dThe rapid rise in DNA sequencing has led to an expansion in the number of glycoside hydrolase (GH) families. The GH43 family currently contains ␣-L-arabinofuranosidase, ␤-D-xylosidase, ␣-L-arabinanase, and ␤-D-galactosidase enzymes for the debranching and degradation of hemicellulose and pectin polymers. Many studies have revealed finer details about members of GH43 that necessitate the division of GH43 into subfamilies, as was done previously for the GH5 and GH13 families. The work presented here is a robust subfamily classification that assigns over 91% of all complete GH43 domains into 37 subfamilies that correlate with conserved sequence residues and results of biochemical assays and structural studies. Furthermore, cooccurrence analysis of these subfamilies and other functional modules revealed strong associations between some GH43 subfamilies and CBM6 and CBM13 domains. Cooccurrence analysis also revealed the presence of proteins containing up to three GH43 domains and belonging to different subfamilies, suggesting significant functional differences for each subfamily. Overall, the subfamily analysis suggests that the GH43 enzymes probably display a hitherto underestimated variety of subtle specificity features that are not apparent when the enzymes are assayed with simple synthetic substrates, such as pNP-glycosides. Carbohydrates serve a range of functional purposes in biological systems, including energy storage, signal transduction, and intracellular trafficking, among others (1). Importantly, carbohydrates are the main end product of plant primary production, representing a large of majority of carbon fixation by plants (2). As a photosynthetically renewable form of fixed carbon, plant biomass represents a prime target for the replacement of petroleumderived fuels for future sustainability efforts. The enzymatic degradation and modification of carbohydrates have thus been cast to the forefront of biofuel production research (3).As functional efforts to discover plant cell wall polysaccharide (PCWP)-degrading enzymes identify novel activities and mechanisms (4, 5), it is important to derive and maintain a concise classification system for these enzymes. A sequence-based classification of carbohydrate-active enzymes (CAZymes) began in 1991 (6), with the classification of 35 families of glycoside hydrolases (GHs). Today the CAZy database (7) comprises 5 separate enzyme classes, namely, the aforementioned GHs, glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs), as well as associated carbohydrate binding modules (CBMs), that together correspond to over 530,000 individual sequences (at the time of submission of this article). The largest of these classes are the glycoside hydrolases, currently represented by over 241,000 sequences classified into 133 families based on amino acid sequence similarity.The rapid advancements in DNA sequencing over the past decade have exponentially increased the number of sequences assigned to each family. Henc...

Microbial communities drive biogeochemical cycles through networks of metabolite exchange that are structured along energetic gradients. As energy yields become limiting, these networks favor co-metabolic interactions to maximize energy disequilibria. Here we apply single-cell genomics, metagenomics, and metatranscriptomics to study bacterial populations of the abundant “microbial dark matter” phylum Marinimicrobia along defined energy gradients. We show that evolutionary diversification of major Marinimicrobia clades appears to be closely related to energy yields, with increased co-metabolic interactions in more deeply branching clades. Several of these clades appear to participate in the biogeochemical cycling of sulfur and nitrogen, filling previously unassigned niches in the ocean. Notably, two Marinimicrobia clades, occupying different energetic niches, express nitrous oxide reductase, potentially acting as a global sink for the greenhouse gas nitrous oxide.

Marine Group A (MGA) is a deeply branching and uncultivated phylum of bacteria. Although their functional roles remain elusive, MGA subgroups are particularly abundant and diverse in oxygen minimum zones and permanent or seasonally stratified anoxic basins, suggesting metabolic adaptation to oxygen-deficiency. Here, we expand a previous survey of MGA diversity in O2-deficient waters of the Northeast subarctic Pacific Ocean (NESAP) to include Saanich Inlet (SI), an anoxic fjord with seasonal O2 gradients and periodic sulfide accumulation. Phylogenetic analysis of small subunit ribosomal RNA (16S rRNA) gene clone libraries recovered five previously described MGA subgroups and defined three novel subgroups (SHBH1141, SHBH391, and SHAN400) in SI. To discern the functional properties of MGA residing along gradients of O2 in the NESAP and SI, we identified and sequenced to completion 14 fosmids harboring MGA-associated 16S RNA genes from a collection of 46 fosmid libraries sourced from NESAP and SI waters. Comparative analysis of these fosmids, in addition to four publicly available MGA-associated large-insert DNA fragments from Hawaii Ocean Time-series and Monterey Bay, revealed widespread genomic differentiation proximal to the ribosomal RNA operon that did not consistently reflect subgroup partitioning patterns observed in 16S rRNA gene clone libraries. Predicted protein-coding genes associated with adaptation to O2-deficiency and sulfur-based energy metabolism were detected on multiple fosmids, including polysulfide reductase (psrABC), implicated in dissimilatory polysulfide reduction to hydrogen sulfide and dissimilatory sulfur oxidation. These results posit a potential role for specific MGA subgroups in the marine sulfur cycle.

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Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.

The North American beaver (Castor canadensis) has long been considered an engineering marvel, transforming landscapes and shaping biological diversity through its dam building behavior. While the beaver possesses conspicuous morphological features uniquely adapted for the use of woody plants as construction materials and dietary staples, relatively little is known about the specialized microorganisms inhabiting the beaver gastrointestinal tract and their functional roles in determining host nutrition. Here we use a combination of shotgun metagenomics, functional screening and carbohydrate biochemistry to chart the community structure and metabolic power of the beaver fecal microbiome. We relate this information to the metabolic capacity of other wood feeding and hindgut fermenting organisms and profile the functional repertoire of glycoside hydrolase (GH) families distributed among and between population genome bins. Metagenomic screening revealed novel mechanisms of xylan oligomer degradation involving GH43 enzymes from uncharacterized subfamilies and divergent polysaccharide utilization loci, indicating the potential for synergistic biomass deconstruction. Together, these results open a functional metagenomic window on less conspicuous adaptations enabling the beaver microbiome to efficiently convert woody plants into host nutrition and point toward rational design of enhanced enzyme mixtures for biorefining process streams.

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Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1 . Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2 , buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5 ). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6 . Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9 . Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10 . The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system. ProtocolBefore starting this protocol, you will need your metagenomic library stored in a 384 well plate format. In our study, we used the pCC1 copy control fosmid vector in combination with phage T1-resistant TransforMax EPI300-T1 R E. coli cells as the library host and stored our plates at -80°C 11 . 3. Prepare LB broth with chloramphenicol at a final concentration of 12.5ug/mL and arabinose at 100ug/mL in a 500mL reagent bottle. Each plate will use approximately 20mL, plus make an additional 50mL to allow for dead volume. 4. Set up the qFill3 as per manufacturer's instructions with the media bottle attached to the manifold via the sterile tubing. Program it for the appropriate amount of media, and set it to fill a 45uL volume in each well. 5. Purge the air from the tubing and manifold using the purge feature of the robot until media is visible coming from each pin of the manifold. 6. Fill the desired number of plates with LB media using the qFill3. Each plate takes approximately 20 seconds to fill. 7. Load the library plates and the fresh plates into the appropriate areas of the qPix2 robot. Fill the cleaning baths with the appropriate reagents; 2% Micro90 in the rear bath, autoclaved distilled water in the middle bath, 80% ethano...