Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher

Gaku Tokudaa,b,1, Aram Mikaelyanc,d, Chiho Fukuia, Yu Matsuuraa, Hirofumi Watanabee, Masahiro Fujishimaf, and Andreas Brunec

aTropical Biosphere Research Center, Center of Molecular Biosciences, University of the Ryukyus, Nishihara, 903-0213 Okinawa, Japan; bGraduate School of Engineering and Science, University of the Ryukyus, Nishihara, 903-0213 Okinawa, Japan; cResearch Group Gut Microbiology and , Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; dDepartment of Entomology and Pathology, North Carolina State University, Raleigh, NC 27607; eBiomolecular Mimetics Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, 305-8634 Ibaraki, Japan; and fDepartment of Sciences, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yoshida 1677-1, 753-8512 Yamaguchi, Japan

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved November 5, 2018 (received for review June 25, 2018) Symbiotic digestion of lignocellulose in wood-feeding higher digestion in the hindgut of higher termites must be attributed to termites (family ) is a two-step process that involves their entirely prokaryotic microbial community (5). endogenous host cellulases secreted in the midgut and a dense The of higher termites comprises more than bacterial community in the hindgut compartment. The genomes of 1,000 bacterial phylotypes, which are organized into distinc- the bacterial gut microbiota encode diverse cellulolytic and hemi- tive communities colonizing the microhabitats provided by the cellulolytic enzymes, but the contributions of host and bacterial compartmentalized intestine, including the highly differentiated symbionts to lignocellulose degradation remain ambiguous. Our hindgut (6, 7). Of particular interest are the associated previous studies of spp. documented that the wood with wood particles in the dilated hindgut paunch of wood- fibers in the hindgut paunch are consistently colonized not only by feeding Nasutitermes ; these bacteria represent less than

uncultured members of , which have been implicated 30% of the total microbial population but contribute more than MICROBIOLOGY in cellulose degradation, but also by unique lineages of Spiro- half of the cellulolytic activity in this compartment (8). Core chaetes. Here, we demonstrate that the degradation of xylan, members of the fiber-associated community are several so-far the major component of hemicellulose, is restricted to the hindgut uncultured lineages of Fibrobacteres and the closely related compartment, where it is preferentially hydrolyzed over cellulose. candidate TG3 (now classified as Fibrobacteria and Metatranscriptomic analysis documented that the majority of gly- coside hydrolase (GH) transcripts expressed by the fiber-associated Chitinivibrionia; ref. 9) and two lineages of uncultured Spiro- bacterial community belong to family GH11, which consists exclu- chaetes ( Ic and If), which represent a separate line of sively of xylanases. The substrate specificity was further confirmed descent that has been found exclusively in higher termites (8). by heterologous expression of the gene encoding the predomi- nant homolog. Although the most abundant transcripts of Significance GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of , immunofluorescence micros- Xylan, the major hemicellulosic component of lignocellulose copy, compositional binning of contigs, and the and the second most abundant polysaccharide after cellulose, genomic context of the homologs indicated that they are encoded contributes to the structural stability of wood and its re- by and were most likely obtained by horizontal gene calcitrance to enzymatic digestion. The present study identifies transfer among the intestinal microbiota. The major role of spiro- Spirochaetes as primary agents of xylan degradation in the chetes in xylan degradation is unprecedented and assigns the hindgut of wood-feeding higher termites, in contrast to the fiber-associated Treponema clades in the hindgut of wood- bovine or the human colon, where are feeding higher termites a prominent part in the breakdown responsible for hydrolysis of xylan in grass or cereals. The of hemicelluloses. presence of distinctive xylanases in Spirochaetes was so far undocumented to our knowledge. Their phylogenetic origin metatranscriptome | xylanase | spirochetes | fiber-associated community | among gut bacteria of other phyla identifies horizontal gene hindgut transfer among the intestinal microbiota as an important driver in the evolutionary adaptation of higher termites to different ignocellulose is the most abundant biopolymer in terrestrial lignocellulosic diets. Lenvironments (1). It consists mainly of cellulose, hemi- Author contributions: G.T., A.M., Y.M., and A.B. designed research; G.T., C.F., Y.M., H.W., cellulose, and lignin, and is remarkably recalcitrant to microbial and M.F. performed research; G.T., A.M., Y.M., and A.B. analyzed data; and G.T., A.M., degradation. The ability of termites to efficiently digest ligno- Y.M., and A.B. wrote the paper. cellulose has a large impact on the global ecosystem (1). The The authors declare no conflict of interest. elucidation of the underlying mechanisms has presented a for- This article is a PNAS Direct Submission. midable challenge to research for almost a century (2). Termites Published under the PNAS license. of phylogenetically basal lineages (“lower termites”) harbor Data deposition: The data reported in this paper have been deposited in the sequence symbiotic in their enlarged hindgut compartment that read archive of the DNA Data Bank Japan (DDBJ) under accession nos. DRA005983 (meta- transcriptomes) and DRA005967 (metagenome). The complete sequence of the cloned phagocytize wood particles and play a crucial role in cellulose GH11 gene (NtSymX11) was deposited in GenBank/European Nucleotide Archive/DDBJ and hemicellulose degradation (3). By contrast, termites of the database under accession no. LC311413. phylogenetically most apical lineage (“higher termites”), which 1To whom correspondence should be addressed. Email: [email protected]. account for approximately two thirds of all termite species, are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. devoid of such eukaryotic symbionts (4). As a consequence, fiber 1073/pnas.1810550115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1810550115 PNAS Latest Articles | 1of9 Downloaded by guest on September 29, 2021 Previous metagenomic analyses (i.e., the comprehensive se- we measured hydrolytic activity against beechwood xylan in sol- quencing of the genetic material of an entire microbial com- uble (supernatant) and particulate (pellet) fractions of homog- munity) had already reported the presence of diverse genes enates of salivary glands and individual gut sections by using a encoding putative cellulases and hemicellulases in the pro- previously described protocol (21, 25). Xylanase activity was karyotic hindgut microbiota of higher termites (10–15). In the essentially confined to the hindgut proper, and the activity in the case of the wood-feeding , many of these particulate fraction (1.42 ± 0.19 U/g termite) exceeded that in genes were tentatively assigned to members of the Fibrobacteres the soluble fraction (0.36 ± 0.32 U/g termite) fourfold (Fig. 1), and Spirochaetes (10). Metagenomic binning revealed that the which suggests that most of the activity is associated with wood genomes of Fibrobacteres encode an abundance of cellulase particles, bacterial cells, or both. Xylanase activities in salivary genes and more hemicellulase genes than the average number in glands, foregut, and mixed segment were below the detection other cellulolytic bacteria. However, these genomes lack the limit (0.02 U/g termite), whereas midgut sections occasionally genes required to metabolize xylose (9), a major component of showed trace activities in some preparations (0.03 ± 0.06 U/g xylan and other hemicelluloses of wood (16). Moreover, none of termite; n = 5). When we compared xylanase activities with the Fibrobacteres from termite guts have been cultured, and cellulolytic activities in the hindgut, we found that xylanase ac- their hemicellulolytic ability remains to be elucidated. Spirochaetes likely play a crucial role in reductive aceto- tivities were in the same range as cellulolytic activities against carboxymethylcellulose (CMC) and more than an order of genesis, the production of acetate from H2 and CO2 by anaerobic bacteria, which is an important reaction in the hindgut of lower magnitude higher than activities against microcrystalline cellu- and higher termites (17–19). Although metagenomes of hindgut lose (Fig. 1). Moreover, activities against CMC and microcrys- contents of Nasutitermes spp. provided evidence that spirochetes talline cellulose were more evenly distributed between the carry genes encoding glycoside hydrolases (GHs) (10), a direct soluble and particulate fractions, which is in agreement with involvement of fiber-associated spirochetes in the degradation of previous reports (8, 21). These results suggest that hindgut cellulose or hemicelluloses has not been demonstrated. Thus, the bacteria play an important role in degrading the xylan backbone major degraders of hemicellulose in the hindgut of higher ter- of hemicellulose. mites remain unidentified. The frequency of genes in the guts of the N. corniger and Amitermes wheeleri elucidated by metagenomic analyses does not necessarily correlate with their expression levels (12). For that 1.8 reason, the role of bacteria in symbiotic digestion of lignocellu- lose inferred by metagenomics of the entire hindgut community Soluble has to be considered with caution. Metatranscriptomic analyses 1.6 (i.e., the comprehensive profiling of the transcripts of a microbial Particulate community) of the bacteria that interact directly with wood fibers 1.4 in the hindgut would help to elucidate the actual role of the bacterial gut microbiota in wood-feeding higher termites. The situation is further obscured by an apparent division of 1.2 the roles in lignocellulose degradation between higher termite hosts and their intestinal bacteria. Higher termites produce en- dogenous cellulases that are secreted in the midgut (20), and 1.0 these activities are considerably higher than those of bacterial origin in the hindgut (21). The cellulase activities in the hindgut of higher termites are also surpassed by those in the hindgut of 0.8 lower termites (22, 23); lower termite hindguts contain the bulk of the cellulolytic activities and most of the xylanolytic activities, but these are attributed to their flagellate protists and not to 0.6 bacterial symbionts (22–25). In the flagellate-free gut of higher termites, the amount and location of hemicellulolytic activities, including xylanases, have not been studied. 0.4 To clarify the role of bacterial symbionts in lignocellulose Enzyme (U/g termite) activity Enzyme degradation in higher termites, we investigated the distribution 0.2 of xylanase activity among the different gut compartments of the wood-feeding higher termite Nasutitermes takasagoensis. By using a previously developed method to isolate wood fibers with their 0.0 attached bacterial microbiota from the total hindgut contents (8), we conducted a meta-analysis of the transcripts of carbohydrate- CMC Cellulose Xylan active enzymes (CAZys) expressed by the fiber-associated bac- terial community. We then used a combination of metagenomics, phylogenetic analyses, and indirect immunofluorescence mi- Substrate croscopy to explore the origin and function of genes encoding the major hemicellulolytic enzymes in the hindgut. Our results Fig. 1. Enzyme activities against CMC, cellulose, and xylan in soluble and shed light on the role in wood decomposition of the fiber- particulate fractions of hindgut homogenates of N. takasagoensis. The ho- associated microbial community in the hindgut of higher ter- mogenates were separated into soluble and particulate fractions by centri- mites and elucidate the unexpected major participants in fugation, and xylanase activities were determined in both fractions after detergent extraction. Values are means of replicate samples from five col- hemicellulose degradation. onies; error bars denote SDs. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of reducing sugar ( equiv- Results alent for cellulose and xylose equivalent for xylan) per minute. “Cellulose” Localization of Xylanase Activity in the Gut of N. takasagoensis. To indicates microcrystalline cellulose (Sigmacell Type 20); “xylan” indicates identify the site of xylan digestion in the gut of N. takasagoensis, beechwood xylan.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810550115 Tokuda et al. Downloaded by guest on September 29, 2021 Table 1. Properties of the metatranscriptomic libraries of the fiber-associated community in the hindgut of N. takasagoensis Raw Clean No. of Total Mean N50 Fraction of No. of distinct Transcripts Genes encoding Sample data, Mbp data, Mbp transcripts length, nt length, nt value mapped reads, % mRNAs encoding CAZys* CAZys*

Library 1 4,770 4,560 94,273 51,566,241 547 731 70.8 —— — Library 2 4,770 4,618 97,454 52,419,831 538 714 70.7 —— — Library 3 4,770 4,619 97,624 52,458,326 537 708 70.5 —— — Merged ——133,206 83,659,303 628 1,024 — 122,671 2,766 2,807

Reads were generated with Illumina HiSeq 2000. *Glycosyltransferases were excluded.

Metatranscriptomic Profiles of Genes Involved in Lignocellulose 1,111 genes), and 3 docking modules (92 genes; Dataset S1). Degradation. To identify genes involved in hemicellulose degra- Transcripts related to lignin degradation and oxidoreductive dation activity detected in the hindgut compartment, we carried cellulases (e.g., auxiliary activity AA10 members; ref. 28) were out a metatranscriptomic analysis. The three mRNA libraries absent in the dataset. The remaining genes were assigned to prepared from replicate fractions of fiber-associated bacteria in another 27 glycosyltransferase families that represent enzymes the hindgut of N. takasagoensis each yielded approximately 4.5 primarily involved in the biosynthesis of polysaccharides rather Gbp of clean sequence reads (Table 1). De novo assembly of the than in their degradation and were excluded from the present reads resulted in 133,206 nonredundant gene clusters (here re- analysis. ferred to as transcripts), from which 122,671 putative mRNA The short reads from each library were mapped onto the sequences were identified. The remaining transcripts are pre- aforementioned identified transcripts, and the expression level of dicted as noncoding RNAs. each transcript was determined as the number of fragments per Database searches for CAZys on dbCAN with amino acid kilobase of transcript per million mapped reads (FPKM), which sequences (26) and dbCAN2 with nucleotide sequences (27) calculates the abundance of paired-end reads and normalizes retrieved 2,766 transcripts with a total of 2,807 genes (Table 1). them to the length of the respective transcript (Fig. 2 and They encoded representatives of 62 GH families (1,553 genes), Dataset S1). In all replicates, the most highly expressed CAZy

12 carbohydrate esterase families (173 genes), 7 polysaccharide family genes were those of CBM36 (8,531 ± 195 FPKM), which MICROBIOLOGY lyase families (39 genes), 3 families with auxiliary activities consists primarily of xylan-binding domains; GH11 (5,548 ± 179 (21 genes), 52 families of carbohydrate-binding modules (CBMs; FPKM), which represents putative endo-β-1,4-xylanases (hereafter

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Fig. 2. Expression levels of CAZy family transcripts in the metatranscriptomic libraries of fiber-associated bacteria of N. takasagoensis grouped by the most relevant functions. Only the most highly expressed families in each group are shown (a full list is provided in Dataset S1). Values are means of replicate samples from three colonies; error bars represent SDs. Numbers in parentheses denotes the total numbers of distinct genes assigned to each CAZy family. Red bars indicate families with high expression levels per gene (Dataset S1).

Tokuda et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 29, 2021 referred to as xylanases); and GH5 (5,037 ± 117 FPKM), which Transcript 30253 includes diverse cellulases and hemicellulases. Most transcripts assigned to GH5 were affiliated with subfamily 2 (74%) and subfamily 4 (14%), which consist predominantly of endo-β-1,4- glucanases or xyloglucanases and lichenases, respectively (29) (SI Transcript 30250 Appendix, Fig. S1). High expression levels were also observed for genes encoding GH130 (1,779 ± 4 FPKM) and GH30 (1,228 ± 43 FPKM), which represent hemicellulases (hydrolases and phos- Transcript 5174 phorylases of mannosyl saccharides in GH130 and mostly β-xylosidases in GH30). The other abundant transcripts encode CBM50 (1,632 ± 62 FPKM), which bind to bacterial Transcript 57501 peptidoglycan and chitin. The expression level of other CAZy family genes were typically far below 1,000 FPKM, and consid- ering that they were represented by a large number of tran- scripts, most of them did not appear very prominent. These Transcript 30249 results indicate that the community of fiber-associated bacteria preferentially expresses genes involved in hydrolysis of hemi- celluloses, particularly xylan, and cellulose. Transcript 29175 Structure and Heterologous Expression of Putative GH11 Xylanases. The 8 most abundant transcripts among the 99 transcripts assigned to family GH11 made up 88.9% of the metatran- Transcript 130482 scriptomic reads assigned to this family (Dataset S2). With the exception of Transcript 57501, which encoded only a single catalytic , each transcript had a single gene that encoded a GH11 catalytic domain and one or two CBM36 domains (Fig. Transcript 30254 3). The deduced amino acid sequences of the catalytic domains showed 95–100% identity to each other and 60–70% identity to those of bona fide GH11 xylanases from Firmicutes (as detailed later). An exception was Transcript 130482, whose catalytic do- main showed only 81–82% identity to those of the other tran- Catalytic domain (GH11) 100 amino scripts and 70–80% identity to those of bona fide GH11 acid residues xylanases from Firmicutes. Carbohydrate-binding module (CBM36) To confirm that the predominantly expressed members of family GH11 encode functional xylanases, we amplified an entire Fig. 3. Domain structure of the GH11 xylanases predominantly expressed GH11 gene from hindgut DNA of N. takasagoensis by using PCR by the fiber-associated bacterial community in the hindgut of N. takasa- primers designed to match the flanking regions of Transcript goensis based on the deduced amino acid sequences of the eight most 30253, the homolog with the highest expression level (30.0% of abundant transcripts. the reads assigned to family GH11). The deduced amino acid sequence encoded by xylanase clone NtSymX11, determined by Supplementary Results Sanger sequencing, showed the same constitution (a single cat- ), we investigated the organismal origin of alytic domain and two CBM36 modules) and an amino acid the predominant xylanases by using a metagenomic approach. identity of 96% in the catalytic domain to the product of Tran- Metagenomic sequencing of the bacterial community associ- script 30253; the overall amino acid identity, including the CBM ated with the fiber fraction yielded 126,962 contigs from more modules and their internal sequences, was approximately 87%. than 40 million reads (SI Appendix, Table S1). A local nucleotide BLAST search against this dataset identified 17 contigs with a Phylogenetic analysis revealed that clone NtSymX11 falls into < −10 the monophyletic group formed by seven of the eight most significant similarity to Transcript 30253 (e-value 10 ). Two abundant transcripts (as detailed later). After removal of a pu- of the contigs contained a complete GH11 gene (83% and 88% tative leader sequence [inferred by using the SignalP 4.1 server nucleotide sequence identity to Transcript 30253), in each case (30)], clone NtSymX11 was heterologously expressed in Escher- surrounded by genes with highest sequence similarity to those in ichia coli. The partially purified polyhistidine-tagged (His-tag) Spirochaetes (SI Appendix, Fig. S3). Most of them were from recombinant enzyme (SI Appendix, Fig. S2A) showed high xyla- Treponema primitia and Treponema azotonutricium, which are nase activity (592 U/mg ) against beechwood xylan (SI the only members of the Treponema I clade that have sequenced Appendix, Fig. S2C). genomes and inhabit the hindgut of lower termites. The remaining contigs contained partial sequences of GH11 and Organismal Origin of the GH11 Xylanases. We raised monoclonal CBM36, and one of the former was flanked as well by a gene antibodies against components of the fiber-associated bacterial with highest sequence similarity to a homolog in T. primitia (SI fraction. In Western blots, only 1 of 15 antibodies cross-reacted Appendix, Fig. S3). with recombinant NtSymX11 xylanase (SI Appendix, Fig. S2B). Compositional binning of all metagenomic contigs with VizBin Indirect immunofluorescence microscopy of the P3 fluid of N. yielded three well-defined clusters (i.e., bins; SI Appendix, Fig. takasagoensis revealed that the antibody bound specifically to S4). BLAST analysis of 31 single-copy marker genes (33) in- bacterial cells with a helical morphology, which occurred in an dicated that bin 1 is composed almost exclusively of members of unattached state or in association with wood fibers (Fig. 4). As Fibrobacteria (phylum Fibrobacteres), whereas bin 2 is com- an undulate or helical morphology is encountered in the mem- posed almost exclusively of Spirochaetes (Dataset S3). In bin 3, bers of Fibrobacteres and Spirochaetes that colonize the hindgut the top BLAST hit of each single-copy marker gene was almost of Nasutitermes spp. (31, 32), and attempts to colocalize the exclusively represented by Chitinivibrio alkaliphilus, the first and immunofluorescence signal with fluorescent rRNA-target ol- only isolate of the Chitinivibrionia (formerly candidate phylum igonucleotide probes gave inconsistent results (SI Appendix, TG3; ref. 34) represented in public databases at the time of

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810550115 Tokuda et al. Downloaded by guest on September 29, 2021 fers of a GH11 xylanase gene from a firmicute to a spirochete. Nasutitermes cluster IV included previously published sequences from other and from a moth [which had been obtained in the same study (36)]. They were most closely related to GH11 homologs from the gut of a beetle larva and loosely embedded in a larger clade of homologs from the gut meta- genomes of various higher termites, which included sequences from the Fibrobacteres and Chitinivibrionia bins (bins 1 and 3) of the fiber fraction of N. takasagoensis. The only GH11 homolog in Nasutitermes cluster IV derived from a cultivated represen- tative is again from a firmicute, lactis cremoris, but the ancestral positions were held by GH11 xylanases from the genome of Fibrobacter succinogenes and from metagenome- assembled genomes of various uncultivated members of Fibro- bacteres in the gut of mammals (35). Discussion Xylans and other hemicelluloses are a major dietary component of the food of herbivorous . Most animals do not produce endogenous enzymes for the depolymerization of hemicelluloses but digest them with the help of their gut microbiota. In the Fig. 4. Indirect immunofluorescence microscopy of the N. takasagoensis bovine rumen and in the human colon, members of the phylum P3 content stained with a monoclonal antibody that cross-reacts with Bacteroidetes play a crucial role in this process (37), mainly recombinant NtSymX11 xylanase (green) and counterstained with DAPI through their GH10 endoxylanases (38). By contrast, the results (blue). (A–C) The same field of view inspected with an epifluorescence mi- of the present study show that, in the guts of higher termites, croscope: (A) DAPI channel, (B) Alexa Fluor 488 channel (antibody), and (C) members of the phylum Spirochaetes are the main players in this merged image. Merged images obtained with a confocal laser microscope at process. This is remarkable for several reasons: (i) a function of

lower (D) and higher (E) magnification show the association of antibody- Spirochaetes in xylan degradation is so far undocumented to our MICROBIOLOGY stained cells with autofluorescent wood particles. (F) Negative control knowledge, (ii) the lineages responsible for the activity belong to – μ – μ without primary antibody treatment. (Scale bars: A C,20 m; D F,100 m.) a monophyletic clade that is specific for termites and that have coevolved with their termite hosts, and (iii) the GH11 xylanase analysis. Hidden Markov model searches for the presence of genes of termite gut spirochetes were most likely acquired by pfam00457, the catalytic domain of GH11, identified a total of horizontal gene transfer from other gut bacteria. Although spirochetes are best known as human pathogens 30 CDSs in the three bins (2 in bin 1, 26 in bin 2, and 2 in bin 3); (39), they are also important pectin degraders in (40, these CDSs encoded putative xylanases of family GH11. CBMs 41) and play a key role in the production of acetate from H and of CBM36, which were identified by using dbCAN because of the 2 CO in termite guts (17–19). An involvement of spirochetes in lack of pfam domains, were detected only in bin 2 (Dataset S4). 2 hemicellulose degradation has not been reported to our knowl- These results indicate that the majority of genes involved in xylan edge, and the genomes of termite gut treponemes isolated from hydrolysis originated from Spirochaetes. lower termites (Treponema Ia) do not encode any xylanases of the GH11 family. Thus, these findings significantly advance our Phylogenetic Analysis of GH11 Xylanases. Phylogenetic analysis understanding of lignocellulose digestion in higher termites and revealed that the GH11 homologs identified in the fiber- shed light on the evolutionary history of termite gut spirochetes. associated community of N. takasagoensis are very closely re- In lower termites, xylanase activity is localized predominantly lated to those of metagenomes of other Nasutitermes species. in the hindgut (42), where it is most likely associated with sym- Together, they form four well-supported clusters (Nasutitermes – biotic protists (25). The present study demonstrates that xylanase clusters I IV; Fig. 5). Nasutitermes cluster I comprises the eight activity is also restricted almost exclusively to the hindgut in predominantly expressed GH11 transcripts in the fiber fraction wood-feeding higher termites. Because higher termites lack such of N. takasagoensis and also several metagenomic sequences protists, the role of xylan (and cellulose) degradation has shifted from the fiber fraction that were taxonomically assigned to Spi- to the symbiotic bacteria colonizing this gut compartment. rochaetes (bin 2) based on binning analysis. Nasutitermes cluster Xylanase activity in the hindgut exceeds cellulase activity against I also included sequences from previously published meta- CMC and microcrystalline cellulose, which is in agreement with genomic analyses of other higher termite species (N. corniger and the predominance of putative xylanases among the GH genes Trinervitermes trinervoides). It formed a larger monophyletic expressed by the fiber-associated community and the greater clade with another cluster of GH11 homologs from the fiber solubility of this substrate. fraction of N. takasagoensis (Nasutitermes cluster II) and pre- Previous metagenomic analyses identified 45–53 families of viously published sequences from metagenomes of A. wheeleri GH genes in the bacterial community in the luminal fluid of the (12) and metagenome-assembled genomes of Fibrobacteres hindgut paunch (P3 compartment) of Nasutitermes spp. (10, 12). [from feces of herbivorous mammals (35)], with a GH11 ho- Our transcriptomic analysis of N. takasagoensis detected tran- molog of a cultivated firmicute, Paenibacillus wynnii, in the most scripts of 62 GH families alone in the fiber fraction, which in- basal position. These sequences comprised a sister group with dicates that the GH repertoire of the fiber-associated bacterial closely related GH11 homologs of other Firmicutes. community was comprehensively sampled. In all metatran- The remaining metagenomic sequences of spirochetal origin scriptomic libraries, this enormous diversity was dominated pri- (bin 2) from N. takasagoensis fell into Nasutitermes clusters III marily by members of family GH11, followed by members of and IV. Nasutitermes cluster III also included metagenomic se- family GH5. The GH11 family consists exclusively of xylanases quences from N. corniger (12), again with a homolog of a culti- that preferentially hydrolyze xylanosic bonds of heteroxylans, vated member of the Firmicutes, Ruminococcus albus,inan such as glucuronoxylans and arabinoxylans (43). The substrate ancestral position, which suggests multiple cross-phylum trans- specificities in the GH5 family are more diverse, but the majority

Tokuda et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 29, 2021 Chitinivibrionia (bin 3) Fibrobacteria (bin 1) Fibrobacteres Firmicutes Spirochaetes (bin 2) Others Actinobacteria, Firmicutes Unclassified Proteobacteria and others Holomastigotoides mirabile cluster Cellvibrio japonicus Ueda107 (ACE85524) Teredinibacter turnerae T7901 (ACR11046) Actinoplanes derwentensis (SDS46682) Firmicutes Firmicutes Elephant cluster I Ruminococcus albus DSM 20455 (AAA85198) Nasutitermes takasagoensis metatranscriptome (Transcript30254AA) N. takasagoensis metatranscriptome (Transcript30249AA) N. takasagoensis metatranscriptome (Transcript30250AA) N. takasagoensis metatranscriptome (Transcript29175AA) N. takasagoensis metatranscriptome (Transcript30253AA) Clone NtSymX11 from N. takasagoensis (LC311413) Nasutitermes N. takasagoensis metatranscriptome (Transcript5174AA) cluster I Nasutitermes corniger metagenome (IMG Gene id: Ga0074247_1086981) N. takasagoensis metatranscriptome (Transcript57501AA) N. takasagoensis metagenome (fiber friction, bin 2) Trinervitermes trinervoides metagenome (AMO13185.1) N. takasagoensis metatranscriptome (Transcript130482AA) N. corniger metagenome (IMG Gene id: NasMGMT1_1189491) Fibrobacteres N. takasagoensis metagenome (fiber fraction, bin 2) Nasutitermes cluster II Amitermes cluster I Paenibacillus wynnii (WP_036655480) Amitermes cluster II N. corniger metagenome (IMG Gene id: Nasutiterm_202150) N. corniger metagenome (IMG Gene id: NasMGMT1_0131151) N. takasagoensis metagenome (fiber fraction, bin 2) Nasutitermes cluster III N. takasagoensis metagenome (fiber fraction, bin 2) Ruminococcus albus 7 (AAA85198) Nasutitermes cluster IV Beetle cluster subsp. cremoris SK1 (WP_011676455) N. corniger metagenome (IMG Gene id: Nasutiterm_192400) Microcerotermes sp. metagenome (ADD82898.1) Microcerotermes sp. metagenome (ADD82900.1) N. takasagoensis metagenomes (fiber fraction, bin1) N. takasagoensis metagenome (fiber fraction, bin 2) N. takasagoensis metagenome (fiber fraction, bin 3) Elephant cluster II Elephant cluster III

Fibrobacteres

Elephant cluster IV uncultured symbiotic of Hodotermopsis sjoestedti (BAF57353.1)

0.10

Fig. 5. Phylogenetic relationship among bacterial GH11 members and phylum-level classification of homologs from fiber-associated bacteria in the hindgut of N. takasagoensis. The unrooted tree is based on 164 alignment positions and represents a consensus phylogeny obtained by using maximum-likelihood (ML) and Bayesian (BA) inference and the WAG model of protein evolution (60). Sequences obtained in this study are shown in bold, and text color indicates the taxonomic affiliation of homologs obtained from bacterial genomes and compositional bins. Node support was determined with the χ2 approximate likelihood ratio test (i.e., ML) and posterior probability (i.e., BA); confidence values are indicated by circles (open, ≥90% support in at least one method; filled, ≥90% support in both methods). The detailed tree, including all accession numbers and confidence values, is provided in SI Appendix, Fig. S5.

of the transcripts were affiliated with GH5 subfamily 2, which luminal content of the P3 compartment of a different Nasuti- consists predominantly of endo-β-1,4-glucanases (29). By con- termes species had been assigned to termite gut treponemes trast, other highly expressed GHs belonged to families GH130 based on phylogenetic binning (10), but neither their expression and GH30 and GH5 subfamily 4, which again comprise various levels nor xylanase activities in the hindgut compartment were hemicellulases (44, 45). This result underscores that the pri- investigated. Our metatranscriptomic and enzymatic results in- mary role of the fiber-associated bacterial community is in the disputably corroborate the spirochetal origin of GH11 xylanases breakdown of hemicelluloses, especially the xylan backbone of in N. takasagoensis. Although phylogenetic analysis underscores wood, which link the cellulose fibrils to the lignin fraction and the close relationship of the GH11 xylanases to homologs in thus impede the access of endoglucanases and other cellulolytic Firmicutes, the reference-independent assignment of the re- enzymes to their substrate. spective contigs, based on their genomic signatures, and the Although a function of spirochetes in xylan degradation has genomic neighborhood of the most highly expressed genes in- never been documented before to our awareness, earlier studies dicate that they are encoded by spirochetes. A misassembly of provided hints that xylanase genes found in the gut of higher DNA fragments originating from Firmicutes with spirochete termites are produced by spirochetes. An endoxylanase gene of DNA in this and probably also previous studies is further ex- family GH11 had been detected in bacterial DNA from a cluded by our localization of the recombinant xylanase on the Nasutitermes sp. almost 15 y ago (35), but its origin remained surface of fiber-associated helical cells by immunofluorescence obscure. Later, 4 of 14 GH11 xylanase genes recovered from the microscopy.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810550115 Tokuda et al. Downloaded by guest on September 29, 2021 The evolutionary origin of GH11 xylanases in termite gut family GH11 (12). It is not clear to which extent this reflects the spirochetes remains obscure. The genomes of Treponema spp. interspecific differences between the microbiota of N. takasa- isolated from lower termites, which belong to the basal Trepo- goensis and N. corniger (12) or the composition of the total and nema Ia subclade (46), do not encode homologs of this enzyme fiber-associated bacterial community in the P3 compartment, family. The GH11 xylanases in Nasutitermes cluster I, which which have been documented for both species (8). Moreover, it encode the most highly expressed transcripts, and their homologs must be taken into account that the proportion of xylanase and in the closely related Nasutitermes clusters II and III, are phy- cellulase activities in P3 compartment and fiber fraction will logenetically situated among xylanase gene sequences of Firmi- depend on the relative abundance of endoglucanases and hem- cutes. They represent separate lines of descent, with homologs icellulases in the transcripts of GH5. Also, the absolute activities from P. wynnii and R. albus as closest, albeit distant, relatives. By of cellulases (determined with microcrystalline cellulose) and contrast, the GH11 xylanases in Nasutitermes cluster IV are xylanases (determined with much more soluble xylan) have to be embedded in a clade that is dominated by homologs of Fibro- taken with caution, as indicated by the considerably higher cel- bacteres, including sequences from the corresponding meta- lulase activities obtained with soluble CMC (23). genomic bins (bins 1 and 3) of N. takasagoensis and from the In view of the large proportion of putative cellulases (e.g., intestinal tracts of mammals (35, 47). In both cases, the most GH5_2, GH8, GH45) in the metatranscriptomic libraries (SI likely explanation for this scenario would be a transfer of the Appendix, Fig. S1 and Dataset S1), the low cellulase activity genes encoding GH11 xylanases from Firmicutes or Fibro- obtained with the particulate fraction might be explained by a bacteres to termite gut treponemes. In the radiation of Nasuti- less efficient recovery of the cellulases of Fibrobacteres and termes clusters I and II, the conspicuous presence of GH11 other fiber-associated microbiota, which are most probably lo- homologs of Fibrobacteres from mammalian guts [recovered calized in the glycocalyx (49) or outer membrane vesicles (50). from metagenome-assembled genomes (35)] suggests that such Therefore, the astonishingly high cellulase activities in the sol- horizontal transfers of xylanase genes has also occurred in the uble fraction of the hindgut paunch may be artificial (Fig. 1). opposite direction. Finally, it is important to consider that the enzyme activities in A horizontal transfer of the xylanase genes from Firmicutes particulate and soluble fractions represent the GHs that are to Spirochaetes (Nasutitermes cluster I) is supported by the re- bound to microbial cells or released into the luminal fluid, sults of metagenomic binning, which assigned the xylan-binding whereas the metagenomic and metatranscriptomic analyses used CBM36 modules associated with the corresponding GH11 only the DNA or RNA of cells obtained by the density- xylanase genes or transcripts exclusively to Spirochaetes (bin 2). dependent enrichment of wood fibers. The fiber-associated MICROBIOLOGY Most of the bacterial CBM36 modules in the CAZy database Spirochaetes in the P3 compartment of Nasutitermes spp. are— (79 of 89) originate from the genomes of Firmicutes. The in contrast to the firmly attached Fibrobacteres—also abun- remaining ones are from Bacteroidetes, Proteobacteria, Dictyoglomi, dantly present in the fiber-free fraction (8). Considering that the or environmental samples, but are not encountered among the most highly expressed GH11 genes from spirochetes encode genomes of Fibrobacteres or the corresponding metagenomic CBMs but no protein structures known to anchor GHs to the cell bins (1, 3). Together with the absence of GH11 and CBM36 surface, it is quite likely that at least some of xylanase activity in homologs from all spirochetal genomes sequenced to date, in- the soluble fraction derives from fiber-associated spirochetes cluding the Treponema I isolates in lower termites, these results (Fig. 1). suggest that the fiber-associated lineages in the gut of higher As in the rumen, where efficient degradation of lignocellulose termites acquired the capacity for the hydrolysis of xylan by involves colonization of dietary fibers by bacteria that hydrolyze horizontal gene transfer. structural polysaccharides to form soluble cellodextrins that are It has been proposed that the ancestral loss of flagellates in subsequently also utilized by the unattached bacterial pop- higher termites was connected with a dietary shift from sound ulations (49, 51), the soluble sugars released by the hydrolytic wood to -infested or humified wood and plant litter, and activity of the fiber-associated community should serve at least in that the return to a wood-feeding lifestyle occurred indepen- part also as carbon and energy sources of other, unattached dently in several subfamilies (48). In the case of Nasutitermitinae, populations. However, the average size of the wood particles in this change in lifestyle might be associated with the acquisition of the hindgut of higher termites is considerably smaller (25 μm) GH11 xylanases by an ancestor of the Treponema Ic and If than that of the forage in the rumen (200 μm) (52), and thereby subclades. This would explain the presence of a GH11 homolog creates a much larger surface area for bacterial colonization. of Nasutitermes cluster I also in the hindgut of the grass-feeding The dietary diversification of higher termites is considered a key T. trinervoides (14), which represents a sister group of wood- element of their ecological and evolutionary success. In wood- feeding Nasutitermes spp. and harbors members of Treponema feeding lineages, this involved the reversal from a detritivorous to Ic (46). The organismal origin of the GH11 homologs from the a xylophagous lifestyle, although the trigger of this evolutionary gut microbiota of Termitinae is more speculative. In the case of transition of feeding habits remains elusive. Based on the corre- the dung-feeding A. wheeleri (12), they may derive from the lation between diet and bacterial community structure in the clostridial lineages that are abundant in the gut of this termite, hindguts of higher termites (53), the present study strongly sug- but, at least in the case of the Amitermes cluster I (a sister group gests that the horizontal acquisition of novel digestive capacities of Nasutitermes cluster II), an ancestral transfer to termite gut by resident gut bacteria provided the termite host with the flexi- treponemes also cannot be excluded. In the case of the wood- bility to exploit additional feeding substrates and eventually resulted feeding Microcerotermes spp. (11, 35), whose gut microbiota in compositional changes in the entire hindgut microbiota. encodes GH11 xylanases that are phylogenetically placed among homologs recovered from the metagenomic bins of fiber- Experimental Procedures associated Spirochaetes and Fibrobacteres from N. takasagoensis, the organismal origin remains to be determined. Colonies of N. takasagoensis were collected on Iriomote Island in Okinawa prefecture, Japan, and maintained as previously described (54, 55). Worker- Although the expression level of GH11 xylanases in the fiber caste termites were used for all experiments. Endo-β-1,4-xylanase (EC fraction of the P3 compartment of N. takasagoensis was slightly 3.2.1.8), endo-β-1,4-glucanase (EC 3.2.1.4), and hydrolytic activities acting on higher than that of GHs of GH5 (as shown in the present study), microcrystalline cellulose in salivary glands and gut sections (pooled preparations a previous meta-analysis of the entire luminal contents of the from 10 individuals each) were measured as previously described (21, 22, 25). hindgut paunch in N. corniger demonstrated that transcripts of For metatranscriptomic analysis, fiber-associated bacteria were prepared family GH5 were considerably more abundant than those of by density-gradient centrifugation as previously described (8) and transferred

Tokuda et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 29, 2021 to RNAprotect bacteria reagent (Qiagen) to stabilize RNA. Total RNA was For metagenomic analysis, DNA was extracted from fiber-associated extracted with the RNeasy Mini Kit (Qiagen) and shipped to BGI for library bacteria and purified by using the ISOPLANTII purification kit (Nippon preparations and sequencing (Illumina HiSeq 2000). We annotated amino Gene). Again, library preparation, sequencing, and assembly were done by acid sequences provided by BGI by using dbCAN (26) to detect CAZys and BGI. We analyzed the contigs/scaffolds provided by BGI by using local BLAST relevant modules. Furthermore, we annotated nucleotide sequences of all search and Genetyx software (Genetyx) and compositional binning with contigs by using dbCAN2 (27) to find transcripts and genes that were missing Vizbin (57). Phylogenetic trees included an alignment of all publicly acces- in the dataset provided by BGI. Expression profiles of CAZy were analyzed by sible GH11 sequences using maximum likelihood (phyml version 3.0.1; ref. using a dataset for all expressed genes provided by BGI. For enzyme profiles, 58) and Bayesian analysis (MrBayes version 3.2.1; ref. 59). Full details of the an amplified fragment of GH11 xylanase gene (NtSymX11) without a pu- experimental procedures are provided in SI Appendix. tative leader sequence was ligated with a pQE-1 expression vector and in- troduced into E. coli strain JM109. The resulting His-tagged enzyme was ACKNOWLEDGMENTS. We thank Karen A. Brune for linguistic improve- partially purified with a Ni-NTA spin column and examined by SDS/PAGE. ments in the manuscript and Aki Kinjo for technical assistance. This study Monoclonal antibodies were raised against the bacteria in the fiber was supported by a research grant from the Institute for Fermentation, fraction following the method previously described (56). Antibodies that Osaka, Japan; by Grants-in-Aid for Scientific Research KAKENHI 26292177, cross-reacted with the recombinant xylanase NtSymX11 were selected by 15K14900, and 17H01510 from the Japan Society for the Promotion of Western blotting. For indirect immunofluorescence microscopy, the entire Science (JSPS); by University of the Ryukyus Research Incentive Grant P3 fluid was mixed with the selected monoclonal antibody, followed by in- 18SP03104; by FY2014 Bilateral Joint Research Program A/14/01075 between cubation with Alexa Fluor 488-labeled anti-mouse IgG, and then with DAPI Germany (Deutscher Akademischer Austauschdienst) and Japan (JSPS); and to visualize DNA. Specimens were observed under a fluorescence microscope by Deutsche Forschungsgemeinschaft in Collaborative Research Center SFB (BX41; Olympus) or a confocal laser microscope (C2; Nikon). 987 (Microbial Diversity in Environmental Signal Response).

1. Cornwell WK, et al. (2009) Plant traits and wood fates across the globe: Rotted, 25. Arakawa G, Watanabe H, Yamasaki H, Maekawa H, Tokuda G (2009) Purification and burned, or consumed? Glob Change Biol 15:2431–2449. molecular cloning of xylanases from the wood-feeding termite, Coptotermes for- 2. Watanabe H, Tokuda G (2001) cellulases. Cell Mol Sci 58:1167–1178. mosanus Shiraki. Biosci Biotechnol Biochem 73:710–718. 3. Ni J, Tokuda G (2013) Lignocellulose-degrading enzymes from termites and their 26. Yin Y, et al. (2012) dbCAN: A web resource for automated carbohydrate-active en- symbiotic microbiota. Biotechnol Adv 31:838–850. zyme annotation. Nucleic Acids Res 40:W445–W451. 4. Lo N, Eggleton P (2011) Termite phylogenetics and co-cladogenesis with symbionts. 27. Zhang H, et al. (2018) dbCAN2: A meta server for automated carbohydrate-active Biology of Termites: A Modern Synthesis, eds Bignell DE, Roisin Y, Lo N (Springer, enzyme annotation. Nucleic Acids Res 46:W95–W101. Dordrecht, The Netherlands), pp 27–50. 28. Book AJ, et al. (2014) Evolution of substrate specificity in bacterial AA10 lytic poly- 5. Brune A (2014) Symbiotic digestion of lignocellulose in termite guts. Nat Rev saccharide monooxygenases. Biotechnol Biofuels 7:109. Microbiol 12:168–180. 29. Aspeborg H, Coutinho PM, Wang Y, Brumer H, 3rd, Henrissat B (2012) Evolution, 6. Köhler T, Dietrich C, Scheffrahn RH, Brune A (2012) High-resolution analysis of gut substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). environment and bacterial microbiota reveals functional compartmentation of the BMC Evol Biol 12:186. gut in wood-feeding higher termites (Nasutitermes spp.). Appl Environ Microbiol 78: 30. Nielsen H (2017) Predicting secretory proteins with SignalP. Methods Mol Biol 1611: – 4691 4701. 59–73. 7. Mikaelyan A, Meuser K, Brune A (2017) Microenvironmental heterogeneity of gut 31. Hongoh Y, et al. (2006) Phylogenetic diversity, localization, and cell morphologies of compartments drives bacterial community structure in wood- and humus-feeding members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, higher termites. FEMS Microbiol Ecol 93:fiw210. recently discovered bacterial groups dominant in termite guts. Appl Environ 8. Mikaelyan A, Strassert JFH, Tokuda G, Brune A (2014) The fiber-associated cellulolytic Microbiol 72:6780–6788. bacterial community in the hindgut of wood-feeding higher termites (Nasutitermes 32. Hongoh Y (2014) Who digests the lignocellulose? Environ Microbiol 16:2644–2645. – spp.). Environ Microbiol 16:2711 2722. 33. Wu M, Eisen JA (2008) A simple, fast, and accurate method of phylogenomic in- 9. Abdul Rahman N, et al. (2016) A phylogenomic analysis of the bacterial phylum Fi- ference. Genome Biol 9:R151. brobacteres. Front Microbiol 6:1469. 34. Sorokin DY, et al. (2014) Genome analysis of Chitinivibrio alkaliphilus gen. nov., sp. 10. Warnecke F, et al. (2007) Metagenomic and functional analysis of hindgut microbiota nov., a novel extremely haloalkaliphilic anaerobic chitinolytic bacterium from the of a wood-feeding higher termite. Nature 450:560–565. candidate phylum Termite Group 3. Environ Microbiol 16:1549–1565. 11. Nimchua T, Thongaram T, Uengwetwanit T, Pongpattanakitshote S, Eurwilaichitr L 35. Parks DH, et al. (2017) Recovery of nearly 8,000 metagenome-assembled genomes (2012) Metagenomic analysis of novel lignocellulose-degrading enzymes from higher substantially expands the tree of life. Nat Microbiol 2:1533–1542. termite guts inhabiting microbes. J Microbiol Biotechnol 22:462–469. 36. Brennan Y, et al. (2004) Unusual microbial xylanases from insect guts. Appl Environ 12. He S, et al. (2013) Comparative metagenomic and metatranscriptomic analysis of Microbiol 70:3609–3617. hindgut paunch microbiota in wood- and dung-feeding higher termites. PLoS One 8: 37. Dodd D, Mackie RI, Cann IKO (2011) Xylan degradation, a metabolic property shared e61126. by rumen and human colonic Bacteroidetes. Mol Microbiol 79:292–304. 13. Poulsen M, et al. (2014) Complementary symbiont contributions to plant de- 38. Zhang M, et al. (2014) Xylan utilization in human gut commensal bacteria is or- composition in a fungus-farming termite. Proc Natl Acad Sci USA 111:14500–14505. chestrated by unique modular organization of polysaccharide-degrading enzymes. 14. Rashamuse K, et al. (2017) Metagenomic mining of glycoside hydrolases from the Proc Natl Acad Sci USA 111:E3708–E3717. hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the char- 39. Gupta RS, Mahmood S, Adeolu M (2013) A phylogenomic and molecular signature acterization of a multimodular β-1,4-xylanase (GH11). Biotechnol Appl Biochem 64: based approach for characterization of the phylum Spirochaetes and its major clades: 174–186. Proposal for a taxonomic revision of the phylum. Front Microbiol 4:217. 15. Liu N, et al. (August 16, 2018) Functional metagenomics reveals abundant 40. Liu J, et al. (2014) Monitoring the rumen pectinolytic bacteria Treponema saccha- polysaccharide-degrading gene clusters and cellobiose utilization pathways within rophilum using real-time PCR. FEMS Microbiol Ecol 87:576–585. gut microbiota of a wood-feeding higher termite. ISME J, 10.1038/s41396-018-0255-1. 41. Svartström O, et al. (2017) Ninety-nine de novo assembled genomes from the moose 16. Albersheim P, Darvill A, Roberts K, Sederoff R, Staehelin A (2011) Plant Cell Walls (Garland Science, New York). (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation. ISME J 11:2538–2551. 17. Leadbetter JR, Schmidt TM, Graber JR, Breznak JA (1999) Acetogenesis from H2 plus 42. Slaytor M (2000) Energy metabolism in the termite and its gut microbiota. Termites: CO2 by spirochetes from termite guts. Science 283:686–689. 18. Rosenthal AZ, Matson EG, Eldar A, Leadbetter JR (2011) RNA-seq reveals cooperative Evolution, Sociality, Symbioses, Ecology, eds Abe T, Bignell DE, Higashi M (Kluwer – metabolic interactions between two termite-gut spirochete species in co-culture. Academic Publishers, Dordrecht, The Netherlands), pp 307 332. ISME J 5:1133–1142. 43. Paës G, Berrin JG, Beaugrand J (2012) GH11 xylanases: Structure/function/properties relationships and applications. Biotechnol Adv 30:564–592. 19. Ohkuma M, et al. (2015) Acetogenesis from H2 plus CO2 and by an endosymbiotic spirochete of a termite-gut cellulolytic protist. Proc Natl Acad Sci USA 44. Cuskin F, et al. (2015) The GH130 family of mannoside phosphorylases contains gly- β 112:10224–10230. coside hydrolases that target -1,2-mannosidic linkages in Candida mannan. J Biol 20. Tokuda G, et al. (2012) Cellulolytic environment in the midgut of the wood-feeding Chem 290:25023–25033. higher termite Nasutitermes takasagoensis. J Insect Physiol 58:147–154. 45. Valenzuela SV, Diaz P, Pastor FIJ (2012) Modular glucuronoxylan-specific xylanase 21. Tokuda G, Watanabe H (2007) Hidden cellulases in termites: Revision of an old hy- with a family CBM35 carbohydrate-binding module. Appl Environ Microbiol 78: pothesis. Biol Lett 3:336–339. 3923–3931. 22. Tokuda G, et al. (2004) Major alteration of the expression site of endogenous cellu- 46. Mikaelyan A, et al. (2015) Classifying the bacterial gut microbiota of termites and lases in members of an apical termite lineage. Mol Ecol 13:3219–3228. cockroaches: A curated phylogenetic reference database (DictDb). Syst Appl Microbiol 23. Tokuda G, Lo N, Watanabe H (2005) Marked variations in patterns of cellulase activity 38:472–482. against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, 47. Suen G, et al. (2011) The complete genome sequence of Fibrobacter succinogenes wood-feeding termites. Physiol Entomol 30:372–380. S85 reveals a cellulolytic and metabolic specialist. PLoS One 6:e18814. 24. Slaytor M, Sugimoto A, Azuma J, Murashima K, Inoue T (1997) Cellulose and xylan 48. Donovan SE, Eggleton P, Bignell DE (2001) Gut content analysis and a new feeding utilisation in the lower termite Reticulitermes speratus. J Insect Physiol 43:235–242. group classification of termites. Ecol Entomol 26:356–366.

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810550115 Tokuda et al. Downloaded by guest on September 29, 2021 49. Weimer PJ (1996) Why don’t ruminal bacteria digest cellulose faster? J Dairy Sci 79: 55. Tokuda G, Miyagi M, Makiya H, Watanabe H, Arakawa G (2009) Digestive β-glucosi- 1496–1502. dases from the wood-feeding higher termite, Nasutitermes takasagoensis: Intestinal 50. Arntzen MØ, Várnai A, Mackie RI, Eijsink VGH, Pope PB (2017) Outer membrane vesicles distribution, molecular characterization, and alteration in sites of expression. Insect from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes Biochem Mol Biol 39:931–937. with versatile polysaccharide-degrading capacity. Environ Microbiol 19:2701–2714. 56. Iwatani K, et al. (2005) Translocation of an 89-kDa periplasmic protein is associated 51. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: with Holospora infection. Biochem Biophys Res Commun 337:1198–1205. Fundamentals and . Microbiol Mol Biol Rev 66:506–577. 57. Laczny CC, et al. (2015) VizBin–An application for reference-independent visualiza- 52. Martz FA, Belyea RL (1986) Role of particle size and forage quality in digestion and tion and human-augmented binning of metagenomic data. Microbiome 3:1. passage by cattle and sheep. J Dairy Sci 69:1996–2008. 58. Guindon S, et al. (2010) New algorithms and methods to estimate maximum-likelihood 53. Mikaelyan A, et al. (2015) Diet is the primary determinant of bacterial community phylogenies: Assessing the performance of PhyML 3.0. Syst Biol 59:307–321. structure in the guts of higher termites. Mol Ecol 24:5284–5295. 59. Ronquist F, et al. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and 54. Tokuda G, Watanabe H, Matsumoto T, Noda H (1997) Cellulose digestion in the model choice across a large model space. Syst Biol 61:539–542. wood-eating higher termite, Nasutitermes takasagoensis (Shiraki): Distribution of 60. Whelan S, Goldman N (2001) A general empirical model of protein evolution derived from cellulases and properties of endo-β-1,4-glucanase. Zool Sci 14:83–93. multiple protein families using a maximum-likelihood approach. Mol Biol Evol 18:691–699. MICROBIOLOGY

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