Analysis of the microbial gene landscape and transcriptome for aromatic pollutants and alkane degradation using a novel internally calibrated microarray system.

Despite various efforts to develop tools to detect and compare the catabolic potential and activity for pollutant degradation in environmental samples, there is still a need for an open-source, curated and reliable array method. We developed a custom array system including a novel normalization strategy that can be applied to any microarray design, allowing the calculation of the reliability of signals and make cross-experimental comparisons. Array probes, which are fully available to the scientific community, were designed from knowledge-based curated databases for key aromatic catabolic gene families and key alkane degradation genes. This design assigns signals to the respective protein subfamilies, thus directly inferring function and substrate specificity. Experimental procedures were optimized using DNA of four genome sequenced biodegradation strains and reliability of signals assessed through a novel normalization procedure, where a plasmid containing four artificial targets in increased copy numbers and co-amplified with the environmental DNA served as an internal calibration curve. The array system was applied to assess the catabolic gene landscape and transcriptome of aromatic contaminated environmental samples, confirming the abundance of catabolic gene subfamilies previously detected by functional metagenomics but also revealing the presence of previously undetected catabolic groups and specifically their expression under pollutant stress.

Diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species.

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-beta-glucanases, beta-glucosidases, beta-cellobiohydrolases, beta-galactosidase, and beta-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of beta-galactosidases/alpha-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional ß-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit ß-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, ß-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, ß-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.

Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

Reactome array: forging a link between metabolome and genome.

We describe a sensitive metabolite array for genome sequence-independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.