Unusual Microbial Xylanases from Insect Guts

Abstract

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted ␤-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.The arthropod gut is a differentiated organ harboring a complex biotope comprising both resident and transient members from protozoal, bacterial, and archaeal genera. Many of these organisms are symbionts that contribute in a concerted way to a complex chemical cycle sustaining the metabolic competence of the host. These symbiotic relationships help to define the metabolic traits of the insect, contributing to efficient sharing of the derived nutrients. In addition to carbon metabolism, data have demonstrated that the microbial consortia contribute to nitrogen cycling, methano-and acetogenesis, and prevention of foreign microbial pathogenesis (29).A complete understanding of the complexities of the gut biotopes is lacking, however, due to the difficulties encountered in culturing the myriad of contributing microbes and in describing the physiologies of the individual species. Studies aimed at a description of microbial complexity have recently been undertaken in termites by using 16S ribosomal DNA analysis and have shown that a number of unique lineages of microorganism inhabit the hindgut (28). These data support the idea that the insect gut represents a contained biome wherein unique species and chemistries evolve.Polysaccharide hydrolysis is a key element in insect nutrition. Since the diet of most arthropods comprises primarily plant matter, digestion of the structural polysaccharides cellulose and hemicellulose is essential for energy metabolism and the ability to obtain carbon from these sources contributes materially to the success of the order. These polysaccharides are resistant to degradation, and the insects themselves do not secrete all of the digestive enzymes to hydrolyze ␤-linkages in the polymer. Rather, much of the hydrolysis of these polysaccharides is carried out by enzymes produced by the microbial symbionts (4,5,36,41).Hemicellulose consists primarily of xylan and it is the second most abundant polymer type in plant materi...

A Symbiont-Independent Endo-1,4-β-Xylanase from the Plant-Parasitic NematodeMeloidogyne incognita

“…For instance, the hydrolysis of xylan polymers of plant cell walls in ruminants depends on enzymes from endosymbiotic bacteria and fungi (Wubah et al 1993). Similarly, bacteria and fungi are known to contribute to the endo-1,4-β-xylanase activity in the digestive tracts of xylophagous wood-boring insects (Brennan et al 2004). Some bacterial and fungal pathogens of plants also secrete endo-1,4-β-xylanase, suggesting that it may contribute to the infection process (Walton 1994).…”

Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes

“…This can be addressed by functional screening of metagenomic libraries, in order to retrieve genes of interest. Numerous studies have provided conclusive evidence on the potential of such an approach for the identification of novel glycoside-hydrolases from various ecosystems such as soil (Rondon et al 2000;Richardson et al 2002;Voget et al 2003;Pang et al 2009), lakes (Rees et al 2003), hot springs (Tang et al 2006, rumen (Ferrer et al 2005;Guo et al 2008;Liu et al 2008;Duan et al 2009), rabbit (Feng et al 2007), and insect guts (Brennan et al 2004; for review, see Ferrer et al 2009;Li et al 2009;Simon and Daniel 2009;Uchiyama and Miyazaki 2009). In all cases, the identification of the gene responsible for the screened activity was carried out by sequencing only a few kilobases of metagenomic DNA.…”

Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio

“…Although one putative cellulase in the GH8 family was predicted in the genome of Pantoea sp. SL1_M5, g-Proteobacteria appear better equipped to deconstruct hemicelluloses, an activity common among gut bacteria of other insects (Brennan et al, 2004;Warnecke et al, 2007).…”

“…Symbioses with cellulose-degrading microorganisms appear to compensate for an inability to produce cellulases in many insects (Martin, 1991;Douglas, 2009) and these associations, whether obligate or facultative, are important drivers in the evolution of woodfeeding insects (Veivers et al, 1983). This group includes prominent invasive pests, such as the wood-boring emerald ash borer, and Formosan termite, among others, which harbors gut microorganisms that contribute to insect nutrition through degradation of cellulose (Delalibera et al, 2005;Warnecke et al, 2007;Vasanthakumar et al, 2008), hemicellulose (Brennan et al, 2004) and lignin (Pasti et al, 1990;Geib et al, 2008).…”