The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer.
ABSTRACT: The primary plant cell wall comprises the most abundant polysaccharides on the Earth and represents a rich source of energy for organisms which have evolved the ability to digest them. Enzymes able to degrade plant cell wall polysaccharides are widely distributed in micro-organisms but are generally absent in animals, although their presence in insects, especially phytophagous beetles from the superfamilies Chrysomeloidea and Curculionoidea, has recently begun to be appreciated. The observed patchy distribution of endogenous genes encoding these enzymes in animals has raised questions about their evolutionary origins. Recent evidence suggests that endogenous plant cell wall degrading enzymes-encoding genes have been acquired by animals through a mechanism known as horizontal gene transfer (HGT). HGT describes how genetic material is moved by means other than vertical inheritance from a parent to an offspring. Here, we provide evidence that the mustard leaf beetle, Phaedon cochleariae, possesses in its genome genes encoding active xylanases from the glycoside hydrolase family 11 (GH11). We also provide evidence that these genes were originally acquired by P. cochleariae from a species of gammaproteobacteria through HGT. This represents the first example of the presence of genes from the GH11 family in animals.
Project description:BACKGROUND: The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. RESULTS: Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (?-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level. CONCLUSIONS: Combining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.
Project description:Horizontal gene transfer (HGT) has had major impacts on the biology of a wide range of organisms from antibiotic resistance in bacteria to adaptations to herbivory in arthropods. A growing body of literature shows that HGT between non-animals and animals is more commonplace than previously thought. In this study, we present a thorough investigation of HGT in the ctenophore Mnemiopsis leidyi. We applied tests of phylogenetic incongruence to identify nine genes that were likely transferred horizontally early in ctenophore evolution from bacteria and non-metazoan eukaryotes. All but one of these HGTs (an uncharacterized protein) are homologous to characterized enzymes, supporting previous observations that genes encoding enzymes are more likely to be retained after HGT events. We found that the majority of these nine horizontally transferred genes were expressed during development, suggesting that they are active and play a role in the biology of M. leidyi. This is the first report of HGT in ctenophores, and contributes to an ever-growing literature on the prevalence of genetic information flowing between non-animals and animals.
Project description:Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.
Project description:Xylophagous long-horned beetles thrive in challenging environments. To access nutrients, they secrete plant-cell-wall-degrading enzymes in their gut fluid; among them are cellulases of the subfamily?2 of glycoside hydrolase family?5 (GH5_2). Recently, we discovered that several beetle-derived GH5_2s use xylan as a substrate instead of cellulose, which is unusual for this family of enzymes. Here, we analyze the substrate specificity of a GH5_2 xylanase from the beetle Apriona japonica (AJAGH5_2-1) using commercially available substrates and synthetic arabinoxylan oligo- and polysaccharides. We demonstrate that AJAGH5_2-1 processes arabinoxylan polysaccharides in a manner distinct from classical xylanase families such as GH10 and GH11. AJAGH5_2-1 is active on long oligosaccharides and cleaves at the non-reducing end of a substituted xylose residue (position +1) only if: 1)?three xylose residues are present upstream and downstream of the cleavage site, and 2)?xylose residues at positions -1, -2, +2 and +3 are not substituted.
Project description:Although prevalent in prokaryotes, horizontal gene transfer (HGT) is rarer in multicellular eukaryotes. Bdelloid rotifers are microscopic animals that contain a higher proportion of horizontally transferred, non-metazoan genes in their genomes than typical of animals. It has been hypothesized that bdelloids incorporate foreign DNA when they repair their chromosomes following double-strand breaks caused by desiccation. HGT might thereby contribute to species divergence and adaptation, as in prokaryotes. If so, we expect that species should differ in their complement of foreign genes, rather than sharing the same set of foreign genes inherited from a common ancestor. Furthermore, there should be more foreign genes in species that desiccate more frequently. We tested these hypotheses by surveying HGT in four congeneric species of bdelloids from different habitats: two from permanent aquatic habitats and two from temporary aquatic habitats that desiccate regularly.Transcriptomes of all four species contain many genes with a closer match to non-metazoan genes than to metazoan genes. Whole genome sequencing of one species confirmed the presence of these foreign genes in the genome. Nearly half of foreign genes are shared between all four species and an outgroup from another family, but many hundreds are unique to particular species, which indicates that HGT is ongoing. Using a dated phylogeny, we estimate an average of 12.8 gains versus 2.0 losses of foreign genes per million years. Consistent with the desiccation hypothesis, the level of HGT is higher in the species that experience regular desiccation events than those that do not. However, HGT still contributed hundreds of foreign genes to the species from permanently aquatic habitats. Foreign genes were mainly enzymes with various annotated functions that include catabolism of complex polysaccharides and stress responses. We found evidence of differential loss of ancestral foreign genes previously associated with desiccation protection in the two non-desiccating species.Nearly half of foreign genes were acquired before the divergence of bdelloid families over 60 Mya. Nonetheless, HGT is ongoing in bdelloids and has contributed to putative functional differences among species. Variation among our study species is consistent with the hypothesis that desiccating habitats promote HGT.
Project description:BACKGROUND: Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. RESULTS: In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. CONCLUSIONS: The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples.
Project description:The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for 1% and 0.1% of the total non-rRNAs, respectively. The majority (98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.
Project description:About half of seaweed biomass is composed of polysaccharides. Most of these complex polymers have a marked polyanionic character. For instance, the red algal cell wall is mainly composed of sulfated galactans, agars and carrageenans, while brown algae contain alginate and fucose-containing sulfated polysaccharides (FCSP) as cell wall polysaccharides. Some marine heterotrophic bacteria have developed abilities to grow on such macroalgal polysaccharides. This is the case of Pseudoalteromonas carrageenovora 9T (ATCC 43555T), a marine gammaproteobacterium isolated in 1955 and which was an early model organism for studying carrageenan catabolism. We present here the genomic analysis of P. carrageenovora. Its genome is composed of two chromosomes and of a large plasmid encompassing 109 protein-coding genes. P. carrageenovora possesses a diverse repertoire of carbohydrate-active enzymes (CAZymes), notably specific for the degradation of macroalgal polysaccharides (laminarin, alginate, FCSP, carrageenans). We confirm these predicted capacities by screening the growth of P. carrageenovora with a large collection of carbohydrates. Most of these CAZyme genes constitute clusters located either in the large chromosome or in the small one. Unexpectedly, all the carrageenan catabolism-related genes are found in the plasmid, suggesting that P. carrageenovora acquired its hallmark capacity for carrageenan degradation by horizontal gene transfer (HGT). Whereas P. carrageenovora is able to use lambda-carrageenan as a sole carbon source, genomic and physiological analyses demonstrate that its catabolic pathway for kappa- and iota-carrageenan is incomplete. This is due to the absence of the recently discovered 3,6-anhydro-D-galactosidase genes (GH127 and GH129 families). A genomic comparison with 52 Pseudoalteromonas strains confirms that carrageenan catabolism has been recently acquired only in a few species. Even though the loci for cellulose biosynthesis and alginate utilization are located on the chromosomes, they were also horizontally acquired. However, these HGTs occurred earlier in the evolution of the Pseudoalteromonas genus, the cellulose- and alginate-related loci being essentially present in one large, late-diverging clade (LDC). Altogether, the capacities to degrade cell wall polysaccharides from macroalgae are not ancestral in the Pseudoalteromonas genus. Such catabolism in P. carrageenovora resulted from a succession of HGTs, likely allowing an adaptation to the life on the macroalgal surface.
Project description:Metabolic networks are responsible for many essential cellular processes, and exhibit a high level of evolutionary conservation from bacteria to eukaryotes. If genes encoding metabolic enzymes are horizontally transferred and are advantageous, they are likely to become fixed. Horizontal gene transfer (HGT) has played a key role in prokaryotic evolution and its importance in eukaryotes is increasingly evident. High levels of endosymbiotic gene transfer (EGT) accompanied the establishment of plastids and mitochondria, and more recent events have allowed further acquisition of bacterial genes. Here, we present the first comprehensive multi-species analysis of E/HGT of genes encoding metabolic enzymes from bacteria to unicellular eukaryotes.The phylogenetic trees of 2,257 metabolic enzymes were used to make E/HGT assertions in ten groups of unicellular eukaryotes, revealing the sources and metabolic processes of the transferred genes. Analyses revealed a preference for enzymes encoded by genes gained through horizontal and endosymbiotic transfers to be connected in the metabolic network. Enrichment in particular functional classes was particularly revealing: alongside plastid related processes and carbohydrate metabolism, this highlighted a number of pathways in eukaryotic parasites that are rich in enzymes encoded by transferred genes, and potentially key to pathogenicity. The plant parasites Phytophthora were discovered to have a potential pathway for lipopolysaccharide biosynthesis of E/HGT origin not seen before in eukaryotes outside the Plantae.The number of enzymes encoded by genes gained through E/HGT has been established, providing insight into functional gain during the evolution of unicellular eukaryotes. In eukaryotic parasites, genes encoding enzymes that have been gained through horizontal transfer may be attractive drug targets if they are part of processes not present in the host, or are significantly diverged from equivalent host enzymes.
Project description:To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6x His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis.