Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N′-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

Midgut transcriptome assessment of the cockroach-hunting wasp Ampulex compressa (Apoidea: Ampulicidae)

The emerald jewel wasp Ampulex compressa (Hymenoptera: Ampulicidae) is a solitary wasp that is widely known for its specialized hunting of cockroaches as larvae provision. Adult wasps mainly feed on pollen and nectar, while their larvae feed on the cockroachs’ body, first as ecto- and later as endoparsitoids. Little is known about the expression of digestive, detoxification and stress-response-related genes in the midgut of A. compressa, or about its transcriptional versatility between life stages. To identify gut-biased genes related to digestion, detoxification, and stress response, we explored the midgut transcriptome of lab-reared A. compressa, for both adults and larvae, by focusing on the top 100 significantly up- and down-regulated genes. From the top 100 significantly differentially expressed genes (DEGs), we identified 39 and 36 DEGs putatively related to digestion and detoxification in the adult wasps and larvae, respectively. The two carbohydrases alpha-glucosidase (containing an alpha-amylase domain) and glycosyl hydrolase family 31, as well as the two proteinases chymotrypsin and trypsin, revealed the highest gene diversity. We identified six significant DEGs related to detoxification, which comprise glutathione S-transferase, cytochrome P450s and UDP-glucuronosyltransferase. The gene expression levels that were significantly expressed in both life stages vary strongly between life stages, as found in genes encoding for chymotrypsin and trypsin or glycosyl hydrolases family 31. The number of genes related to alpha-glucosidase, glycosyl hydrolase family 31, and cytochrome P450s was found to be similar across nine reference hymenopteran species, except for the identified glycosyl hydrolase family 31 gene, which was absent in all reference bee species. Phylogenetic analyses of the latter candidate genes revealed that they cluster together with their homologous genes found in the reference hymenopteran species. These identified candidate genes provide a basis for future comparative genomic and proteomic studies on (ontogenetic) dietary transitions in Hymenoptera.

Expression, Characterization and Its Deinking Potential of a Thermostable Xylanase From Planomicrobium glaciei CHR43

Genome mining is more and more widely used in identifying new enzymes from database. In the present study, we reported a putative xylanase, Pg-Xyn (WP_053166147.1), which originated from a psychrotolerant strain Planomicrobium glaciei CHR 43, and was identified from Genbank by genome mining. Sequence analysis and homology modeling showed that Pg-Xyn belongs to glycosyl hydrolase family 10. On the basis of heterologous expression in E. coli and biochemical characterization, we found Pg-Xyn was most active at pH 9.0 and 80°C and exhibited good stability from pH 5.0 to 12.0 and below 90°C. Pg-Xyn was slightly activated in the presence of Ca2+ and Mg2+, while it was strongly inhibited by Mn2+. The analysis of hydrolysis products showed that Pg-Xyn was an endo-β-1,4-xylanase. In addition, Pg-Xyn performed good deinking ability in a paper deinking test. In consideration of its unique properties, Pg-Xyn might be a promising candidate for application in the paper and pulp industries.

Genome Assembly and Analyses of the Macrofungus Macrocybe gigantea

Macrocybe gigantea (M. gigantea) is a macrofungus genus that contains a big number of fairly fleshy gilled mushrooms with white spores. This macrofungus produces diverse bioactive compounds, antioxidants, and water-soluble polysaccharides. However, the genomic resources of this species remain unknown. Here, we assembled the genome of M. gigantea (41.23 Mb) into 336 scaffolds with a N50 size of 374,455 bp and compared it with the genomes of eleven other macrofungi. Comparative genomics study confirmed that M. gigantea belonged to the Macrocybe genus, a stand-alone genus different from the Tricholoma genus. In addition, we found that glycosyl hydrolase family 28 (GH28) in M. gigantea shared conserved motifs that were significantly different from their counterparts in Tricholoma. The genomic resource uncovered by this study will enhance our understanding of fungi biology, especially the differences in their growth rates and energy metabolism.

Phylogenetic Analyses of Glycosyl Hydrolase Family 6 Genes in Tunicates: Possible Horizontal Transfer

Horizontal gene transfer (HGT) is the movement of genetic material between different species. Although HGT is less frequent in eukaryotes than in bacteria, several instances of HGT have apparently shaped animal evolution. One well-known example is the tunicate cellulose synthase gene, CesA, in which a gene, probably transferred from bacteria, greatly impacted tunicate evolution. A Glycosyl Hydrolase Family 6 (GH6) hydrolase-like domain exists at the C-terminus of tunicate CesA, but not in cellulose synthases of other organisms. The recent discovery of another GH6 hydrolase-like gene (GH6-1) in tunicate genomes further raises the question of how tunicates acquired GH6. To examine the probable origin of these genes, we analyzed the phylogenetic relationship of GH6 proteins in tunicates and other organisms. Our analyses show that tunicate GH6s, the GH6-1 gene, and the GH6 part of the CesA gene, form two independent, monophyletic gene groups. We also compared their sequence signatures and exon splice sites. All tunicate species examined have shared splice sites in GH6-containing genes, implying ancient intron acquisitions. It is likely that the tunicate CesA and GH6-1 genes existed in the common ancestor of all extant tunicates.