Dataset Information

Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

ABSTRACT: The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-β-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-β-1,4-mannosidase [EC 3.2.1.25] that is highly specific for β-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Å resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family β-galactosidases and the GH164-family exo-β-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 β-galactosidase-type homotrimeric structure.

SUBMITTER: Kalyani DC

PROVIDER: S-EPMC8233224 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Two morphologically distinct, H2- and CO2-utilizing methanogens were isolated from gut homogenates of the subterranean termite, Reticulitermes-flavipes (Kollar) (Rhinotermitidae). Strain RFM-1 was a short straight rod (0.4 by 1.2 micron), whereas strain RFM-2 was a slightly curved rod (0.34 by 1.6 microns) that possessed polar fibers. Their morphology, gram-positive staining reaction, resistance to cell lysis by chemical agents, and narrow range of utilizable substracts were typical of species belonging to the family Methanobacteriaceae. Analysis of the nearly complete sequences of the small-subunit rRNA-encoding genes confirmed this affiliation and supported their recognition as new species of Methanobrevibacter: M. cuticularis (RFM-1) and M. curvatus (RFM-2). The per cell rates of methanogenesis by strains RFM-1 and RFM-2 in vitro, taken together with their in situ population densities (ca. 10(6) cells.gut-1; equivalent to 10(9) cells . ml of gut fluid-1), could fully account for the rate of methane emission by the live termites. UV epifluorescence and electron microscopy confirmed that RFM-1- and RFM-2-type cells were the dominant methanogens in R.flavipes collected in Michigan (but were not the only methanogens associated with this species) and that they colonized the peripheral, microoxic region of the hindgut, i.e., residing on or near the hindgut epithelium and also attached to filamentous prokaryotes associated with the gut wall. An examination of their oxygen tolerance revealed that both strains possessed catalase-like activity. Moreover, when dispersed in tubes or agar medium under H2-CO2-O2 (75: 18.8:6.2, vol/vol/vol), both strains grew to form a thin plate about 6 mm below the meniscus, just beneath the oxic-anoxic interface. Such growth plates were capable of mediating a net consumption of O2 that otherwise penetrated much deeper into uninoculated control tubes. Similar results were obtained with an authentic strain of Methanobrevibacter arboriphilicus. This is the first detailed description of an important and often cited but poorly understood component of the termite gut microbiota.

Project description:BACKGROUND:Termite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing. RESULTS:Over 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding. CONCLUSION:To our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a single termite species. This sequence database represents an important new genomic resource for use in further studies of collaborative host-symbiont termite digestion, as well as development of coevolved host and symbiont-derived biocatalysts for use in industrial biomass-to-bioethanol applications. Additionally, this study demonstrates that: (i) phenoloxidase activities are prominent in the R. flavipes gut and are not symbiont derived, (ii) expands the known number of host and symbiont glycosyl hydrolase families in Reticulitermes, and (iii) supports previous models of lignin degradation and host-symbiont collaboration in cellulose/hemicellulose digestion in the termite gut. All sequences in this paper are available publicly with the accession numbers FL634956-FL640828 (Termite Gut library) and FL641015-FL645753 (Symbiont library).

Dataset Information

Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets