNA gene tree illustrating the relationship of strain RmG30 (red) to other lineages of termite cluster I and other members of the family *Treponemataceae*. Type strains that represent genus-level taxa in the newly proposed family *Termitinemataceae* are shown in bold. Members of *Spirochaetaceae* were used as outgroup. Bullets indicate node support (● ≥ 95%; ◦ ≥ 70%). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

turbidity within 10 days. Cells precultivated on glucose showed a lag phase of 7–14 days when transferred on D-mannose, D-ribose and D-trehalose, and a lag phase of 14–28 days on D-galactose, D-fructose, L-arabinose and N-acetyl-glucosamine, indicating that the corresponding pathways require induction. No growth was observed on L-rhamnose, D-mannitol, D-gluconic acid, D-glucuronic acid, D-cellobiose, D-maltose, D-sucrose, D-lactose, starch, cellulose, xylan, pyruvate, L-lactate, formate and H₂ + CO₂.

Lactate was the only product detected from all sugars tested. Acetate was formed only from N-acetyl-glucosamine and in stoichiometric amounts, indicating that it stems exclusively from deacetylation (Table 1). Ethanol, formate, or H₂ were not detected, and carbon and electron recoveries were complete on all substrates.

The lactate-to-substrate stoichiometries on glucose (1.96), N-acetyl-glucosamine (2.08) and xylose (1.73) matched those expected from homolactic fermentation of hexoses (2.00) and pentoses (1.67). Growth rates on xylose and N-acetyl-glucosamine were close to those on glucose, but growth yields were higher on N-acetyl-glucosamine and lower on xylose (Table 1).

Fumarate was not reduced to succinate in the presence of glucose, formate, or H₂. The addition of H₂ to the headspace did not affect growth and product formation on glucose. Cells did not grow in mineral medium without yeast extract and Casamino acids. Higher concentrations of yeast extract (0.2%) slightly stimulated the growth.

Strain RmG30 grew in non-reduced anoxic medium under N₂/CO₂, but no growth occurred if 0.5% O₂ was added to the headspace. Catalase and oxidase activity
<figure><img src="image_4.png" /><figcaption>**Fig 3.** Phylogenomic tree illustrating the relationship of strain RmG30 (red) to termite cluster I and other members of the family *Treponemataceae*. Type strains that represent genus-level lineages in the newly proposed family ‘Termitinemataceae’ are shown in bold. All nodes in the tree are fully supported (>99%). The tree was rooted using other lineages of *Spirochaeta* as outgroup. Taxon names that differ in the GTDB taxonomy are shown in parentheses. For a fully expanded version of the tree, including genome accession numbers, see Supplementary Fig. S1. The detailed results of the GTDB-tk classification are shown in Supplementary Table S1. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>16S rRNA gene sequence identity (%)</figcaption></figure>

<caption>**Fig 4.** Pairwise comparison of sequence identity of the 16S rRNA genes and average nucleotide identity (ANI) of the genomes between strain RmG30 and its relatives in the proposed family ‘Termitinemataceae’. The phylogenetic relationship was taken from Fig. 2. The colour depth of each cell was adjusted according to the respective value. ANI values <76% are below the cut-off of the FastANI tool. [Color figure can be viewed at wileyonlinelibrary.com]</caption>

were not detected in cells grown on standard medium. However, cultures amended with hemin showed catalase activity. In washed cell suspensions incubated in the presence of oxygen, the turnover rate of glucose was

significantly higher (0.99 ± 0.03 µmol mg⁻¹ h⁻¹) than under anoxic conditions (0.70 ± 0.08 µmol mg⁻¹ h⁻¹), and acetate accumulated (0.12 ± 0.01 µmol mg⁻¹ h⁻¹) in addition to lactate (Fig. 6). The amount of air that had to
<figure><img src="image_5.png" /><figcaption>**Fig 3.** Phylogenomic tree illustrating the relationship of strain RmG30 (red) to termite cluster I and other members of the family *Treponemataceae*. Type strains that represent genus-level lineages in the newly proposed family ‘Termitinemataceae’ are shown in bold. All nodes in the tree are fully supported (>99%). The tree was rooted using other lineages of *Spirochaeta* as outgroup. Taxon names that differ in the GTDB taxonomy are shown in parentheses. For a fully expanded version of the tree, including genome accession numbers, see Supplementary Fig. S1. The detailed results of the GTDB-tk classification are shown in Supplementary Table S1. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>16S rRNA gene sequence identity (%)</figcaption></figure>

<caption>**Fig 4.** Pairwise comparison of sequence identity of the 16S rRNA genes and average nucleotide identity (ANI) of the genomes between strain RmG30 and its relatives in the proposed family ‘Termitinemataceae’. The phylogenetic relationship was taken from Fig. 2. The colour depth of each cell was adjusted according to the respective value. ANI values <76% are below the cut-off of the FastANI tool. [Color figure can be viewed at wileyonlinelibrary.com]</caption>

were not detected in cells grown on standard medium. However, cultures amended with hemin showed catalase activity. In washed cell suspensions incubated in the presence of oxygen, the turnover rate of glucose was

significantly higher (0.99 ± 0.03 µmol mg⁻¹ h⁻¹) than under anoxic conditions (0.70 ± 0.08 µmol mg⁻¹ h⁻¹), and acetate accumulated (0.12 ± 0.01 µmol mg⁻¹ h⁻¹) in addition to lactate (Fig. 6). The amount of air that had to
be added to the tubes to maintain microoxic conditions indicated a considerable rate of O₂ consumption (roughly estimated at 0.2–0.3 µmol mg⁻¹ h⁻¹).

## Genome analysis

Genome assembly of strain RmG30 resulted in a circular genome with a genome size of 4 646 109 bp and a G + C content of 52.9 mol.%. Exploration of the annotated genes (Supplementary Table S2) fully explained the substrate spectrum of the strain and revealed further details of its homolactic metabolism (Fig. 7).

The genome encodes two possible mechanisms for the uptake and activation of glucose: (i) via glucose permease (GlcU) plus hexokinase, and (ii) via a phosphotransferase system (PTS) of the Man family, whose members have been shown to transport and phosphorylate glucose, mannose, fructose, N-acetyl-glucosamine and other substrates in *E. coli* (Plumbridge and Vimr, 1999). In addition, the genome contains numerous genes encoding putative transporters, including putative ATP-binding cassette transporters for galactose, xylose, ribose and trehalose, and putative major facilitator super-family transporters for arabinose.

The glycolytic pathway oxidizing hexose phosphates to pyruvate is complete. The absence of glucose 6-phosphate dehydrogenase and phosphoketolase agree with the homolactic fermentation of both hexoses and pentoses. The absence of transaldolase, which catalyses a key step in the pentose phosphate pathway, indicates that pentoses are most likely shuttled into glycolysis via a bypass of the classic pentose phosphate pathway, which has been experimentally documented in *E. coli* mutants lacking transaldolase (Nakahigashi et al., 2009). It involves the phosphorylation of sedoheptulose 7-phosphate to sedoheptulose 1,7-bisphosphate and its subsequent cleavage to erythrose 4-phosphate and dihydroxyacetone phosphate. These activities are side reactions of 6-phosphofructokinase and fructose 1,6-bisphosphate aldolase, which are represented in the genome of strain RmG30 in multiple copies (Supplementary Table S2).

The genome encodes a D-lactate dehydrogenase required to reduce pyruvate to lactate. In addition, there are homologues encoding pyruvate formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), which catalyse the conversion of pyruvate to acetyl-CoA, enzymes for the production of ethanol (acetaldehyde dehydrogenase, alcohol dehydrogenase) and acetate (phosphate acetyltransferase, acetate kinase), and two [FeFe] hydrogenases, namely, a ferredoxin- and NAD⁺-dependent electron-bifurcating hydrogenase (HydABC) of group A and a ferredoxin-dependent homologue of group B (HydA2). The genome also encodes a ferredoxin:

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Influence of temperature and pH on doubling time and growth yield (OD₅₀₇₈) of strain RmG30. Cells were grown in basal medium on 8 mM glucose. For temperature dependence (A), the initial pH was 7.0. For pH dependence (B), the initial pH was adjusted by adding different volumes of CO₂ to the culture headspace. The final pH of the cultures was recorded in the stationary phase. Values are means of three cultures (±standard deviation). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
be added to the tubes to maintain microoxic conditions indicated a considerable rate of O₂ consumption (roughly estimated at 0.2–0.3 µmol mg⁻¹ h⁻¹).

## Genome analysis

Genome assembly of strain RmG30 resulted in a circular genome with a genome size of 4 646 109 bp and a G + C content of 52.9 mol.%. Exploration of the annotated genes (Supplementary Table S2) fully explained the substrate spectrum of the strain and revealed further details of its homolactic metabolism (Fig. 7).

The genome encodes two possible mechanisms for the uptake and activation of glucose: (i) via glucose permease (GlcU) plus hexokinase, and (ii) via a phosphotransferase system (PTS) of the Man family, whose members have been shown to transport and phosphorylate glucose, mannose, fructose, N-acetyl-glucosamine and other substrates in *E. coli* (Plumbridge and Vimr, 1999). In addition, the genome contains numerous genes encoding putative transporters, including putative ATP-binding cassette transporters for galactose, xylose, ribose and trehalose, and putative major facilitator super-family transporters for arabinose.

The glycolytic pathway oxidizing hexose phosphates to pyruvate is complete. The absence of glucose 6-phosphate dehydrogenase and phosphoketolase agree with the homolactic fermentation of both hexoses and pentoses. The absence of transaldolase, which catalyses a key step in the pentose phosphate pathway, indicates that pentoses are most likely shuttled into glycolysis via a bypass of the classic pentose phosphate pathway, which has been experimentally documented in *E. coli* mutants lacking transaldolase (Nakahigashi et al., 2009). It involves the phosphorylation of sedoheptulose 7-phosphate to sedoheptulose 1,7-bisphosphate and its subsequent cleavage to erythrose 4-phosphate and dihydroxyacetone phosphate. These activities are side reactions of 6-phosphofructokinase and fructose 1,6-bisphosphate aldolase, which are represented in the genome of strain RmG30 in multiple copies (Supplementary Table S2).

The genome encodes a D-lactate dehydrogenase required to reduce pyruvate to lactate. In addition, there are homologues encoding pyruvate formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), which catalyse the conversion of pyruvate to acetyl-CoA, enzymes for the production of ethanol (acetaldehyde dehydrogenase, alcohol dehydrogenase) and acetate (phosphate acetyltransferase, acetate kinase), and two [FeFe] hydrogenases, namely, a ferredoxin- and NAD⁺-dependent electron-bifurcating hydrogenase (HydABC) of group A and a ferredoxin-dependent homologue of group B (HydA2). The genome also encodes a ferredoxin:

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Influence of temperature and pH on doubling time and growth yield (OD₅₀₇₈) of strain RmG30. Cells were grown in basal medium on 8 mM glucose. For temperature dependence (A), the initial pH was 7.0. For pH dependence (B), the initial pH was adjusted by adding different volumes of CO₂ to the culture headspace. The final pH of the cultures was recorded in the stationary phase. Values are means of three cultures (±standard deviation). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
<table><caption>**Table 1.** Growth parameters and fermentation balance of strain RmG30 on different substrates.</caption><thead><tr><th rowspan="2">Substrate</th><th>Substrate</th><th rowspan="2">Doubling time (h)</th><th>Turbidity</th><th>Yield</th><th>Substrate</th><th colspan="2">Products formed (mM)</th><th>Carbon and</th></tr><tr><th>consumed (mM)</th><th>(OD<sub>578</sub>)</th><th>coefficient (g mol<sup>-1</sup>)<sup>a</sup></th><th>assim. (mM)<sup>b</sup></th><th>Lactate</th><th>Acetate</th><th>electron recovery (%)<sup>c</sup></th></tr></thead><tbody><tr><td>No substrate</td><td></td><td></td><td>0.023<sup>d</sup></td><td></td><td></td><td>1.5<sup>d</sup></td><td>-</td><td></td></tr><tr><td>Glucose</td><td>8.0</td><td>16</td><td>0.202</td><td>13.5</td><td>0.7</td><td>15.8</td><td>-</td><td>99</td></tr><tr><td>Xylose</td><td>8.0</td><td>14</td><td>0.142</td><td>9.0</td><td>0.6</td><td>14.3</td><td>-</td><td>104</td></tr><tr><td>N-Acetyl-glucosamine<sup>e</sup></td><td>8.0</td><td>15</td><td>0.247</td><td>16.8</td><td>0.9<sup>f</sup></td><td>16.3</td><td>8.1</td><td>105</td></tr></tbody></table>

Unless indicated otherwise, precultures were grown on glucose. Values are means of results obtained with duplicate cultures (less than 10% deviation).

<sup>a</sup>Based on consumed substrate and the conversion factor determined for glucose-grown cultures (60 ± 2 mg L<sup>-1</sup> at OD<sub>578</sub> = 0.1; n = 2).

<sup>b</sup>Assuming an elemental composition of C<sub>4</sub>H<sub>8</sub>O<sub>2</sub>N for bacterial cell mass (Mayberry *et al.*, 1968).

<sup>c</sup>Based on dissimilated substrate.

<sup>d</sup>Turbidity and products formed in basal medium without glucose were subtracted in the subsequent calculations.

<sup>e</sup>Preculture grown on N-acetyl-glucosamine.

<sup>f</sup>Calculated as assimilated glucose.

<figure><img src="image_7.png" /><figcaption>**Fig 6.** Effect of oxygen on glucose turnover by cell suspensions of strain RmG30. Washed cells (0.3 mg ml<sup>-1</sup>) were incubated under a headspace of N<sub>2</sub>/CO<sub>2</sub> under anoxic (1 mM DTT) or microoxic (0.2%–0.4% O<sub>2</sub>) conditions. Values are means of four cultures (±standard deviation). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

NADP<sup>+</sup> oxidoreductase (FNR) and a Na<sup>+</sup>-translocating ferredoxin:NAD<sup>+</sup> oxidoreductase (Rnf) complex.

Key enzymes of the Wood–Ljungdahl pathway (hydrogen-dependent CO<sub>2</sub> reductase (HDCR), carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS)) are absent, confirming the inability of strain RmG30 to catalyse reductive acetogenesis. Genes indicating the capacity for aerobic respiration or anaerobic respiration with sulfate or nitrate as electron acceptor were not detected. The genome encodes several homologues of succinate dehydrogenase/fumarate reductase, although we found no evidence for fumarate respiration in strain RmG30 (see above). Genes encoding pyruvate oxidase,

catalase and peroxiredoxin are present, but homologues of superoxide dismutase and glutathione peroxidase were not found.

Strain RmG30 possesses transporters for peptides, amino acids and ammonia (Supplementary Table S2). It encodes the biosynthetic pathways for almost all proteinogenic amino acids (Supplementary File 1). The pathway for tryptophan synthesis is absent. Meso-diaminopimelate, an essential precursor of lysin and peptidoglycan, is probably synthesized directly from tetra-hydrodipicolinate via diaminopimelate aminotransferase (DapL) (McCoy *et al.*, 2006), but all possible pathway variants lack at least one coding gene, it is unclear whether strain RmG30 can synthesize lysine (Supplementary Fig. S2). In the case of methionine synthesis, the acylhomoserine pathway that is used by most bacteria is incomplete, but strain RmG30 might produce homocysteine via an aspartate semialdehyde sulfur transferase (Ast, Supplementary Fig. S2), as experimentally documented in methanogenic archaea (Allen *et al.*, 2015). The pathways for the synthesis and breakdown of glycogen are complete, which indicates that strain RmG30 uses glycogen as an intracellular reserve compound. Genes encoding homologues of group IV nitrogenases (structural subunits D, H and K) are most likely not involved in dinitrogen fixation but seem to be part of an Fe<sup>3+</sup>-siderophore transport system (Supplementary Table S2; Ghebreamlak and Mansoorabadi, 2020).

## Discussion

The isolation of the most basal member of termite cluster I from a cockroach provides important insights into the metabolic diversity of this clade and the evolutionary history of termite gut treponemes. Like the termite gut
<table><caption>**Table 1.** Growth parameters and fermentation balance of strain RmG30 on different substrates.</caption><thead><tr><th rowspan="2">Substrate</th><th>Substrate</th><th rowspan="2">Doubling time (h)</th><th>Turbidity</th><th>Yield</th><th>Substrate</th><th colspan="2">Products formed (mM)</th><th>Carbon and</th></tr><tr><th>consumed (mM)</th><th>(OD<sub>578</sub>)</th><th>coefficient (g mol<sup>-1</sup>)<sup>a</sup></th><th>assim. (mM)<sup>b</sup></th><th>Lactate</th><th>Acetate</th><th>electron recovery (%)<sup>c</sup></th></tr></thead><tbody><tr><td>No substrate</td><td></td><td></td><td>0.023<sup>d</sup></td><td></td><td></td><td>1.5<sup>d</sup></td><td>—</td><td></td></tr><tr><td>Glucose</td><td>8.0</td><td>16</td><td>0.202</td><td>13.5</td><td>0.7</td><td>15.8</td><td>—</td><td>99</td></tr><tr><td>Xylose</td><td>8.0</td><td>14</td><td>0.142</td><td>9.0</td><td>0.6</td><td>14.3</td><td>—</td><td>104</td></tr><tr><td>N-Acetyl-glucosamine<sup>e</sup></td><td>8.0</td><td>15</td><td>0.247</td><td>16.8</td><td>0.9<sup>f</sup></td><td>16.3</td><td>8.1</td><td>105</td></tr></tbody></table>

Unless indicated otherwise, precultures were grown on glucose. Values are means of results obtained with duplicate cultures (less than 10% deviation).

<sup>a</sup>Based on consumed substrate and the conversion factor determined for glucose-grown cultures (60 ± 2 mg L<sup>-1</sup> at OD<sub>578</sub> = 0.1; n = 2).

<sup>b</sup>Assuming an elemental composition of C<sub>4</sub>H<sub>8</sub>O<sub>2</sub>N for bacterial cell mass (Mayberry et al., 1968).

<sup>c</sup>Based on dissimilated substrate.

<sup>d</sup>Turbidity and products formed in basal medium without glucose were subtracted in the subsequent calculations.

<sup>e</sup>Preculture grown on N-acetyl-glucosamine.

<sup>f</sup>Calculated as assimilated glucose.

<figure><img src="image_8.png" /><figcaption>**Fig 6.** Effect of oxygen on glucose turnover by cell suspensions of strain RmG30. Washed cells (0.3 mg ml<sup>-1</sup>) were incubated under a headspace of N<sub>2</sub>/CO<sub>2</sub> under anoxic (1 mM DTT) or microoxic (0.2%–0.4% O<sub>2</sub>) conditions. Values are means of four cultures (±standard deviation). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

NADP<sup>+</sup> oxidoreductase (FNR) and a Na<sup>+</sup>-translocating ferredoxin:NAD<sup>+</sup> oxidoreductase (Rnf) complex.

Key enzymes of the Wood–Ljungdahl pathway (hydrogen-dependent CO<sub>2</sub> reductase (HDCR), carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS)) are absent, confirming the inability of strain RmG30 to catalyse reductive acetogenesis. Genes indicating the capacity for aerobic respiration or anaerobic respiration with sulfate or nitrate as electron acceptor were not detected. The genome encodes several homologues of succinate dehydrogenase/fumarate reductase, although we found no evidence for fumarate respiration in strain RmG30 (see above). Genes encoding pyruvate oxidase,

catalase and peroxiredoxin are present, but homologues of superoxide dismutase and glutathione peroxidase were not found.

Strain RmG30 possesses transporters for peptides, amino acids and ammonia (Supplementary Table S2). It encodes the biosynthetic pathways for almost all proteinogenic amino acids (Supplementary File 1). The pathway for tryptophan synthesis is absent. Meso-diaminopimelate, an essential precursor of lysin and peptidoglycan, is probably synthesized directly from tetra-hydrodipicolinate via diaminopimelate aminotransferase (DapL) (McCoy et al., 2006), but all possible pathway variants lack at least one coding gene, it is unclear whether strain RmG30 can synthesize lysine (Supplementary Fig. S2). In the case of methionine synthesis, the acylhomoserine pathway that is used by most bacteria is incomplete, but strain RmG30 might produce homocysteine via an aspartate semialdehyde sulfur transferase (Ast, Supplementary Fig. S2), as experimentally documented in methanogenic archaea (Allen et al., 2015). The pathways for the synthesis and breakdown of glycogen are complete, which indicates that strain RmG30 uses glycogen as an intracellular reserve compound. Genes encoding homologues of group IV nitrogenases (structural subunits D, H and K) are most likely not involved in dinitrogen fixation but seem to be part of an Fe<sup>3+</sup>-siderophore transport system (Supplementary Table S2; Ghebreamlak and Mansoorabadi, 2020).

Discussion

The isolation of the most basal member of termite cluster I from a cockroach provides important insights into the metabolic diversity of this clade and the evolutionary history of termite gut treponemes. Like the termite gut
<figure><img src="image_8.png" /><figcaption>**Fig 7.** Metabolic map of strain RmG30. Substrates and products that were experimentally verified are shown in bold. The hypothetical bypass in the pentose phosphate pathway compensating for the absence of transaldolase (see text) is shown in orange. The pathway for the oxygen-dependent production of acetate is shown in blue. Missing reactions are shown in grey. Important cosubstrates in energy metabolism are shown in red. Amino acids (AAs) and their biosynthetic pathways are outlined in green. Non-standard abbreviations (non-IUPAC) for metabolites: Ac, acetate; AcCoA, acetyl coenzyme A; AcP, acetyl phosphate; Ara, arabinose; CoB₁₂, Coenzyme B₁₂; DAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; Fru, fructose; F6P, fructose 6-phosphate; Fum, fumarate; Gal, galactose; Gal1P, galactose 1-phosphate; GAP, glyceraldehyde 3-phosphate; Glc, glucose; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; GlcNAc, N-acetyl-glucosamine; GlcNAc6P, N-acetyl-glucosamine 6-phosphate; GlcN6P, glucosamine 6-phosphate; Hcy, homocysteine; Mal, malate; Man, mannose; M6P, mannose 6-phosphate; 2OG, 2-oxoglutaric acid; OxAc, oxaloacetate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Pyr, pyruvate; Rib, ribose; R5P, ribose 6-phosphate; Rul, ribulose; Ru5P, ribulose 5-phosphate; SAM, S-adenosyl methionine; S7P, sedoheptulose 7-phosphate; S1,7BP, sedoheptulose 1,7-bisphosphate; Succ, succinate; SuccCoA, succinate coenzyme A; THF, tetrahydrofolate; Tre, trehalose; Xul, xylulose; Xu5P, xylulose 5-phosphate; Xyl, xylose; for enzymes and transporters: ABC, ATP-binding cassette transport system; Amt, ammonium transporter; FNR, ferredoxin:NADP⁺ oxidoreductase; GlcU, glucose uptake protein; HydABC, ferredoxin- and NAD⁺-dependent electron-bifurcating [FeFe]-hydrogenase; HydA2, ferredoxin-dependent [FeFe]-hydrogenase; MFS, major facilitator superfamily transporter; Nha, Na⁺/H⁺ antiporter; PTS, phosphotransferase system; Rnf, Na⁺-translocating ferredoxin:NADP⁺ oxidoreductase complex. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

isolates *T. azotonutricium* and *T. isoptericolens*, strain RmG30 possesses a purely fermentative metabolism and lacks the ability to grow lithotrophically on H₂ + CO₂ (or homoacetogenically on glucose), corroborating the hypothesis that the capacity for reductive acetogenesis in *T. primitia* has been acquired by lateral gene transfer from *Firmicutes* (Salmassi and Leadbetter, 2003; Matson et al., 2011; Ikeda-Ohtsubo et al., 2016). Moreover, the inability to grow on xylan agrees with the hypothesis that also the hemicellulolytic capacity attributed to uncultured members in the apical lineages of termite cluster I, which occur exclusively in higher termites, was acquired by lateral gene transfer (Tokuda et al., 2018).

## Evolutionary history of termite cluster I

Members of termite cluster I are present in all termite species investigated so far and - unlike the cellulolytic flagellates - even maintained a prominent place in the microbial community after the evolutionary transition from a protist-based (lower termites) to a bacteria-based (higher termites) digestion of lignocellulose (Brune and Dietrich, 2015; Chouvenc et al., 2021). The internal phylogeny of the clade provides a strong coevolutionary signal with the major host groups (Mikaelyan et al., 2015b; Bourguignon et al., 2018), but their evolutionary origin is entirely obscure.
<figure><img src="image_9.png" /><figcaption>**Fig 7.** Metabolic map of strain RmG30. Substrates and products that were experimentally verified are shown in bold. The hypothetical bypass in the pentose phosphate pathway compensating for the absence of transaldolase (see text) is shown in orange. The pathway for the oxygen-dependent production of acetate is shown in blue. Missing reactions are shown in grey. Important cosubstrates in energy metabolism are shown in red. Amino acids (AAs) and their biosynthetic pathways are outlined in green. Non-standard abbreviations (non-IUPAC) for metabolites: Ac, acetate; AcCoA, acetyl coenzyme A; AcP, acetyl phosphate; Ara, arabinose; CoB₁₂, Coenzyme B₁₂; DAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; Fru, fructose; F6P, fructose 6-phosphate; Fum, fumarate; Gal, galactose; Gal1P, galactose 1-phosphate; GAP, glyceraldehyde 3-phosphate; Glc, glucose; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; GlcNAc, N-acetyl-glucosamine; GlcNAc6P, N-acetyl-glucosamine 6-phosphate; GlcN6P, glucosamine 6-phosphate; Hcy, homocysteine; Mal, malate; Man, mannose; M6P, mannose 6-phosphate; 2OG, 2-oxoglutaric acid; OxAc, oxaloacetate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Pyr, pyruvate; Rib, ribose; R5P, ribose 6-phosphate; Rul, ribulose; Ru5P, ribulose 5-phosphate; SAM, S-adenosyl methionine; S7P, sedoheptulose 7-phosphate; S1,7BP, sedoheptulose 1,7-bisphosphate; Succ, succinate; SuccCoA, succinate coenzyme A; THF, tetrahydrofolate; Tre, trehalose; Xul, xylulose; Xu5P, xylulose 5-phosphate; Xyl, xylose; for enzymes and transporters: ABC, ATP-binding cassette transport system; Amt, ammonium transporter; FNR, ferredoxin:NADP⁺ oxidoreductase; GlcU, glucose uptake protein; HydABC, ferredoxin- and NAD⁺-dependent electron-bifurcating [FeFe]-hydrogenase; HydA2, ferredoxin-dependent [FeFe]-hydrogenase; MFS, major facilitator superfamily transporter; Nha, Na⁺/H⁺ antiporter; PTS, phosphotransferase system; Rnf, Na⁺-translocating ferredoxin:NADP⁺ oxidoreductase complex. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

isolates *T. azotonutricium* and *T. isoptericolens*, strain RmG30 possesses a purely fermentative metabolism and lacks the ability to grow lithotrophically on H₂ + CO₂ (or homoacetogenically on glucose), corroborating the hypothesis that the capacity for reductive acetogenesis in *T. primitia* has been acquired by lateral gene transfer from *Firmicutes* (Salmassi and Leadbetter, 2003; Matson et al., 2011; Ikeda-Ohtsubo et al., 2016). Moreover, the inability to grow on xylan agrees with the hypothesis that also the hemicellulolytic capacity attributed to uncultured members in the apical lineages of termite cluster I, which occur exclusively in higher termites, was acquired by lateral gene transfer (Tokuda et al., 2018).

## Evolutionary history of termite cluster I

Members of termite cluster I are present in all termite species investigated so far and - unlike the cellulolytic flagellates - even maintained a prominent place in the microbial community after the evolutionary transition from a protist-based (lower termites) to a bacteria-based (higher termites) digestion of lignocellulose (Brune and Dietrich, 2015; Chouvenc et al., 2021). The internal phylogeny of the clade provides a strong coevolutionary signal with the major host groups (Mikaelyan et al., 2015b; Bourguignon et al., 2018), but their evolutionary origin is entirely obscure.
It is widely accepted that termites evolved from wood-
feeding, subsocial cockroaches (Bourguignon
et al., 2015). Although the gut microbiota of cockroaches
and termites shows a strong coevolutionary pattern
(Dietrich et al., 2014) and the presence of particular taxa
is linked to host diet (Mikaelyan et al., 2015a), concrete
evidence for an origin of termite gut treponemes in an
ancestral cockroach is so far lacking.

Hence, the genome sequence of strain RmG30 is of
great significance for the reconstruction of the evolution-
ary history of termite cluster I. The monophyly of the clus-
ter, its exclusive presence in termites, and the striking
host specificity of its members (the phylotypes from a
particular termite species are, in general, more closely
related to each other than to any phylotype from other
termites), had been recognized already in the early stud-
ies of termite gut treponemes (Lilburn et al., 1999;
Ohkuma et al., 1999). Our phylogenomic analysis, cor-
roborated by the corresponding 16S rRNA-based phylog-
eny, provides unequivocal support for a basal position of
strain RmG30 (from a cockroach), an intermediate posi-
tion of the isolates from lower termites, and an apical
position of clones (there are no isolates) from higher ter-
mites, which is in full agreement with a coevolutionary
scenario (Fig. 2).

An interesting exception is the single clone (AB234368) obtained from *Macrotermes gilvus* (Hongoh et al., 2006) and a corresponding MAG (g\_UBA937) from *Macrotermes natalensis* (Parks et al., 2017), which are more closely related to strain RmG30 than to all other members of termite cluster I. Apparently, the fungus-feeding *Macrotermes* spp. have independently acquired a lineage of termite cluster I that is separate from the main line of descent - possibly from a cockroach. This was corroborated by phylogenetic placement of the short reads previously obtained from amplicon libraries of cockroach gut microbiota (Lampert et al., 2019), where reads from several cockroaches that had been classified as *Treponema caldarium* or termite cluster I clustered with the 16S rRNA gene of strain RmG30 and the clone from *Macrotermes gilvus* (details not shown). Such a horizontal acquisition of termite gut microbiota from the environment, including the guts of other animals, is not unusual (Bourguignon et al., 2018), and agrees with the observation that the gut microbiota of Macrotermitinae (fungus-feeding higher termites) is more similar to that of cockroaches than to termites from other diet groups (Dietrich et al., 2014; Otani et al., 2014; Lampert et al., 2019).

These observations provide strong evidence that mem-
bers of termite cluster I were already present among the
gut microbiota of omnivorous cockroaches before
the common ancestor of termites and Cryptocercidae
acquired cellulolytic flagellates. Since *Rhinarobia mad-
erae* (superfam. Blaberoidea), the host of strain RmG30, is

only distantly related to the lineage that gave rise to ter-
mites (superfam. Blattoidea) (Djernæs et al., 2020), host
specificity and transmission mode of the basal members
of termite cluster I in cockroaches remain unclear. The
same is true for the ultimate origin of the cluster and their
relationship to the isolates from freshwater sediments (T.
stenostreptum and T. caldarium). Clearly, more informa-
tion is needed on the diversity and distribution of the clus-
ter in cockroaches, other insects and their environments.

Energy metabolism

Since the isolation of *T. primitia* (Leadbetter et al., 1999; Graber et al., 2004), evidence has accumulated that mem-
bers of termite cluster I are responsible for the high rates
of reductive acetogenesis in termite guts. However, *T.
primita* and possibly also the closely related strain NkOx-
clu11, an uncultured ectosymbiont of a termite gut flagel-
late, are the only representatives of the cluster that pos-
sess a complete Wood–Ljungdahl pathway (Rosenthal
et al., 2011; Utami et al., 2019). Their energy metabolism
resembles that of *Acetobacterium woodii* and involves an
HDCR, a CODH/ACS complex, an electron-bifurcating
hydrogenase (HydABC), and an Rnf complex, which bal-
ance the reducing equivalents and generate the mem-
brane potential for ATP synthesis (Schuchmann and
Müller, 2014).

The presence of a fermentative metabolism in the
ancestral *T. caldarium* and *T. stenostreptum*, in strain
RmG30, and in two of the three species isolated from ter-
mite guts is a strong indication that reductive
acetogenesis in *T. primitia* and possibly other uncultured
members of termite cluster I is an apomorphic trait. This
agrees with the absence of HDCR and CODH/ACS from
*T. azotonutricium* and strain RmG30 - the only other iso-
lates of termite cluster I with sequenced genomes - and
from the MAG from *Macrotermes natalensis*, the closest
relative of strain RmG30 in the phylogenomic tree
(Fig. 3). The gene functions of the Wood-Ljungdahl path-
way that are encoded by strain RmG30 (Fig. 7) and all
other basal members of the clade probably serve in the
interconversion of C₁ compounds and the provision of
formate and methyl groups for the biosynthesis of purines
(Sah et al., 2015), methionine and serine (Zhuang
et al., 2014) and were most likely present already in the
common ancestor of termite cluster I. The Wood-
Ljungdahl pathway was most likely complemented during
the radiation of the clade by the lateral acquisition of
HDCR and CODH/ACS from acetogenic *Firmicutes*, ren-
dering certain lineages acetogenic. It remains to be clari-
fied whether this has happened more than once.

There is circumstantial evidence that also the apical
lineages of termite cluster I, which occur exclusively in
higher termites (Fig. 3), may not be acetogenic.
It is widely accepted that termites evolved from wood-
feeding, subsocial cockroaches (Bourguignon
et al., 2015). Although the gut microbiota of cockroaches
and termites shows a strong coevolutionary pattern
(Dietrich et al., 2014) and the presence of particular taxa
is linked to host diet (Mikaelyan et al., 2015a), concrete
evidence for an origin of termite gut treponemes in an
ancestral cockroach is so far lacking.

Hence, the genome sequence of strain RmG30 is of
great significance for the reconstruction of the evolution-
ary history of termite cluster I. The monophyly of the clus-
ter, its exclusive presence in termites, and the striking
host specificity of its members (the phylotypes from a
particular termite species are, in general, more closely
related to each other than to any phylotype from other
termites), had been recognized already in the early stud-
ies of termite gut treponemes (Lilburn et al., 1999;
Ohkuma et al., 1999). Our phylogenomic analysis, cor-
roborated by the corresponding 16S rRNA-based phylog-
eny, provides unequivocal support for a basal position of
strain RmG30 (from a cockroach), an intermediate posi-
tion of the isolates from lower termites, and an apical
position of clones (there are no isolates) from higher ter-
mites, which is in full agreement with a coevolutionary
scenario (Fig. 2).

An interesting exception is the single clone (AB234368) obtained from *Macrotermes gilvus* (Hongoh et al., 2006) and a corresponding MAG (g\_UBA937) from *Macrotermes natalensis* (Parks et al., 2017), which are more closely related to strain RmG30 than to all other members of termite cluster I. Apparently, the fungus-feeding *Macrotermes* spp. have independently acquired a lineage of termite cluster I that is separate from the main line of descent - possibly from a cockroach. This was corroborated by phylogenetic placement of the short reads previously obtained from amplicon libraries of cockroach gut microbiota (Lampert et al., 2019), where reads from several cockroaches that had been classified as *Treponema caldarium* or termite cluster I clustered with the 16S rRNA gene of strain RmG30 and the clone from *Macrotermes gilvus* (details not shown). Such a horizontal acquisition of termite gut microbiota from the environment, including the guts of other animals, is not unusual (Bourguignon et al., 2018), and agrees with the observation that the gut microbiota of Macrotermitinae (fungus-feeding higher termites) is more similar to that of cockroaches than to termites from other diet groups (Dietrich et al., 2014; Otani et al., 2014; Lampert et al., 2019).

These observations provide strong evidence that mem-
bers of termite cluster I were already present among the
gut microbiota of omnivorous cockroaches before
the common ancestor of termites and Cryptocercidae
acquired cellulolytic flagellates. Since *Rhinarobia mad-
erae* (superfam. Blaberoidea), the host of strain RmG30, is

only distantly related to the lineage that gave rise to ter-
mites (superfam. Blattoidea) (Djernæs et al., 2020), host
specificity and transmission mode of the basal members
of termite cluster I in cockroaches remain unclear. The
same is true for the ultimate origin of the cluster and their
relationship to the isolates from freshwater sediments (T.
stenostreptum and T. caldarium). Clearly, more informa-
tion is needed on the diversity and distribution of the clus-
ter in cockroaches, other insects and their environments.

Energy metabolism

Since the isolation of *T. primitia* (Leadbetter et al., 1999; Graber et al., 2004), evidence has accumulated that mem-
bers of termite cluster I are responsible for the high rates
of reductive acetogenesis in termite guts. However, *T.
primita* and possibly also the closely related strain NkOx-
clu11, an uncultured ectosymbiont of a termite gut flagel-
late, are the only representatives of the cluster that pos-
sess a complete Wood–Ljungdahl pathway (Rosenthal
et al., 2011; Utami et al., 2019). Their energy metabolism
resembles that of *Acetobacterium woodii* and involves an
HDCR, a CODH/ACS complex, an electron-bifurcating
hydrogenase (HydABC), and an Rnf complex, which bal-
ance the reducing equivalents and generate the mem-
brane potential for ATP synthesis (Schuchmann and
Müller, 2014).

The presence of a fermentative metabolism in the
ancestral *T. caldarium* and *T. stenostreptum*, in strain
RmG30, and in two of the three species isolated from ter-
mite guts is a strong indication that reductive
acetogenesis in *T. primitia* and possibly other uncultured
members of termite cluster I is an apomorphic trait. This
agrees with the absence of HDCR and CODH/ACS from
*T. azotonutricium* and strain RmG30 - the only other iso-
lates of termite cluster I with sequenced genomes - and
from the MAG from *Macrotermes natalensis*, the closest
relative of strain RmG30 in the phylogenomic tree
(Fig. 3). The gene functions of the Wood-Ljungdahl path-
way that are encoded by strain RmG30 (Fig. 7) and all
other basal members of the clade probably serve in the
interconversion of C₁ compounds and the provision of
formate and methyl groups for the biosynthesis of purines
(Sah et al., 2015), methionine and serine (Zhuang
et al., 2014) and were most likely present already in the
common ancestor of termite cluster I. The Wood-
Ljungdahl pathway was most likely complemented during
the radiation of the clade by the lateral acquisition of
HDCR and CODH/ACS from acetogenic *Firmicutes*, ren-
dering certain lineages acetogenic. It remains to be clari-
fied whether this has happened more than once.

There is circumstantial evidence that also the apical
lineages of termite cluster I, which occur exclusively in
higher termites (Fig. 3), may not be acetogenic.
Metagenome analysis of the hindgut fluid of a *Nasutitermes* sp. revealed numerous gene homologues encoding FTHFS and CODH/ACS that were assigned to treponemes, but homologues of HDCR were conspicuously absent (Warnecke *et al.*, 2007). Comparative analysis of the gene functions encoded by the uncultured representatives of termite cluster I, including also MAGs from lower termites, will allow to clarify the situation (Y. Song, V. Hervé, and A. Brune; unpublished results).

An unusual feature of strain RmG30 is the homolactic fermentation of pentoses. While homolactic fermentation which uses the glycolytic pathway and yields two lactate per sugar, is typical for hexoses, pentoses are usually fermented heterolactically via the phosphoketolase pathway, which yields one lactate and one acetate (Kandler, 1983). Homolactic fermentation of pentoses is quite rare in nature but common in biotechnological applications, where a high yield of lactate is achieved by genetic engineering (Tarraran and Mazzoli, 2018), which usually involves the substitution of phosphoketolase with a heterologous expressed transketolase (Okano et al., 2009).

The absence of both phosphoketolase and transaldolase from the genome of strain RmG30 and the formation of about 1.7 lactate per xylose, which matches the theoretical stoichiometry of a homolactic fermentation, indicates that pentoses are converted to hexoses via a modified pentose phosphate pathway that involves a sedoheptulose 1,7-bisphosphate shunt (Nakahigashi et al., 2009; see above). While the few lactic acid bacteria that ferment pentoses via pentose phosphate pathway/glycolysis still form appreciable amounts of acetate, strain RmG30 is the first strain that conducts a purely homolactic fermentation of pentoses. Although the strain has the genomic capacity to convert pyruvate to acetyl-CoA (via PFL or PFOR) and to form acetate, formate, hydrogen or ethanol, these enzymes are obviously not involved in catabolic reactions.

## Relationship to oxygen

Insect guts are gradient systems characterized by the continuous influx of oxygen across the gut epithelium (Brune, 2014). Therefore, even strictly anaerobic members of the gut microbiota may be at least temporarily exposed to oxygen. Typical adaptations to microoxic conditions are pathways for a non-respiratory reduction of oxygen and the removal of reactive oxygen species, which can be found even in strict anaerobes (e.g. Condon, 1987; Brioukhanov and Netrusov, 2007; Imlay, 2019). Strain RmG30 is no exception. Although it has a purely fermentative metabolism and does not grow under microoxic conditions (0.5% O₂, vol./vol.), resting cells consume oxygen and accumulate acetate at

appreciable rates. An oxygen-dependent accumulation of acetate is common among lactic acid bacteria, which reduce oxygen in non-respiratory pathways, which shifts fermentation products from lactate to acetate and allows to gain additional ATP (e.g. Condon, 1987; Bauer et al., 2000). In strain RmG30, oxygen consumption is most likely catalysed by pyruvate oxidase; homologues of lactate oxidase or NADH oxidase were not detected.

Like most non-respiratory oxidases, pyruvate oxidase produces acetyl-CoA and H₂O₂. The latter is cytotoxic but may be detoxified by catalase, which ameliorates the effects of H₂O₂ production and increases oxygen tolerance (Whittenbury, 1960; Engesser and Hammes, 1994). Also, the genome of strain RmG30 encodes the apoenzyme, catalase activity requires the presence of hemin. The dependence of catalase activity on an external hemin supply is common in lactic acid bacteria (Baureder and Hederstedt, 2013) but has been reported also for methanogens (Brioukhanov and Netrusov, 2012). In strain RmG30, hemin auxotrophy is explained by the absence of genes required for porphyrin biosynthesis, which indicates a requirement also for vitamin B₁₂. Such cross-feeding of growth factors is common in gut environments (Sokolovskaya et al., 2020), exemplified by the dependence of *T. primitia* on the provision of folinate by other members of the termite gut microbiota (Graber and Breznak, 2005).

Except for the microaerophilic *Treponema pallidum*, which requires low levels of oxygen for survival and multiplication, all other members of the genus *Treponema* are considered strict anaerobes (Norris et al., 2015). Nevertheless, cultures of *T. primitia* tolerate exposure to low levels of oxygen, and cell extracts of strain ZAS-2 show relatively high levels of NADH oxidase and NADH peroxidase but no catalase activity (Graber and Breznak, 2004). In addition, and quite surprisingly, *T. primitia* encodes a catechol-2,3-dioxygenase (Lucey and Leadbetter, 2014), an enzyme that typically occurs in aerobic bacteria degrading aromatic compounds; it is absent from the other members of the clade. The genomes of *T. primitia* ZAS-2 and *T. azotonutricium* encode homologues of NADH oxidase, but strain RmG30 is the only isolate of termite cluster I (with sequenced genome) that encodes catalase and pyruvate oxidase. It remains to be investigated whether external hemin affects the aerotolerance of strain RmG30, its capacity for fumarate reduction, or its cell yields on glucose.

## Taxonomic implications

For the longest time, all members of the phylum *Spirochaetes* were classified in a single order (*Spirochaetales*) (Paster, 2015). However, 16S rRNA-based and phylogenomic analyses indicated that the
Metagenome analysis of the hindgut fluid of a *Nasutitermes* sp. revealed numerous gene homologues encoding FTHFS and CODH/ACS that were assigned to treponemes, but homologues of HDCR were conspicuously absent (Warnecke *et al.*, 2007). Comparative analysis of the gene functions encoded by the uncultured representatives of termite cluster I, including also MAGs from lower termites, will allow to clarify the situation (Y. Song, V. Hervé, and A. Brune; unpublished results).

An unusual feature of strain RmG30 is the homolactic fermentation of pentoses. While homolactic fermentation which uses the glycolytic pathway and yields two lactate per sugar, is typical for hexoses, pentoses are usually fermented heterolactically via the phosphoketolase pathway, which yields one lactate and one acetate (Kandler, 1983). Homolactic fermentation of pentoses is quite rare in nature but common in biotechnological applications, where a high yield of lactate is achieved by genetic engineering (Tarraran and Mazzoli, 2018), which usually involves the substitution of phosphoketolase with a heterologous expressed transketolase (Okano et al., 2009).

The absence of both phosphoketolase and transaldolase from the genome of strain RmG30 and the formation of about 1.7 lactate per xylose, which matches the theoretical stoichiometry of a homolactic fermentation, indicates that pentoses are converted to hexoses via a modified pentose phosphate pathway that involves a sedoheptulose 1,7-bisphosphate shunt (Nakahigashi et al., 2009; see above). While the few lactic acid bacteria that ferment pentoses via pentose phosphate pathway/glycolysis still form appreciable amounts of acetate, strain RmG30 is the first strain that conducts a purely homolactic fermentation of pentoses. Although the strain has the genomic capacity to convert pyruvate to acetyl-CoA (via PFL or PFOR) and to form acetate, formate, hydrogen or ethanol, these enzymes are obviously not involved in catabolic reactions.

## Relationship to oxygen

Insect guts are gradient systems characterized by the continuous influx of oxygen across the gut epithelium (Brune, 2014). Therefore, even strictly anaerobic members of the gut microbiota may be at least temporarily exposed to oxygen. Typical adaptations to microoxic conditions are pathways for a non-respiratory reduction of oxygen and the removal of reactive oxygen species, which can be found even in strict anaerobes (e.g. Condon, 1987; Brioukhanov and Netrusov, 2007; Imlay, 2019). Strain RmG30 is no exception. Although it has a purely fermentative metabolism and does not grow under microoxic conditions (0.5% O₂, vol./vol.), resting cells consume oxygen and accumulate acetate at

appreciable rates. An oxygen-dependent accumulation of acetate is common among lactic acid bacteria, which reduce oxygen in non-respiratory pathways, which shifts fermentation products from lactate to acetate and allows to gain additional ATP (e.g. Condon, 1987; Bauer et al., 2000). In strain RmG30, oxygen consumption is most likely catalysed by pyruvate oxidase; homologues of lactate oxidase or NADH oxidase were not detected.

Like most non-respiratory oxidases, pyruvate oxidase produces acetyl-CoA and H₂O₂. The latter is cytotoxic but may be detoxified by catalase, which ameliorates the effects of H₂O₂ production and increases oxygen tolerance (Whittenbury, 1960; Engesser and Hammes, 1994). Also, the genome of strain RmG30 encodes the apoenzyme, catalase activity requires the presence of hemin. The dependence of catalase activity on an external hemin supply is common in lactic acid bacteria (Baureder and Hederstedt, 2013) but has been reported also for methanogens (Brioukhanov and Netrusov, 2012). In strain RmG30, hemin auxotrophy is explained by the absence of genes required for porphyrin biosynthesis, which indicates a requirement also for vitamin B₁₂. Such cross-feeding of growth factors is common in gut environments (Sokolovskaya et al., 2020), exemplified by the dependence of *T. primitia* on the provision of folinate by other members of the termite gut microbiota (Graber and Breznak, 2005).

Except for the microaerophilic *Treponema pallidum*, which requires low levels of oxygen for survival and multiplication, all other members of the genus *Treponema* are considered strict anaerobes (Norris et al., 2015). Nevertheless, cultures of *T. primitia* tolerate exposure to low levels of oxygen, and cell extracts of strain ZAS-2 show relatively high levels of NADH oxidase and NADH peroxidase but no catalase activity (Graber and Breznak, 2004). In addition, and quite surprisingly, *T. primitia* encodes a catechol-2,3-dioxygenase (Lucey and Leadbetter, 2014), an enzyme that typically occurs in aerobic bacteria degrading aromatic compounds; it is absent from the other members of the clade. The genomes of *T. primitia* ZAS-2 and *T. azotonutricium* encode homologues of NADH oxidase, but strain RmG30 is the only isolate of termite cluster I (with sequenced genome) that encodes catalase and pyruvate oxidase. It remains to be investigated whether external hemin affects the aerotolerance of strain RmG30, its capacity for fumarate reduction, or its cell yields on glucose.

## Taxonomic implications

For the longest time, all members of the phylum *Spirochaetes* were classified in a single order (*Spirochaetales*) (Paster, 2015). However, 16S rRNA-based and phylogenomic analyses indicated that the
taxonomic ranks did not appropriately reflect the diversity
of the phylum, and numerous taxa have been subse-
quently elevated to higher ranks (Gupta *et al.*, 2013;
Yarza *et al.*, 2014; Hördt *et al.*, 2020). In that course, the
family *Treponemataceae* has been reinstated; it currently
contains the genera *Treponema* and *Rectinema* (Hördt
*et al.*, 2020).

However, members of the genus *Treponema* are phy-
logenetically highly divergent (Norris *et al.*, 2015;
Paster, 2018). Based on the clear separation of termite
cluster I (together with *T. stenostreptum* and *T. caldarium*) from all other members of the genus *Treponema*, Lilburn *et al.* (1999) already considered it inevita-
ble that ‘a new genus or genera will ultimately be created
to accommodate spirochaetes of the termite cluster.’ This
notion is substantiated by the current GTDB taxonomy,
which takes into account phylogeny, average nucleotide
identity and relative evolutionary distance (Parks
*et al.*, 2018; Parks *et al.*, 2020). The GTDB taxonomy dis-
tinguishes two separate, family-level lineages in the order
‘Treponematales’: (i) *Treponemataceae*, which comprise
the majority of all *Treponema* species but distinguishes
several genus-level taxa, and (ii) ‘Treponemataceae_B’,
which include *T. caldarium*, strain RmG30, and members
of termite cluster I as separate genus-level taxa (Fig. 3).
The membership of *T. stenostrepta* and *T. isoptericolens*,
whose genomes have not been sequenced, in
‘Treponemataceae_B’ is highly supported by the 16S
rRNA-based phylogeny (Fig. 2).

The nucleotide identities of the genomes and/or the
respective 16S rRNA genes (Fig. 4) corroborate that
each species in the family ‘Treponemataceae_B’ repre-
sents separate genus-level lineages. This agrees with
the considerable phenotypic differences between the
species, concerning not only cell shape, flagellar
arrangement, genome size and G + C content but also
metabolic traits such as substrate and product range,
response to oxygen, and the capacity for reductive
acetogenesis (Tables 2 and 3).

Based on these results, we describe strain RmG30 as
the type strain of *Breznakiella homolactica* gen. nov.
sp. nov. and propose classification of all members of the
‘Treponemataceae_B’ in the new family ‘Termitinemataceae’.
The formal description of this family and the
reclassification of all *Treponema* spp. in its radiation into
separate genera, with ‘Termitinema’ [*Treponema primitia*]
as type genus, will be addressed in a separate publica-
tion. In addition, the taxonomic ranks suggested by
GTDB will require further taxonomic acts, such as eleva-
tion of the genus *Rectinema* to family level (‘*Rectine-
mataceae*’), and the reclassification of *Trepone
mataceae*, *Termitinemataceae* and ‘*Rectinemataceae*’
in the new order ‘*Treponematales*’.

Description of Breznakiella gen. nov.

Etymology: Brez.na.ki.el'la. N. L. dimin. fem. n. *Breznakiella*, named after the American microbiologist John A. Breznak, in recognition of his fundamental contributions to the studies on termite gut treponemes.

The description is as given for *Breznakiella homolactica* sp. nov., which is the type species. The genus is monospecific and has been separated from other members of the family 'Termitinemataceae' based on physiology and phylogenetic analyses of genome and 16S rRNA gene sequences.

Description of *Breznakiella homolactica* sp. nov.

Etymology: ho.mo.lac'ti.ca. Gr. adj. *homoios*, the same,
similar; N.L. neut. n. *acidum lacticum*, lactic acid;
N.L. fem. adj. *homolactica*, referring to a metabolic anal-
ogy to other bacteria with homolactic fermentation.

Cells are helical, with a diameter of 0.2 µm, a length of 10–25 µm, and a wavelength of about 1.0 µm. Motile by two periplasmic flagella inserted at opposite ends of the cytoplasmic cylinder. Spherical bodies with a diameter of 2–4 µm are formed in stationary phase cultures. Mesophilic, grows optimally at 35°C (range 20–37°C); no growth at 40°C. Optimum pH for growth is 7.3–7.8 (range 6.5–7.8). Fermentative metabolism. Energy sources include D-glucose, D-mannose, D-galactose, D-fructose, D-xylose, L-arabinose, D-ribose, D-trehalose and N-acetylglucosamine. No growth on L-rhamnose, D-mannitol, D-gluconic acid, D-glucuronic acid, D-cellobiose, D-maltose, D-sucrose, D-lactose, starch, cellulose, xylan, pyruvate, lactate, formate, or H₂ + CO₂. Lactate is the only product from all sugars tested. Requires yeast extract and Casamino acids. Obligately anaerobic, but resting cells reduce oxygen. Genome size is 4.65 Mbp, G + C content is 52.9 mol.% (based on the type strain).

Source: The intestinal tract of the Madeira cockroach,
*Rhyparobia maderae* (Fabricius 1781).

Type strain: strain RmG30 = DSM 111054 = JCM 39135. GenBank accession numbers: MW396710 (16S rRNA gene); CP067089 (genome).

**Experimental procedures**

Microbiological media

Cultures were routinely grown in medium AM-5, an
anoxic, bicarbonate-buffered mineral medium sup-
plemented with vitamins and other growth factors
(Tegtmeier *et al.*, 2016), which was amended with yeast
extract and Casamino acids (0.1% each), cysteine and
DTT (1 mM each) as reducing agents, and resazurin
taxonomic ranks did not appropriately reflect the diversity
of the phylum, and numerous taxa have been subse-
quently elevated to higher ranks (Gupta *et al.*, 2013;
Yarza *et al.*, 2014; Hördt *et al.*, 2020). In that course, the
family *Treponemataceae* has been reinstated; it currently
contains the genera *Treponema* and *Rectinema* (Hördt
*et al.*, 2020).

However, members of the genus *Treponema* are phy-
logenetically highly divergent (Norris *et al.*, 2015;
Paster, 2018). Based on the clear separation of termite
cluster I (together with *T. stenostreptum* and *T. caldarium*) from all other members of the genus *Treponema*, Lilburn *et al.* (1999) already considered it inevita-
ble that ‘a new genus or genera will ultimately be created
to accommodate spirochaetes of the termite cluster.’ This
notion is substantiated by the current GTDB taxonomy,
which takes into account phylogeny, average nucleotide
identity and relative evolutionary distance (Parks
*et al.*, 2018; Parks *et al.*, 2020). The GTDB taxonomy dis-
tinguishes two separate, family-level lineages in the order
‘Treponematales’: (i) *Treponemataceae*, which comprise
the majority of all *Treponema* species but distinguishes
several genus-level taxa, and (ii) ‘Treponemataceae_B’,
which include *T. caldarium*, strain RmG30, and members
of termite cluster I as separate genus-level taxa (Fig. 3).
The membership of *T. stenostrepta* and *T. isoptericolens*,
whose genomes have not been sequenced, in
‘Treponemataceae_B’ is highly supported by the 16S
rRNA-based phylogeny (Fig. 2).

The nucleotide identities of the genomes and/or the
respective 16S rRNA genes (Fig. 4) corroborate that
each species in the family ‘Treponemataceae_B’ repre-
sents separate genus-level lineages. This agrees with
the considerable phenotypic differences between the
species, concerning not only cell shape, flagellar
arrangement, genome size and G + C content but also
metabolic traits such as substrate and product range,
response to oxygen, and the capacity for reductive
acetogenesis (Tables 2 and 3).

Based on these results, we describe strain RmG30 as
the type strain of *Breznakiella homolactica* gen. nov.
sp. nov. and propose classification of all members of the
‘Treponemataceae_B’ in the new family ‘Termitinemataceae’.
The formal description of this family and the
reclassification of all *Treponema* spp. in its radiation into
separate genera, with ‘Termitinema’ [*Treponema primitia*]
as type genus, will be addressed in a separate publica-
tion. In addition, the taxonomic ranks suggested by
GTDB will require further taxonomic acts, such as eleva-
tion of the genus *Rectinema* to family level (‘*Rectine-
mataceae*’), and the reclassification of *Trepone
mataceae*, *Termitinemataceae* and ‘*Rectinemataceae*’
in the new order ‘*Treponematales*’.

Description of Breznakiella gen. nov.

Etymology: Brez.na.ki.el'la. N. L. dimin. fem. n. *Breznakiella*, named after the American microbiologist John A. Breznak, in recognition of his fundamental contributions to the studies on termite gut treponemes.

The description is as given for *Breznakiella homolactica* sp. nov., which is the type species. The genus is monospecific and has been separated from other members of the family 'Termitinemataceae' based on physiology and phylogenetic analyses of genome and 16S rRNA gene sequences.

Description of *Breznakiella homolactica* sp. nov.

Etymology: ho.mo.lac'ti.ca. Gr. adj. *homoios*, the same,
similar; N.L. neut. n. *acidum lacticum*, lactic acid;
N.L. fem. adj. *homolactica*, referring to a metabolic anal-
ogy to other bacteria with homolactic fermentation.

Cells are helical, with a diameter of 0.2 µm, a length of
10–25 µm, and a wavelength of about 1.0 µm. Motile by
two periplasmic flagella inserted at opposite ends of the
cytoplasmic cylinder. Spherical bodies with a diameter of
2–4 µm are formed in stationary phase cultures. Meso-
philic, grows optimally at 35°C (range 20–37°C); no
growth at 40°C. Optimum pH for growth is 7.3–7.8 (range
6.5–7.8). Fermentative metabolism. Energy sources
include D-glucose, D-mannose, D-galactose, D-fructose, D-
xylose, L-arabinose, D-ribose, D-trehalose and N-acetyl-
glucosamine. No growth on L-rhamnose, D-mannitol, D-
gluconic acid, D-glucuronic acid, D-cellobiose, D-maltose,
D-sucrose, D-lactose, starch, cellulose, xylan, pyruvate,
lactate, formate, or H₂ + CO₂. Lactate is the only product
from all sugars tested. Requires yeast extract and
Casamino acids. Obligately anaerobic, but resting cells
reduce oxygen. Genome size is 4.65 Mbp, G + C content
is 52.9 mol.% (based on the type strain).

Source: The intestinal tract of the Madeira cockroach,
*Rhyparobia maderae* (Fabricius 1781).

Type strain: strain RmG30 = DSM 111054 = JCM
39135. GenBank accession numbers: MW396710 (16S
rRNA gene); CP067089 (genome).

**Experimental procedures**

Microbiological media

Cultures were routinely grown in medium AM-5, an
anoxic, bicarbonate-buffered mineral medium sup-
plemented with vitamins and other growth factors
(Tegtmeier *et al.*, 2016), which was amended with yeast
extract and Casamino acids (0.1% each), cysteine and
DTT (1 mM each) as reducing agents, and resazurin
<table><caption>**Table 2.** Characteristics that differentiate strain RmG30 from other species in the radiation of the new family 'Termitinemataceae'.</caption><thead><tr><th>Characteristics</th><th>Strain<br>RmG30</th><th>Treponema<br>primitia<sup>a</sup></th><th>Treponema<br>azotonutricium<sup>a</sup></th><th>Treponema<br>isoptericolens<sup>b</sup></th><th>Treponema<br>caldarium<sup>c</sup></th><th>Treponema<br>stenostreptum<sup>d</sup></th></tr></thead><tbody><tr><td>Habitat</td><td>Cockroach<br>gut</td><td>Termite gut</td><td>Termite gut</td><td>Termite gut</td><td>Hot spring<br>sediment</td><td>Freshwater sediment</td></tr><tr><td>Cell diameter (µm)</td><td>0.2</td><td>0.2–0.3</td><td>0.2–0.3</td><td>0.4–0.5</td><td>0.2–0.3</td><td>0.2–0.3</td></tr><tr><td>Cell length (µm)</td><td>10–25</td><td>3–7</td><td>10–12</td><td>12–20</td><td>15–45</td><td>15–45</td></tr><tr><td>Wavelength (µm)</td><td>1.0</td><td>2.3</td><td>1.2</td><td>6.5</td><td>0.6<sup>e</sup></td><td>1.2</td></tr><tr><td>Flagellar arrangement</td><td>1:2:1</td><td>1:2:1</td><td>1:2:1</td><td>3:6:3</td><td>1:2:1</td><td>1:2:1</td></tr><tr><td>Genome size (Mbp)</td><td>4.65</td><td>4.06</td><td>3.86</td><td>NA</td><td>3.24</td><td>NA</td></tr><tr><td>G + C content (mol.%)<sup>f</sup></td><td>52.9</td><td>50.8</td><td>49.8</td><td>47.7<sup>g</sup></td><td>45.6</td><td>60.2<sup>h</sup></td></tr><tr><td>pH optimum</td><td>7.3–7.8</td><td>7.2</td><td>ND</td><td>7.2–7.4</td><td>7.2–7.5</td><td>7.0–7.5</td></tr><tr><td>Temp. optimum (°C)</td><td>35</td><td>30</td><td>30</td><td>30</td><td>48–52</td><td>35–37</td></tr><tr><td>Catalase</td><td><sup>l</sup></td><td>–</td><td>–</td><td>–</td><td>ND<sup>j</sup></td><td>–</td></tr><tr><td>Fermentation products<sup>k</sup></td><td>Lactate</td><td>Acetate</td><td>Acetate, ethanol,<br>CO<sub>2</sub>, H<sub>2</sub></td><td>Ethanol, CO<sub>2</sub></td><td>Acetate, lactate,<br>CO<sub>2</sub>, H<sub>2</sub></td><td>Ethanol, acetate,<br>lactate, CO<sub>2</sub>, H<sub>2</sub></td></tr><tr><td>Growth on H<sub>2</sub> + CO<sub>2</sub></td><td>–</td><td>+</td><td>–</td><td>–</td><td>ND</td><td>ND</td></tr></tbody></table>

–, no activity; NA, not available; ND, not determined.

<sup>a</sup>Data from Graber and Breznak (2004), Graber *et al.* (2004), Norris *et al.* (2015).

<sup>b</sup>Data from Dröge *et al.* (2008).

<sup>c</sup>Data from Pohlschroeder *et al.* (1994).

<sup>d</sup>Data from Zuelzer (1912) and Norris *et al.* (2015).

<sup>e</sup>Estimated from Fig. 1 in Pohlschroeder *et al.* (1994).

<sup>f</sup>Based on genome sequences.

<sup>g</sup>Determined by HPLC.

<sup>h</sup>Estimated from buoyant density of DNA in CsCl (Canale-Parola *et al.*, 1968).

<sup>i</sup>Expressed only in the presence of hemin.

<sup>j</sup>Not encoded in genome.

<sup>k</sup>Product(s) from glucose (maltose in *T. isoptericolens*), in stoichiometric order.

<table><caption>**Table 3.** Differences in substrate utilization by strain RmG30 and other species in the new family 'Termitinemataceae'.</caption><thead><tr><th>Substrate</th><th>Strain<br>RmG30</th><th>Treponema<br>primitia<sup>a</sup></th><th>Treponema<br>azotonutricium<sup>a</sup></th><th>Treponema<br>isoptericolens<sup>b</sup></th><th>Treponema<br>caldarium<sup>c</sup></th><th>Treponema<br>stenostreptum<sup>d</sup></th></tr></thead><tbody><tr><td>Glucose</td><td>+</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Mannose</td><td>+</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Fructose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Galactose</td><td>+</td><td>+</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Xylose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Arabinose</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Ribose</td><td>+</td><td>–</td><td>+</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Mannitol</td><td>–</td><td>+</td><td>–</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Maltose</td><td>–</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Cellobiose</td><td>–</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Trehalose</td><td>+</td><td>–</td><td>–</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Sucrose</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Lactose</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Starch</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr></tbody></table>

+, utilized; –, not utilized.

<sup>a</sup>Data from Graber and Breznak (2004) and Graber *et al.* (2004).

<sup>b</sup>Data from Dröge *et al.* (2008).

<sup>c</sup>Data from Pohlschroeder *et al.* (1994).

<sup>d</sup>Data from Zuelzer (1912) and Norris *et al.* (2015).

(0.8 mg L⁻¹) as redox indicator. Non-reduced anoxic medium received cystine (0.5 mM) from an acidic stock solution (250 mM in 1 M HCl) as sulfur source. Unless otherwise indicated, this ‘basal medium’ was amended with glucose (8 mM), dispensed (5 ml) into 16-ml rubber-stoppered culture tubes, gassed with a headspace of N₂/CO₂, and inoculated with a fresh preculture (0.1 ml).

To assess the pH range of growth, the initial pH of the medium was adjusted by varying the mixing ratio of N₂ and CO₂ in the headspace gas; Widdel and Bak, 1992). The N₂/CO₂ ratio routinely used during isolation and initial experiments was 80:20 (vol./vol.), resulting in a medium pH of 7.0. It was changed to 90:10 (vol./vol.), resulting in a medium pH of 7.3, after we realized that
<table><caption>**Table 2.** Characteristics that differentiate strain RmG30 from other species in the radiation of the new family *Termitinemataceae*.</caption><thead><tr><th>Characteristics</th><th>Strain<br>RmG30</th><th>Treponema<br>primitia<sup>a</sup></th><th>Treponema<br>azotonutricium<sup>a</sup></th><th>Treponema<br>isoptericolens<sup>b</sup></th><th>Treponema<br>caldarium<sup>c</sup></th><th>Treponema<br>stenostreptum<sup>d</sup></th></tr></thead><tbody><tr><td>Habitat</td><td>Cockroach<br>gut</td><td>Termite gut</td><td>Termite gut</td><td>Termite gut</td><td>Hot spring<br>sediment</td><td>Freshwater sediment</td></tr><tr><td>Cell diameter (µm)</td><td>0.2</td><td>0.2–0.3</td><td>0.2–0.3</td><td>0.4–0.5</td><td>0.2–0.3</td><td>0.2–0.3</td></tr><tr><td>Cell length (µm)</td><td>10–25</td><td>3–7</td><td>10–12</td><td>12–20</td><td>15–45</td><td>15–45</td></tr><tr><td>Wavelength (µm)</td><td>1.0</td><td>2.3</td><td>1.2</td><td>6.5</td><td>0.6<sup>e</sup></td><td>1.2</td></tr><tr><td>Flagellar arrangement</td><td>1:2:1</td><td>1:2:1</td><td>1:2:1</td><td>3:6:3</td><td>1:2:1</td><td>1:2:1</td></tr><tr><td>Genome size (Mbp)</td><td>4.65</td><td>4.06</td><td>3.86</td><td>NA</td><td>3.24</td><td>NA</td></tr><tr><td>G + C content (mol.%)<sup>f</sup></td><td>52.9</td><td>50.8</td><td>49.8</td><td>47.7<sup>g</sup></td><td>45.6</td><td>60.2<sup>h</sup></td></tr><tr><td>pH optimum</td><td>7.3–7.8</td><td>7.2</td><td>ND</td><td>7.2–7.4</td><td>7.2–7.5</td><td>7.0–7.5</td></tr><tr><td>Temp. optimum (°C)</td><td>35</td><td>30</td><td>30</td><td>30</td><td>48–52</td><td>35–37</td></tr><tr><td>Catalase</td><td>+<sup>i</sup></td><td>–</td><td>–</td><td>–</td><td>ND<sup>j</sup></td><td>–</td></tr><tr><td>Fermentation products<sup>k</sup></td><td>Lactate</td><td>Acetate</td><td>Acetate, ethanol,<br>CO<sub>2</sub>, H<sub>2</sub></td><td>Ethanol, CO<sub>2</sub></td><td>Acetate, lactate,<br>CO<sub>2</sub>, H<sub>2</sub></td><td>Ethanol, acetate,<br>lactate, CO<sub>2</sub>, H<sub>2</sub></td></tr><tr><td>Growth on H<sub>2</sub> + CO<sub>2</sub></td><td>–</td><td>+</td><td>–</td><td>–</td><td>ND</td><td>ND</td></tr></tbody></table>

–, no activity; NA, not available; ND, not determined.

<sup>a</sup>Data from Graber and Breznak (2004), Graber *et al.* (2004), Norris *et al.* (2015).

<sup>b</sup>Data from Dröge *et al.* (2008).

<sup>c</sup>Data from Pohlschroeder *et al.* (1994).

<sup>d</sup>Data from Zuelzer (1912) and Norris *et al.* (2015).

<sup>e</sup>Estimated from Fig. 1 in Pohlschroeder *et al.* (1994).

<sup>f</sup>Based on genome sequences.

<sup>g</sup>Determined by HPLC.

<sup>h</sup>Estimated from buoyant density of DNA in CsCl (Canale-Parola *et al.*, 1968).

<sup>i</sup>Expressed only in the presence of hemin.

<sup>j</sup>Not encoded in genome.

<sup>k</sup>Product(s) from glucose (maltose in *T. isoptericolens*), in stoichiometric order.

<table><caption>**Table 3.** Differences in substrate utilization by strain RmG30 and other species in the new family *Termitinemataceae*.</caption><thead><tr><th>Substrate</th><th>Strain<br>RmG30</th><th>Treponema<br>primitia<sup>a</sup></th><th>Treponema<br>azotonutricium<sup>a</sup></th><th>Treponema<br>isoptericolens<sup>b</sup></th><th>Treponema<br>caldarium<sup>c</sup></th><th>Treponema<br>stenostreptum<sup>d</sup></th></tr></thead><tbody><tr><td>Glucose</td><td>+</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Mannose</td><td>+</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Fructose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Galactose</td><td>+</td><td>+</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Xylose</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Arabinose</td><td>+</td><td>+</td><td>–</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Ribose</td><td>+</td><td>–</td><td>+</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Mannitol</td><td>–</td><td>+</td><td>–</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Maltose</td><td>–</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Cellobiose</td><td>–</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Trehalose</td><td>+</td><td>–</td><td>–</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Sucrose</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Lactose</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr><tr><td>Starch</td><td>–</td><td>–</td><td>–</td><td>–</td><td>+</td><td>+</td></tr></tbody></table>

+, utilized; –, not utilized.

<sup>a</sup>Data from Graber and Breznak (2004) and Graber *et al.* (2004).

<sup>b</sup>Data from Dröge *et al.* (2008).

<sup>c</sup>Data from Pohlschroeder *et al.* (1994).

<sup>d</sup>Data from Zuelzer (1912) and Norris *et al.* (2015).

(0.8 mg L⁻¹) as redox indicator. Non-reduced anoxic medium received cystine (0.5 mM) from an acidic stock solution (250 mM in 1 M HCl) as sulfur source. Unless otherwise indicated, this ‘basal medium’ was amended with glucose (8 mM), dispensed (5 ml) into 16-ml rubber-stoppered culture tubes, gassed with a headspace of N₂/CO₂, and inoculated with a fresh preculture (0.1 ml).

To assess the pH range of growth, the initial pH of the medium was adjusted by varying the mixing ratio of N₂ and CO₂ in the headspace gas; Widdel and Bak, 1992). The N₂/CO₂ ratio routinely used during isolation and initial experiments was 80:20 (vol./vol.), resulting in a medium pH of 7.0. It was changed to 90:10 (vol./vol.), resulting in a medium pH of 7.3, after we realized that
strain RmG30 grew better if the medium was slightly alkaline. For growth tests at pH 8.5, bicarbonate buffer was replaced with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid at a final concentration of 20 mM, and N₂ was the headspace gas.

## Enrichment and isolation

Rhyparobia maderae was obtained from a commercial breeder (Jörg Bernhardt, Helbigsdorf, Germany; http://www.schaben-spinnen.de) and maintained as previously described (Schauer et al., 2012). An adult female cockroach was dissected, and the whole gut was placed in a culture tube containing 2-mm glass beads (2 g). After addition of 5 ml basal medium, the tube was closed with a rubber stopper, the headspace was gassed with N₂/CO₂ (80:20, vol./vol.), and the gut was homogenized by vortexing for 2 min. The gut homogenate was passed through a cellulose ester membrane filter (Merck Milipore) with pore diameter of 0.3 µm, and the filtrate was serially diluted in deep-agar tubes containing basal medium with 1% agar under an N₂/CO₂ headspace. The pure culture was isolated by picking single colonies from the ultimate dilution step that showed growth.

## Growth and physiology

Growth was measured photometrically by following the increase in optical density at 578 nm (OD₅₇₈) using a culture tube photometer (Spectronic 20⁺, Milton Roy; path length 1.3 cm). Dry weight was determined with replicate cultures grown on glucose (8 mM) in 1-L glass vessels containing 500 ml basal medium. After OD measurement, the cells were harvested by centrifugation (10 000g; 20 min), washed with ammonium acetate solution (20 mM), and dried at 60°C until weight constancy.

Growth on other substrates was tested in basal medium supplemented with the respective substrates (8 mM, except 4 mM for disaccharides); carboxylic acids were supplied as sodium salts. Polysaccharides (6 mg ml⁻¹) were autoclaved in the tubes before basal medium was added. Growth on H₂ + CO₂ was tested by adding 5 ml H₂ to the headspace of culture tubes with (bicarbonate-buffered) basal medium. Fumarate reduction was tested by adding 16 mM fumarate to basal medium with glucose, sodium formate, or H₂.

Oxygen tolerance was tested in culture tubes with non-reduced basal medium with 8 mM glucose under N₂/CO₂, which received different volumes of air in the headspace and were incubated on a roller mixer (60 rpm). The effect of oxygen on glucose turnover was tested with washed cell suspensions. Cells were grown on the same medium (100 ml) in rubber-stopped, centrifugable glass bottles (150 ml) under N₂/CO₂, harvested in the exponential

growth phase by centrifugation (3000g; 30 min) and resuspended in 10 ml anoxic buffer (30 mM NaHCO₃, 40 mM NaCl, pH 7.0) under N₂/CO₂. The cell suspensions (0.3 mg ml⁻¹) were amended with 8 mM glucose and divided into two cohorts: one was supplemented with 1 mM DTT and was incubated under anoxic conditions, the other was supplemented with air at a headspace concentration of 0.4% O₂ to create microoxic conditions. The cell suspensions were incubated on a shaker (100 rpm) at 30°C; O₂ concentration was monitored with a Fibox 4 trace meter and SP-PSt6-NAU sensor spots (PreSens; https://www.presens.de/) and maintained between 0.2% and 0.4% throughout the incubation period by adding defined volumes of air at regular intervals.

Oxidase activity was tested with glucose-grown cultures in basal medium using oxidase test strips (Bactident, Merck, Darmstadt, Germany); *Bacillus subtilis* (oxidase-positive) and *Escherichia coli* (oxidase-negative) were used as controls. Catalase activity was tested by checking the formation of gas bubbles after adding a drop of H₂O₂ (3%) to cell pellets of glucose-grown cultures; *E. coli* (catalase-positive) and *Elusimicrobium minutum* (catalase-negative; Geissinger et al., 2009) were used as controls. The effect of hemin on catalase expression was tested by adding hemin (2 µg ml⁻¹; Sigma-Aldrich) from a stock solution (5 mg ml⁻¹ in 50 mM NaOH). To avoid false-positive reactions, the suspended cells were separated from precipitated hemin before centrifugation and washed twice with phosphate-buffered saline (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.2).

## Metabolic products

Hydrogen was analysed in the culture headspace by gas chromatography, using a molecular sieve column and a thermal conductivity detector (Schuler and Conrad, 1990). Other fermentation products were analysed in the culture supernatant by high-performance liquid chromatography after centrifugation at 10 000g for 10 min and acidification with H₂SO₄ to 50 mM final concentration, using a system equipped with an ion-exclusion column and a refractive index detector (Schauer et al., 2012). For the calculation of electron recoveries, all metabolites were formally oxidized to CO₂, and the number of valence electrons theoretically released during product formation was compared with that of the dissimilated substrate (Tholen et al., 1997).

## Light and electron microscopy

Cultures were examined by light microscopy using an Axiophot photomicroscope (Zeiss, Oberkochen,
strain RmG30 grew better if the medium was slightly alkaline. For growth tests at pH 8.5, bicarbonate buffer was replaced with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid at a final concentration of 20 mM, and N₂ was the headspace gas.

## Enrichment and isolation

Rhyparobia maderae was obtained from a commercial breeder (Jörg Bernhardt, Helbigsdorf, Germany; http://www.schaben-spinnen.de) and maintained as previously described (Schauer et al., 2012). An adult female cockroach was dissected, and the whole gut was placed in a culture tube containing 2-mm glass beads (2 g). After addition of 5 ml basal medium, the tube was closed with a rubber stopper, the headspace was gassed with N₂/CO₂ (80:20, vol./vol.), and the gut was homogenized by vortexing for 2 min. The gut homogenate was passed through a cellulose ester membrane filter (Merck Milipore) with pore diameter of 0.3 µm, and the filtrate was serially diluted in deep-agar tubes containing basal medium with 1% agar under an N₂/CO₂ headspace. The pure culture was isolated by picking single colonies from the ultimate dilution step that showed growth.

## Growth and physiology

Growth was measured photometrically by following the increase in optical density at 578 nm (OD₅₇₈) using a culture tube photometer (Spectronic 20⁺, Milton Roy; path length 1.3 cm). Dry weight was determined with replicate cultures grown on glucose (8 mM) in 1-L glass vessels containing 500 ml basal medium. After OD measurement, the cells were harvested by centrifugation (10 000g; 20 min), washed with ammonium acetate solution (20 mM), and dried at 60°C until weight constancy.

Growth on other substrates was tested in basal medium supplemented with the respective substrates (8 mM, except 4 mM for disaccharides); carboxylic acids were supplied as sodium salts. Polysaccharides (6 mg ml⁻¹) were autoclaved in the tubes before basal medium was added. Growth on H₂ + CO₂ was tested by adding 5 ml H₂ to the headspace of culture tubes with (bicarbonate-buffered) basal medium. Fumarate reduction was tested by adding 16 mM fumarate to basal medium with glucose, sodium formate, or H₂.

Oxygen tolerance was tested in culture tubes with non-reduced basal medium with 8 mM glucose under N₂/CO₂, which received different volumes of air in the headspace and were incubated on a roller mixer (60 rpm). The effect of oxygen on glucose turnover was tested with washed cell suspensions. Cells were grown on the same medium (100 ml) in rubber-stopped, centrifugable glass bottles (150 ml) under N₂/CO₂, harvested in the exponential

growth phase by centrifugation (3000g; 30 min) and resuspended in 10 ml anoxic buffer (30 mM NaHCO₃, 40 mM NaCl, pH 7.0) under N₂/CO₂. The cell suspensions (0.3 mg ml⁻¹) were amended with 8 mM glucose and divided into two cohorts: one was supplemented with 1 mM DTT and was incubated under anoxic conditions, the other was supplemented with air at a headspace concentration of 0.4% O₂ to create microoxic conditions. The cell suspensions were incubated on a shaker (100 rpm) at 30°C; O₂ concentration was monitored with a Fibox 4 trace meter and SP-PSt6-NAU sensor spots (PreSens; https://www.presens.de/) and maintained between 0.2% and 0.4% throughout the incubation period by adding defined volumes of air at regular intervals.

Oxidase activity was tested with glucose-grown cultures in basal medium using oxidase test strips (Bactident, Merck, Darmstadt, Germany); *Bacillus subtilis* (oxidase-positive) and *Escherichia coli* (oxidase-negative) were used as controls. Catalase activity was tested by checking the formation of gas bubbles after adding a drop of H₂O₂ (3%) to cell pellets of glucose-grown cultures; *E. coli* (catalase-positive) and *Elusimicrobium minutum* (catalase-negative; Geissinger et al., 2009) were used as controls. The effect of hemin on catalase expression was tested by adding hemin (2 µg ml⁻¹; Sigma-Aldrich) from a stock solution (5 mg ml⁻¹ in 50 mM NaOH). To avoid false-positive reactions, the suspended cells were separated from precipitated hemin before centrifugation and washed twice with phosphate-buffered saline (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.2).

## Metabolic products

Hydrogen was analysed in the culture headspace by gas chromatography, using a molecular sieve column and a thermal conductivity detector (Schuler and Conrad, 1990). Other fermentation products were analysed in the culture supernatant by high-performance liquid chromatography after centrifugation at 10 000g for 10 min and acidification with H₂SO₄ to 50 mM final concentration, using a system equipped with an ion-exclusion column and a refractive index detector (Schauer et al., 2012). For the calculation of electron recoveries, all metabolites were formally oxidized to CO₂, and the number of valence electrons theoretically released during product formation was compared with that of the dissimilated substrate (Tholen et al., 1997).

## Light and electron microscopy

Cultures were examined by light microscopy using an Axiophot photomicroscope (Zeiss, Oberkochen,
Germany). Non-stained cultures were routinely examined using phase-contrast illumination (100× objective).

For electron microscopy, cells were fixed with glutaraldehyde and postfixed with osmium tetroxide before dehydrating in a graded series of ethanol and embedding in Spurr's resin (Zheng et al., 2016). Alternatively, 2-µl samples of concentrated cell suspensions were high-pressure frozen, freeze-substituted with HUGA (0.5% uranyl acetate, 0.5% glutaraldehyde, 5% H₂O in acetone), and embedded in Epon 812 substitute resin, as previously described (Renicke et al., 2017). Ultrathin sections were cut with a microtome equipped with a diamond knife and contrasted with uranyl acetate and lead citrate. The sections were examined with a Philips EM 208 transmission electron microscope.

## Genome sequencing and annotation

Genomic DNA was prepared using cetyltrimethylammonium bromide extraction (Winnepenninckx et al., 1993) and commercially sequenced (GATC-Eurofins, Konstanz, Germany) on a PacBio RS platform using one SMRT cell (insert size up to 10 kbp). Reads were assembled with the PacBio SMRT Portal software (version 2.3.0) using the Hierarchical Genome Assembly Process for assembly and Quiver for polishing (Chin et al., 2013). The polished single contig was circularized with Circulator (Hunt et al., 2015).

The genome sequence of strain RmG30 has been submitted to GenBank (https://www.ncbi.nlm.nih.gov; accession number CP067089; circularized) and to the Integrated Microbial Genomes and Microbiomes database (IMG/M) of the Joint Genome Institute (JGI) (https://img.jgi.doe.gov; taxon ID 2772190975; not circularized). The genome was annotated via the IMG annotation pipeline (v.4.16.1; Chen et al., 2019). For the analysis of the metabolic pathways, annotation results were verified, and missing functions were identified using Blast with a threshold E-value of 1E-5. Hydrogenases were classified using the HydDB reference database (https://services.birc.au.dk/hyddb/; Søndergaard et al., 2016).

## Phylogenetic analyses

The 16S rRNA gene of strain RmG30 was amplified with Bacteria-specific primers and sequenced by Sanger sequencing as previously described (Strassert et al., 2010); the sequence has been submitted to GenBank (ID: MW396710). The sequence was aligned with the SINA aligner (Pruesse et al., 2012) and imported into the reference alignment of the Silva database (version 132; Pruesse et al., 2007); additional sequences were downloaded from GenBank. The alignments were manually curated using the ARB software package (version 6.0.6; Ludwig et al., 2004). A maximum-likelihood

tree of the 16S rRNA genes was inferred from 1275 unambiguous aligned positions (sites with more than 50% gaps were masked) using the PhyML algorithm (version 3.3; Guindon et al., 2010) with GTR model and aBayes branch supports (Anisimova et al., 2011) included in ARB. Pairwise sequence identities of 16S rRNA genes are based on a distance matrix of the unfiltered alignment generated in ARB.

The genomes of strain RmG30 and other members of termite cluster I were phylogenetically classified within the taxonomic framework of the Genome Taxonomy Database (GTDB, release 89) using the GTDB toolkit (GTDB-tk, version 1.3.0; Chaumeil et al., 2020). A maximum-likelihood tree based on the genomes was inferred from a concatenated alignment of 120 bacterial single-copy genes (5040 amino acid positions) using the PhyML algorithm with LG model and aBayes branch supports. The average nucleotide identities of the genomes were calculated with FastANI (version 1.3; Jain et al., 2018).

## Acknowledgements

This study was funded by the Deutsche Forschungsgemeinschaft (DFG) in the collaborative research center SFB 987 (Microbial Diversity in Environmental Signal Response) and by the Max Planck Society (MPG). Yulin Song was supported by a fellowship of the China Scholarship Council (CSC). We thank Karen A. Brune for linguistic comments on the manuscript.

## References

Abt, B., Göker, M., Scheuner, C., Han, C., Lu, M., Misra, M., et al. (2013) Genome sequence of the thermophilic freshwater bacterium *Spirochaeta caldaria* type strain (H1T), reclassification of *Spirochaeta caldaria*, *Spirochaeta stenostrepta*, and *Spirochaeta zuelzerae* in the genus *Treponema* as *Treponema caldaria* comb. nov., *Treponema stenostrepta* comb. nov., and *Treponema zuelzerae* comb. nov., and emendation of the genus *Treponema*. *Stand Genomic Sci* **8**: 88–105.

Allen, K.D., Miller, D.V., Rauch, B.J., Perona, J.J., and White, R.H. (2015) Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea. *Biochemistry* **54**: 3129–3132.

Anisimova, M., Gil, M., Dufayard, J.F., Dessimoz, C., and Gascuel, O. (2011) Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. *Syst Biol* **60**: 685–699.

Bauer, E., Lampert, N., Mikaelyan, A., Köhler, T., Maekawa, K., and Brune, A. (2015) Physicochemical conditions, metabolites and community structure of the bacterial microbiota in the gut of wood-feeding cockroaches (Blaberidae: Panesthiinae). *FEMS Microbiol Ecol* **91**: 1–14.

Bauer, S., Tholen, A., Overmann, J., and Brune, A. (2000) Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termites
Germany). Non-stained cultures were routinely examined using phase-contrast illumination (100× objective).

For electron microscopy, cells were fixed with glutaraldehyde and postfixed with osmium tetroxide before dehydrating in a graded series of ethanol and embedding in Spurr's resin (Zheng et al., 2016). Alternatively, 2-µl samples of concentrated cell suspensions were high-pressure frozen, freeze-substituted with HUGA (0.5% uranyl acetate, 0.5% glutaraldehyde, 5% H₂O in acetone), and embedded in Epon 812 substitute resin, as previously described (Renicke et al., 2017). Ultrathin sections were cut with a microtome equipped with a diamond knife and contrasted with uranyl acetate and lead citrate. The sections were examined with a Philips EM 208 transmission electron microscope.

## Genome sequencing and annotation

Genomic DNA was prepared using cetyltrimethylammonium bromide extraction (Winnepenninckx et al., 1993) and commercially sequenced (GATC-Eurofins, Konstanz, Germany) on a PacBio RS platform using one SMRT cell (insert size up to 10 kbp). Reads were assembled with the PacBio SMRT Portal software (version 2.3.0) using the Hierarchical Genome Assembly Process for assembly and Quiver for polishing (Chin et al., 2013). The polished single contig was circularized with Circlator (Hunt et al., 2015).

The genome sequence of strain RmG30 has been submitted to GenBank (https://www.ncbi.nlm.nih.gov; accession number CP067089; circularized) and to the Integrated Microbial Genomes and Microbiomes database (IMG/M) of the Joint Genome Institute (JGI) (https://img.jgi.doe.gov; taxon ID 2772190975; not circularized). The genome was annotated via the IMG annotation pipeline (v.4.16.1; Chen et al., 2019). For the analysis of the metabolic pathways, annotation results were verified, and missing functions were identified using Blast with a threshold E-value of 1E-5. Hydrogenases were classified using the HydDB reference database (https://services.birc.au.dk/hyddb/; Søndergaard et al., 2016).

## Phylogenetic analyses

The 16S rRNA gene of strain RmG30 was amplified with Bacteria-specific primers and sequenced by Sanger sequencing as previously described (Strassert et al., 2010); the sequence has been submitted to GenBank (ID: MW396710). The sequence was aligned with the SINA aligner (Pruesse et al., 2012) and imported into the reference alignment of the Silva database (version 132; Pruesse et al., 2007); additional sequences were downloaded from GenBank. The alignments were manually curated using the ARB software package (version 6.0.6; Ludwig et al., 2004). A maximum-likelihood tree of the 16S rRNA genes was inferred from 1275 unambiguous aligned positions (sites with more than 50% gaps were masked) using the PhyML algorithm (version 3.3; Guindon et al., 2010) with GTR model and aBayes branch supports (Anisimova et al., 2011) included in ARB. Pairwise sequence identities of 16S rRNA genes are based on a distance matrix of the unfiltered alignment generated in ARB.

The genomes of strain RmG30 and other members of termite cluster I were phylogenetically classified within the taxonomic framework of the Genome Taxonomy Database (GTDB, release 89) using the GTDB toolkit (GTDB-tk, version 1.3.0; Chaumeil et al., 2020). A maximum-likelihood tree based on the genomes was inferred from a concatenated alignment of 120 bacterial single-copy genes (5040 amino acid positions) using the PhyML algorithm with LG model and aBayes branch supports. The average nucleotide identities of the genomes were calculated with FastANI (version 1.3; Jain et al., 2018).

## Acknowledgements

This study was funded by the Deutsche Forschungsgemeinschaft (DFG) in the collaborative research center SFB 987 (Microbial Diversity in Environmental Signal Response) and by the Max Planck Society (MPG). Yulin Song was supported by a fellowship of the China Scholarship Council (CSC). We thank Karen A. Brune for linguistic comments on the manuscript.

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Bauer, E., Lampert, N., Mikaelyan, A., Köhler, T., Maekawa, K., and Brune, A. (2015) Physicochemical conditions, metabolites and community structure of the bacterial microbiota in the gut of wood-feeding cockroaches (Blaberidae: Panesthiinae). *FEMS Microbiol Ecol* 91: 1–14.

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Berlanga, M., Llorens, C., Comas, J., and Guerrero, R. (2016) Gut bacterial community of the xylophagous cockroaches *Cryptocercus punctulatus* and *Parasphaeria boleiriana*. *PLoS One* **11**: e0152400.

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by molecular and culture-dependent techniques. *Arch Microbiol* **173**: 126-137.

Baureder, M., and Hederstedt, L. (2013) Heme proteins in lactic acid bacteria. *Adv Microb Physiol* **62**: 1-43.

Berchtold, M., Ludwig, W., and König, H. (1994) 16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite *Mastotermes darwiniensis* Froggatt. *FEMS Microbiol Lett* **123**: 269-273.

Berlanga, M., Llorens, C., Comas, J., and Guerrero, R. (2016) Gut bacterial community of the xylophagous cockroaches *Cryptocercus punctulatus* and *Parasphaeria boleiriana*. *PLoS One* **11**: e0152400.

Bourguignon, T., Lo, N., Cameron, S.L., Šobotník, J., Hayashi, Y., Shigenobu, S., et al. (2015) The evolutionary history of termites as inferred from 66 mitochondrial genomes. *Mol Biol Evol* **32**: 406-421.

Bourguignon, T., Lo, N., Dietrich, C., Šobotník, J., Sidek, S., Roisin, Y., et al. (2018) Rampant host switching shaped the termite gut microbiome. *Curr Biol* **28**: 649-654.

Breznak, J.A. (2002) Phylogenetic diversity and physiology of termite gut spirochetes. *Integr Comp Biol* **4**: 313-318.

Breznak, J.A., and Leadbetter, J.R. (2006) Termite gut spirochetes. In *The Prokaryotes*, 3rd ed, Vol. 7, Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., and Stackebrandt, E. (eds). New York: Springer, pp. 318-329.

Brioukhanov, A.L., and Netrusov, A.I. (2007) Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review. *Appl Biochem Microbiol* **43**: 567-582.

Brioukhanov, A.L., and Netrusov, A.I. (2012) The positive effect of exogenous hemin on a resistance of strict anaerobic archaeon *Methanobrevibacter arboriphilus* to oxidative stresses. *Curr Microbiol* **65**: 375-383.

Brune, A. (2014) Symbiotic digestion of lignocellulose in termite guts. *Nat Rev Microbiol* **12**: 168-180.

Brune, A., and Dietrich, C. (2015) The gut microbiota of termites: digesting the diversity in the light of ecology and evolution. *Annu Rev Microbiol* **69**: 145-166.

Canale-Parola, E., Udris, Z., and Mandel, M. (1968) The classification of free-living spirochetes. *Arch Mikrobiol* **63**: 385-397.

Chaumeil, P.A., Mussig, A.J., Hugenholtz, P., and Parks, D. H. (2020) GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. *Bioinformatics* **36**: 1925-1927.

Chen, I.A., Chu, K., Palaniappan, K., Pillay, M., Ratner, A., Huang, J., et al. (2019) IMG/M v.5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. *Nucleic Acids Res* **47**: D666-D677.

Chin, C.S., Alexander, D.H., Marks, P., Klammer, A.A., Drake, J., Heiner, C., et al. (2013) Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. *Nat Methods* **10**: 563-569.

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**Supporting Information**

Additional Supporting Information may be found in the online
version of this article at the publisher's web-site:

**Fig. S1.** Expanded version of the phylogenomic tree in the manuscript (Fig. 3), illustrating the relationship of strain RmG30 (red) and other members of the proposed ‘Termitinemataceae’ to other lineages in the *Spirochaetales*. Type species of current genera are marked in bold. The tree was rooted using other lineages of *Spirochaetia* as out-group. Genome sequences were downloaded from GenBank (accession numbers are given), except for strain NkOx-clu11 (Utami *et al.*, 2019) and the genomes in UBA8932 (Dong *et al.*, 2018), which were provided by the authors.

**Fig. S2.** The pathways for lysine and methionine biosynthesis in the genome of strain RmG30. Missing genes and the respective reactions are shown in grey. Gene abbreviations and the encoded functions: asd, aspartate-semialdehyde dehydrogenase; ast, L-aspartate semialdehyde sulfurtransferase; dapA, 4-hydroxy-tetrahydrodipicolinate synthase; dapB, dihydrodipicolinate reductase; dapC, succinyl-diaminopimelate aminotransferase; dapD, tetrahydrodipicolinate succinylase; dapE, succinyl-diaminopimelate desuccinylase; dapF, diaminopimelate epimerase; dapH, tetrahydrodipicolinate acetylase; dapL, LL-diaminopimelate aminotransferase; ddh, diaminopimelate dehydrogenase; lysA, LL-diaminopimelate decarboxylase; lysC, aspartate kinase; metA, homoserine O-succinyltransferase/O-acetyltransferase; metB, cystathionine gamma-synthase; metC, cystathionine beta-lyase; metH, 5-methyltetrahydrofolate-homocysteine methyltransferase; metK, S-adenosylmethionine synthase; metX, homoserine O-acetyltransferase; metY, O-acetylhomoserine aminocarboxypropyltransferase; metZ, O-succinylhomoserine sulfhydrylase; nifS, cysteine desulfurase; patA, N-acetyl-LL-diaminopimelate aminotransferase; ykuR, N-acetyl-LL-diaminopimelate deacetylase.

**File S1.** KEGG maps showing detailed results on the pathways for the biosynthesis of amino acids in RmG30.

**Table S1.** GTDB-tk classification of strain RmG30 and other members of termite cluster I (shown in blue).

**Table S2.** Annotation details of the genes encoding metabolic pathways of strain RmG30 (Fig. 7). The genes are sorted by functional groups (in blue) and subgroups (in grey). Genes without gene ID were not found in the genome.
associated spirochetes are major agents of hemicellulose
degradation in the hindgut of wood-feeding higher ter-
mites. Proc Natl Acad Sci U S A 115: 11996–12004.

Utami, Y.D., Kuwahara, H., Igai, K., Murakami, T., Sugaya, K., Morikawa, T., et al. (2019) Genome analyses of uncultured TG2/ZB3 bacteria in 'Margulisbacteria' specifically attached to ectosymbiotic spirochetes of protists in the termite gut. *ISME J* 13: 455–467.

Warnecke, F., Luginbuhl, P., Ivanova, N., Ghassemian, M., Richardson, T.H., Stege, J.T., et al. (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. *Nature* **450**: 560–565.

Whittenbury, R. (1960) Two types of catalase-like activity in lactic acid bacteria. *Nature* **187**: 433–434.

Widdel, F., and Bak, F. (1992) Gram-negative mesophilic sulfate-reducing bacteria. In *The Prokaryotes*, Balows, A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer, K.H. (eds). *New York: Springer*, pp. 3352–3378.

Winnepenninckx, B., Backeljau, T., and De Wachter, R. (1993) Extraction of high molecular weight DNA from mol-luscs. *Trends Genet* **9**: 407.

Yarza, P., Yilmaz, P., Pruesse, E., Glöckner, F.O., Ludwig, W., Schleifer, K.H., et al. (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. *Nat Rev Microbiol* **12**: 635–645.

Zhang, X., and Leadbetter, J.R. (2012) Evidence for cascades of perturbation and adaptation in the metabolic genes of higher termite gut symbionts. *mBio* 3: e00223-12.

Zhang, X., Matson, E.G., and Leadbetter, J.R. (2011) Genes for selenium dependent and independent formate dehydrogenase in the gut microbial communities of three lower, wood-feeding termites and a wood-feeding roach. *Environ Microbiol* 13: 307–323.

Zheng, H., Dietrich, C., Radek, R., and Brune, A. (2016) *Endomicrobium proavitum*, the first isolate of *Endomicrobia* class. nov. (phylum *Elusimicrobia*) – an ultramicrobacterium with an unusual cell cycle that fixes nitrogen with a group IV nitrogenase. *Environ Microbiol* 18: 191–204.

Zhuang, W.Q., Yi, S., Bill, M., Brisson, V.L., Feng, X., Men, Y., et al. (2014) Incomplete wood–Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring *Dehalococcoides mccartyi*. Proc Natl Acad Sci U S A 111: 6419–6424.

Zuelzer, M. (1912) Über *Spirochaeta plicatilis* Ehrenberg und deren Verwandtschaftsbeziehungen. Arch Protistenkd 24: 1–59.

**Supporting Information**

Additional Supporting Information may be found in the online
version of this article at the publisher's web-site:

**Fig. S1.** Expanded version of the phylogenomic tree in the manuscript (Fig. 3), illustrating the relationship of strain RmG30 (red) and other members of the proposed ‘Termitinemataceae’ to other lineages in the *Spirochaetales*. Type species of current genera are marked in bold. The tree was rooted using other lineages of *Spirochaetia* as out-group. Genome sequences were downloaded from GenBank (accession numbers are given), except for strain NkOx-clu11 (Utami *et al.*, 2019) and the genomes in UBA8932 (Dong *et al.*, 2018), which were provided by the authors.

**Fig. S2.** The pathways for lysine and methionine biosynthesis in the genome of strain RmG30. Missing genes and the respective reactions are shown in grey. Gene abbreviations and the encoded functions: asd, aspartate-semialdehyde dehydrogenase; ast, L-aspartate semialdehyde sulfurtransferase; dapA, 4-hydroxy-tetrahydrodipicolinate synthase; dapB, dihydrodipicolinate reductase; dapC, succinyl-diaminopimelate aminotransferase; dapD, tetrahydrodipicolinate succinylase; dapE, succinyl-diaminopimelate desuccinylase; dapF, diaminopimelate epimerase; dapH, tetrahydrodipicolinate acetylase; dapL, LL-diaminopimelate aminotransferase; ddh, diaminopimelate dehydrogenase; lysA, LL-diaminopimelate decarboxylase; lysC, aspartate kinase; metA, homoserine O-succinyltransferase/O-acetyltransferase; metB, cystathionine gamma-synthase; metC, cystathionine beta-lyase; metH, 5-methyltetrahydrofolate-homocysteine methyltransferase; metK, S-adenosylmethionine synthase; metX, homoserine O-acetyltransferase; metY, O-acetylhomoserine aminocarboxypropyltransferase; metZ, O-succinylhomoserine sulfhydrylase; nifS, cysteine desulfurase; patA, N-acetyl-LL-diaminopimelate aminotransferase; ykuR, N-acetyl-LL-diaminopimelate deacetylase.

**File S1.** KEGG maps showing detailed results on the pathways for the biosynthesis of amino acids in RmG30.

**Table S1.** GTDB-tk classification of strain RmG30 and other members of termite cluster I (shown in blue).

**Table S2.** Annotation details of the genes encoding metabolic pathways of strain RmG30 (Fig. 7). The genes are sorted by functional groups (in blue) and subgroups (in grey). Genes without gene ID were not found in the genome.