Dataset Information

Screening of multimeric ?-xylosidases from the gut microbiome of a higher termite, Globitermes brachycerastes.

ABSTRACT: Termite gut microbiome is a rich reservoir for glycoside hydrolases, a suite of enzymes critical for the degradation of lignocellulosic biomass. To search for hemicellulases, we screened 12,000 clones from a fosmid gut library of a higher termite, Globitermes brachycerastes. As a common Southeastern Asian genus, Globitermes distributes predominantly in tropical rain forests and relies on the lignocellulases from themselves and bacterial symbionts to digest wood. In total, 22 positive clones with ?-xylosidase activity were isolated, in which 11 representing different restriction fragment length polymorphism (RFLP) patterns were pooled and subjected to 454 pyrosequencing. As a result, eight putative ?-xylosidases were cloned and heterologously expressed in Escherichia coli BL21 competent cells. After purification using Ni-NTA affinity chromatography, recombinant G. brachycerastes symbiotic ?-xylosidases were characterized enzymatically, including their pH and temperature optimum. In addition to ?-xylosidase activity, four of them also exhibited either ?-glucosidase or ?-arabinosidases activities, suggesting the existence of bifunctional hemicellulases in the gut microbiome of G. brachycerastes. In comparison to multimeric protein engineering, the involvement of naturally occurring multifunctional biocatalysts streamlines the genetic modification procedures and simplifies the overall production processes. Alternatively, these multimeric enzymes could serve as the substitutes for ?-glucosidase, ?-xylosidase and ?-arabinosidase to facilitate a wide range of industrial applications, including food processing, animal feed, environment and waste management, and biomass conversion.

SUBMITTER: Liu C

PROVIDER: S-EPMC6001650 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Screening of multimeric β-xylosidases from the gut microbiome of a higher termite, Globitermes brachycerastes.

Liu Chunyan C Zou Gen G Yan Xing X Zhou Xuguo X

International journal of biological sciences 20180426 6

Termite gut microbiome is a rich reservoir for glycoside hydrolases, a suite of enzymes critical for the degradation of lignocellulosic biomass. To search for hemicellulases, we screened 12,000 clones from a fosmid gut library of a higher termite, Globitermes brachycerastes. As a common Southeastern Asian genus, Globitermes distributes predominantly in tropical rain forests and relies on the lignocellulases from themselves and bacterial symbionts to digest wood. In total, 22 positi ...[more]

PMID: 29904275

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Project description:BackgroundThanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging.MethodsIn this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy.ResultsWe showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit "Epidemiology" in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines.ConclusionsWe have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.

Project description:Termites and their gut microbes engage in fascinating dietary mutualisms. Less is known about how these complex symbioses have evolved after first emerging in an insect ancestor over 120 million years ago. Here we examined a bacterial gene, formate dehydrogenase (fdhF), that is key to the mutualism in 8 species of "higher" termite (members of the Termitidae, the youngest and most biomass-abundant and species-rich termite family). Patterns of fdhF diversity in the gut communities of higher termites contrasted strongly with patterns in less-derived (more-primitive) insect relatives (wood-feeding "lower" termites and roaches). We observed phylogenetic evidence for (i) the sweeping loss of several clades of fdhF that may reflect extinctions of symbiotic protozoa and, importantly, bacteria dependent on them in the last common ancestor of all higher termites and (ii) a radiation of genes from the (possibly) single allele that survived. Sweeping gene loss also resulted in (iii) the elimination of an entire clade of genes encoding selenium (Se)-independent enzymes from higher termite gut communities, perhaps reflecting behavioral or morphological innovations in higher termites that relaxed preexisting environmental limitations of Se, a dietary trace element. Curiously, several higher termite gut communities may have subsequently reencountered Se limitation, reinventing genes for Se-independent proteins via convergent evolution. Lastly, the presence of a novel fdhF lineage within litter-feeding and subterranean higher (but not other) termites may indicate recent gene "invasion" events. These results imply that cascades of perturbation and adaptation by distinct evolutionary mechanisms have impacted the evolution of complex microbial communities in a highly successful lineage of insects. Since patterns of relatedness between termite hosts are broadly mirrored by the relatedness of their symbiotic gut microbiota, coevolution between hosts and gut symbionts is rightly considered an important force that has shaped dietary mutualism since its inception over 120 million years ago. Apart from that concerning lateral gene or symbiont transfer between termite gut communities (for which no evidence yet exists), there has been little discussion of alternative mechanisms impacting the evolution of mutualism. Here we provide strong gene-based evidence for past environmental perturbations creating significant upheavals that continue to reverberate throughout the gut communities of species comprising a single termite lineage. We suggest that symbiont extinction events, sweeping gene losses, evolutionary radiations, relaxation and reemergence of key nutritional pressures, convergent evolution of similar traits, and recent gene invasions have all shaped gene composition in the symbiotic gut microbial communities of higher termites, currently the most dominant and successful termite family on Earth.

Project description:BackgroundTermites are among the most successful insects on Earth and can feed on a broad range of organic matter at various stages of decomposition. The termite gut system is often referred to as a micro-reactor and is a complex structure consisting of several components. It includes the host, its gut microbiome and fungal gardens, in the case of fungi-growing higher termites. The digestive tract of soil-feeding higher termites is characterised by radial and axial gradients of physicochemical parameters (e.g. pH, O2 and H2 partial pressure), and also differs in the density and structure of residing microbial communities. Although soil-feeding termites account for 60% of the known termite species, their biomass degradation strategies are far less known compared to their wood-feeding counterparts.ResultsIn this work, we applied an integrative multi-omics approach for the first time at the holobiont level to study the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis. We relied on 16S rRNA gene community profiling, metagenomics and (meta)transcriptomics to uncover the distribution of functional roles, in particular those related to carbohydrate hydrolysis, across different gut compartments and among the members of the bacterial community and the host itself. We showed that the Labiotermes gut was dominated by members of the Firmicutes phylum, whose abundance gradually decreased towards the posterior segments of the hindgut, in favour of Bacteroidetes, Proteobacteria and Verrucomicrobia. Contrary to expectations, we observed that L. labralis gut microbes expressed a high diversity of carbohydrate active enzymes involved in cellulose and hemicelluloses degradation, making the soil-feeding termite gut a unique reservoir of lignocellulolytic enzymes with considerable biotechnological potential. We also evidenced that the host cellulases have different phylogenetic origins and structures, which is possibly translated into their different specificities towards cellulose. From an ecological perspective, we could speculate that the capacity to feed on distinct polymorphs of cellulose retained in soil might have enabled this termite species to widely colonise the different habitats of the Amazon basin.ConclusionsOur study provides interesting insights into the distribution of the hydrolytic potential of the highly compartmentalised higher termite gut. The large number of expressed enzymes targeting the different lignocellulose components make the Labiotermes worker gut a relevant lignocellulose-valorising model to mimic by biomass conversion industries.

Dataset Information

Screening of multimeric ?-xylosidases from the gut microbiome of a higher termite, Globitermes brachycerastes.

Publications

Screening of multimeric β-xylosidases from the gut microbiome of a higher termite, <i>Globitermes brachycerastes</i>.

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