Project description:Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides since they produce a vast variety of glycoside hydrolases. The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharide or glycans. This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares and microarrays were directly synthetized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, previously characterized by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota. The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems.
Project description:This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth of at least one of the two studied strains. Short-chain fatty acid (SCFA) analysis by HPLC corroborated the suitability of most of the studied novel oligosaccharides as growth substrates for the two bifidobacterial strains, showing that acetate is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and protein transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of β-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.
Project description:Understanding the biogeographical patterns and underlying drivers of microbial functional diversity is essential for anticipating climate change impacts on ecosystem functioning worldwide. However, this matter remains scarcely addressed in freshwater ecosystems. Using the high-throughput gene array GeoChip 4.0, we show that functional gene alpha diversity and compositon differ across mountains, alpha diversity declines towards high elevations and compositional turnover increases with larger elevational distances. Both continental- and mountain-scale patterns were primarily driven by climatic variables.
Project description:To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. Acid mine drainage (AMD) presents numerous problems to the aquatic life and surrounding ecosystems. However, little is known about the geographic distribution, diversity, composition, structure and function of AMD microbial communities. In this study, we analyzed the geographic distribution of AMD microbial communities from twenty sites using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, and the results showed that AMD microbial communities were geographically distributed and had high variations among different sites. Then an AMD-specific microarray was used to further analyze nine AMD microbial communities, and showed that those nine AMD microbial communities had high variations measured by the number of detected genes, overlapping genes between samples, unique genes, and diversity indices. Statistical analyses indicated that the concentrations of Fe, S, Ca, Mg, Zn, Cu and pH had strong impacts on both phylogenetic and functional diversity, composition, and structure of AMD microbial communities. This study provides insights into our understanding of the geographic distribution, diversity, composition, structure and functional potential of AMD microbial communities and key environmental factors shaping them. This study investigated the geographic distribution of Acid Mine Drainages microbial communities using a 16S rRNA gene-based RFLP method and the diversity, composition and structure of AMD microbial communities phylogenetically and functionally using an AMD-specific microarray which contained 1,072 probes ( 571 related to 16S rRNA and 501 related to functional genes). The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11).
Project description:Brown algae synthesize various polysaccharides that are ultimately catabolized by marine heterotrophic bacteria. Complex cell wall polysaccharides such as sulfated fucans are considered recalcitrant to microbial degradation and their pathways remain elusive. The branched structure of fucans sterically constraints enzyme-substrate interaction and also, fucan structure varies depending on algae and season challenging adaptation of microbial pathways. Here we show how Lentimonas specialized to overcome the complexity and diversity of sulfated fucans. The strain acquired a 0.9 mbp plasmid with over 200 glycoside hydrolases and sulfatases for the degradation of at least six different sulfated fucans. Per fucan, 100 enzymes are induced and we identified three structural types of fucans with similar pathways depending on their galactose, acetate and sulfate content. The highly decorated structure sulfated fucans expands the copy number and diversity of few key enzyme families, namely GH29, GH95, GH141 and sulfatases S1_15, S1_16 and S1_17. Those enzymes are co-regulated in large operons to step-wise degrade sulfated fucans. Fucose metabolism places additional burden as the conversion of toxic intermediates into lactate and propanediol occurs in a proteome-costly bacterial microcompartment. Through analyzing available genomes and metagenomes, we emphasize that Verrucomicrobia are abundant, yet specialized degraders of complex polysaccharides.
Project description:Distal gut bacteria play a pivotal role in the digestion of dietary polysaccharides by producing a large number of carbohydrate-active enzymes (CAZymes) that the host otherwise does not produce. We report here the design of a high density custom microarray that we used to spot non-redundant DNA probes for more than 6,500 genes encoding glycoside hydrolases and lyases selected from 174 reference genomes from distal gut bacteria. The custom microarray was tested and validated by the hybridization of bacterial DNA extracted from the stool samples of lean, obese and anorexic individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the finding that a gene encoding a GH6-family cellulase was present in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding CAZome core containing 46 families of glycoside hydrolases and polysaccharide lyases, which suggests the functional stability of the gut microbiota despite large taxonomical variations between individuals.
Project description:To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. Acid mine drainage (AMD) presents numerous problems to the aquatic life and surrounding ecosystems. However, little is known about the geographic distribution, diversity, composition, structure and function of AMD microbial communities. In this study, we analyzed the geographic distribution of AMD microbial communities from twenty sites using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, and the results showed that AMD microbial communities were geographically distributed and had high variations among different sites. Then an AMD-specific microarray was used to further analyze nine AMD microbial communities, and showed that those nine AMD microbial communities had high variations measured by the number of detected genes, overlapping genes between samples, unique genes, and diversity indices. Statistical analyses indicated that the concentrations of Fe, S, Ca, Mg, Zn, Cu and pH had strong impacts on both phylogenetic and functional diversity, composition, and structure of AMD microbial communities. This study provides insights into our understanding of the geographic distribution, diversity, composition, structure and functional potential of AMD microbial communities and key environmental factors shaping them.
Project description:The genome of the lignocellulose-degrading, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus encodes genes comprising clusters of glycoside hydrolases, ABC transporters and metabolic enzymes that are transcriptionally responsive to carbohydrates. Transcriptomic and biosolubilization analyses were used to determine if C. saccharolyticus could be deployed as a probe to assess the characteristics of plant biomass feedstocks and efficacy of pre-treatment methods, as these both relate to deconstruction strategies for biofuels production. Based on the response of C. saccharolyticus to plant cell wall polysaccharides, genomic loci were identified that reflected the availability of cellulose, glucomannan, pectin and xylan in biomass to microbial degradation. Furthermore, these loci were useful in assessing how various plant biomass feedstocks (genetically and chemically modified Populus sp., unpretreated Populus sp., and chemically modified switchgrass) were amenable C. saccharolyticus solubilization.