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

Abstract

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

Keywords: fiber-associated community; metatranscriptome; spirochetes; termite hindgut; xylanase.

Fig. 1.

Enzyme activities against CMC, cellulose, and xylan in soluble and particulate fractions of hindgut homogenates of N. takasagoensis. The homogenates were separated into soluble and particulate fractions by centrifugation, and xylanase activities were determined in both fractions after detergent extraction. Values are means of replicate samples from five colonies; error bars denote SDs. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of reducing sugar (glucose equivalent for cellulose and xylose equivalent for xylan) per minute. “Cellulose” indicates microcrystalline cellulose (Sigmacell Type 20); “xylan” indicates beechwood xylan.

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).

Fig. 3.

Domain structure of the GH11 xylanases predominantly expressed by the fiber-associated bacterial community in the hindgut of N. takasagoensis based on the deduced amino acid sequences of the eight most abundant transcripts.

Fig. 4.

Indirect immunofluorescence microscopy of the N. takasagoensis P3 content stained with a monoclonal antibody that cross-reacts with recombinant NtSymX11 xylanase (green) and counterstained with DAPI (blue). (A–C) The same field of view inspected with an epifluorescence microscope: (A) DAPI channel, (B) Alexa Fluor 488 channel (antibody), and (C) merged image. Merged images obtained with a confocal laser microscope at lower (D) and higher (E) magnification show the association of antibody-stained cells with autofluorescent wood particles. (F) Negative control without primary antibody treatment. (Scale bars: A–C, 20 µm; D–F, 100 μm.)

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.