Nonribosomal Peptide Synthetases in Animals.

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize a range of bio-active secondary metabolites including antibiotics and siderophores. They are widespread among both prokaryotes and eukaryotes but are considered rare among animals. Recently, several novel NRPS genes have been described in nematodes, schistosomes, and arthropods, which led us to investigate how prevalent NRPS genes are in the animal kingdom. We screened 1059 sequenced animal genomes and showed that NRPSs were present in 7 out of the 19 phyla analyzed. A phylogenetic analysis showed that the identified NRPSs form clades distinct from other adenylate-forming enzymes that contain similar domains such as fatty acid synthases. NRPSs show a remarkably scattered distribution over the animal kingdom. They are especially abundant in rotifers and nematodes. In rotifers, we found a large variety of domain architectures and predicted substrates. In the nematode Plectus sambesii, we identified the beta-lactam biosynthesis genes L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase that catalyze the formation of beta-lactam antibiotics in fungi and bacteria. These genes are also present in several species of Collembola, but not in other hexapods analyzed so far. In conclusion, our survey showed that NRPS genes are more abundant and widespread in animals than previously known.

Moderate and transient impact of antibiotic use on the gut microbiota in a rural Vietnamese cohort.

The human gut microbiota has been shown to be significantly perturbed by antibiotic use, while recovering to the pre-treatment state several weeks after short antibiotic exposure. The effects of antibiotics on the gut microbiota have however been mainly documented in high-income settings with lower levels of antibiotic resistance as compared to lower and middle income countries (LMIC). This study aimed to examine the long-term consequences of repeated exposure to commonly use antibiotics on the fecal microbiota of residents living in a low income setting with high prevalence of antibiotic resistance. Fecal samples from household individuals (n = 63) participating in a rural cohort in northern Vietnam were collected monthly for a period of 6 months. Using 16S V4 rRNA gene region amplicon sequencing and linear mixed-effects models analysis, we observed only a minor and transient effect of antibiotics on the microbial richness (ß = - 31.3, 95%CI = - 55.3, - 7.3, p = 0.011), while the microbial diversity was even less affected (ß = - 0.298, 95%CI - 0.686, 0.090, p = 0.132). Principal Component Analyses (PCA) did not reveal separation of samples into distinct microbiota-based clusters by antibiotics use, suggesting the microbiota composition was not affected by the antibiotics commonly used in this population. Additionally, the fecal microbial diversity of the subjects in our study cohort was lower when compared to that of healthy Dutch adults (median 3.95 (IQR 3.72-4.13) vs median 3.69 (IQR3.31-4.11), p = 0.028, despite the higher dietary fiber content in the Vietnamese as compared to western diet. Our findings support the hypothesis that frequent antibiotic exposure may push the microbiota to a different steady state that is less diverse but more resilient to disruption by subsequent antibiotic use.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.

Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats' Rumen by Metagenomic Deep Sequencing.

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats' rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5-6.5, temperature 40-50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

Antimicrobial activity and carbohydrate metabolism in the bacterial metagenome of the soil-living invertebrate Folsomia candida.

The microbiome associated with an animal's gut and other organs is considered an integral part of its ecological functions and adaptive capacity. To better understand how microbial communities influence activities and capacities of the host, we need more information on the functions that are encoded in a microbiome. Until now, the information about soil invertebrate microbiomes is mostly based on taxonomic characterization, achieved through culturing and amplicon sequencing. Using shotgun sequencing and various bioinformatics approaches we explored functions in the bacterial metagenome associated with the soil invertebrate Folsomia candida, an established model organism in soil ecology with a fully sequenced, high-quality genome assembly. Our metagenome analysis revealed a remarkable diversity of genes associated with antimicrobial activity and carbohydrate metabolism. The microbiome also contains several homologs to F. candida genes that were previously identified as candidates for horizontal gene transfer (HGT). We suggest that the carbohydrate- and antimicrobial-related functions encoded by Folsomia's metagenome play a role in the digestion of recalcitrant soil-born polysaccharides and the defense against pathogens, thereby significantly contributing to the adaptation of these animals to life in the soil. Furthermore, the transfer of genes from the microbiome may constitute an important source of new functions for the springtail.

Metagenomic insights into lignocellulose-degrading genes through Illumina-based de novo sequencing of the microbiome in Vietnamese native goats' rumen.

The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats' rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes' modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter.

Metagenomic analysis of bacterial community structure and diversity of lignocellulolytic bacteria in Vietnamese native goat rumen.

OBJECTIVE: In a previous study, analysis of Illumina sequenced metagenomic DNA data of bacteria in Vietnamese goats' rumen showed a high diversity of putative lignocellulolytic genes. In this study, taxonomy speculation of microbial community and lignocellulolytic bacteria population in the rumen was conducted to elucidate a role of bacterial structure for effective degradation of plant materials. METHODS: The metagenomic data had been subjected into Basic Local Alignment Search Tool (BLASTX) algorithm and the National Center for Biotechnology Information non-redundant sequence database. Here the BLASTX hits were further processed by the Metagenome Analyzer program to statistically analyze the abundance of taxa. RESULTS: Microbial community in the rumen is defined by dominance of Bacteroidetes compared to Firmicutes. The ratio of Firmicutes versus Bacteroidetes was 0.36:1. An abundance of Synergistetes was uniquely identified in the goat microbiome may be formed by host genotype. With regard to bacterial lignocellulose degraders, the ratio of lignocellulolytic genes affiliated with Firmicutes compared to the genes linked to Bacteroidetes was 0.11:1, in which the genes encoding putative hemicellulases, carbohydrate esterases, polysaccharide lyases originated from Bacteroidetes were 14 to 20 times higher than from Firmicutes. Firmicutes seem to possess more cellulose hydrolysis capacity showing a Firmicutes/Bacteroidetes ratio of 0.35:1. Analysis of lignocellulolytic potential degraders shows that four species belonged to Bacteroidetes phylum, while two species belonged to Firmicutes phylum harbouring at least 12 different catalytic domains for all lignocellulose pretreatment, cellulose, as well as hemicellulose saccharification. CONCLUSION: Based on these findings, we speculate that increasing the members of Bacteroidetes to keep a low ratio of Firmicutes versus Bacteroidetes in goat rumen has resulted most likely in an increased lignocellulose digestion.

Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida.

BACKGROUND: Folsomia candida is a model in soil biology, belonging to the family of Isotomidae, subclass Collembola. It reproduces parthenogenetically in the presence of Wolbachia, and exhibits remarkable physiological adaptations to stress. To better understand these features and adaptations to life in the soil, we studied its genome in the context of its parthenogenetic lifestyle. RESULTS: We applied Pacific Bioscience sequencing and assembly to generate a reference genome for F. candida of 221.7 Mbp, comprising only 162 scaffolds. The complete genome of its endosymbiont Wolbachia, was also assembled and turned out to be the largest strain identified so far. Substantial gene family expansions and lineage-specific gene clusters were linked to stress response. A large number of genes (809) were acquired by horizontal gene transfer. A substantial fraction of these genes are involved in lignocellulose degradation. Also, the presence of genes involved in antibiotic biosynthesis was confirmed. Intra-genomic rearrangements of collinear gene clusters were observed, of which 11 were organized as palindromes. The Hox gene cluster of F. candida showed major rearrangements compared to arthropod consensus cluster, resulting in a disorganized cluster. CONCLUSIONS: The expansion of stress response gene families suggests that stress defense was important to facilitate colonization of soils. The large number of HGT genes related to lignocellulose degradation could be beneficial to unlock carbohydrate sources in soil, especially those contained in decaying plant and fungal organic matter. Intra- as well as inter-scaffold duplications of gene clusters may be a consequence of its parthenogenetic lifestyle. This high quality genome will be instrumental for evolutionary biologists investigating deep phylogenetic lineages among arthropods and will provide the basis for a more mechanistic understanding in soil ecology and ecotoxicology.