Phylogenetic Analysis of Cellulolytic Enzyme Genes from Representative Lineages of Termites and a Related Cockroach

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1▿ †

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1

Multiple alignments of symbiotic protist cellulase and catalytic domains of the members of the glycoside hydrolase family 7.

<p>Rs, <i>Reticulitermes speratus symbiotic protists</i>; Hs, <i>Hodotermopsis sjostedti symbiotic protists</i>; Nk, <i>Neotermes koshunensis symbiotic protists</i>; Md, <i>Mastotermes darwiniensis symbiotic protists</i>; Cp, <i>Cryptocercus punctulatus symbiotic protists</i>; Numbered clones (for example SM2038A27) are clone names of cellulase genes identified in this study from the symbiotic protists of termites; CBH, cellobiohydrolase; EG, endoglucanase; TrCel7A, a cellobiohydrolase component, <i>Trichoderma reesei</i> Cel7A [Uni Prot. P00725]; PgCBH-homo, <i>Pseudotrichonympha grassii</i> PgCBH-homo1 [Q95YH1]; TrCel7B, an endo-β-1,4-glucanase (EG) component, <i>T. reesei</i> Cel7B [P07981]; FoCel7B, an EG component, <i>Fusarium oxysporum</i> Cel7B [P46237]; HiCel7B, an EG component, <i>Humicola insolens</i> Cel7B [P56680]. The alignments were performed using CLUSTAL_W and subsequent manual refinement based on the three-dimensional structures of reference sequences. Arabic numerals denote the number of residues from each N terminal end. Solid and open circles under the column indicate the sites of putative proton donors and general acids/bases, respectively. Shaded columns represent conserved positions within the sequences. White letters with black shading denote cysteine residues composing the disulfide bond of <i>T. reesei</i> Cel7A. The asterisks represent the putative protistan GHF7 CBH homologue cysteine residue sites corresponding to the cysteine residue sites of <i>T. reesei</i> Cel7A. The underlined sequences in TrCel7A indicate the loop-forming regions covering the catalytic tunnel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008636#pone.0008636-Slomovic1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008636#pone.0008636-Wang1" target="_blank">[23]</a>.</p

Phylogenetic tree of GHF7. Numbered clones (for example SM2734B07) are cellulase genes identified in this study from the symbiotic protists of termites.

<p>Letters in parentheses after each clone denote host termite species (Rs  =  <i>Reticulitermes speratus</i>, Nk  =  <i>Neotermes koshunensis</i>, Hs  =  <i>Hodotermopsis sjostedti</i>, Md  =  <i>Mastotermes darwiniensis</i>, Cp  =  <i>Cryptocercus punctulatus</i>). Accession numbers of reference sequences are denoted after species names.</p

Phylogenetic tree of GHF5. Numbered clones (for example SM2030B48) are cellulase genes identified in this study from the symbiotic protists of termites.

<p>Letters in parentheses after each clone denote host termite species (Rs  =  <i>Reticulitermes speratus</i>, Nk  =  <i>Neotermes koshunensis</i>, Hs  =  <i>Hodotermopsis sjostedti</i>, Md  =  <i>Mastotermes darwiniensis</i>, Cp  =  <i>Cryptocercus punctulatus</i>). Accession numbers of reference sequences are denoted after species names. SF are indicated the sub-family (21) and SSFs are indicated sub-sub families that were used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008636#pone-0008636-g003" target="_blank">Fig. 3</a>. In sub-family 1 and 2, gray boxed sub-sub-family indicated that composed by symbiont's sequences and other sub-sub-families composed by bacterial sequences.</p

The number of GHF clones from EST analysis.

<p>The number of GHF clones from EST analysis.</p

Phylogenetic tree of GHF45. Numbered clones (for example NT0285A89) are cellulase genes identified in this study from the symbiotic protists of termites.

<p>Letters in paretheses after each clone denote host termite species (Rs  =  <i>Reticulitermes speratus</i>, Nk  =  <i>Neotermes koshunensis</i>, Hs  =  <i>Hodotermopsis sjostedti</i>, Md  =  <i>Mastotermes darwiniensis</i>, Cp  =  <i>Cryptocercus punctulatus</i>). Accession numbers of reference sequences are denoted after species names.</p

Phylogenetic tree of GHF11. Numbered clones (for example SM2038A64) are cellulase genes identified in this study from the symbiotic protists of termites.

<p>Letters in paretheses after each clone denote host termite species (Rs  =  <i>Reticulitermes speratus</i>, Nk  =  <i>Neotermes koshunensis</i>, Hs  =  <i>Hodotermopsis sjostedti</i>, Md  =  <i>Mastotermes darwiniensis</i>, Cp  =  <i>Cryptocercus punctulatus</i>). Accession numbers of reference sequences are denoted after species names.</p