High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies.

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30-40 kb) genomic or metagenomic DNA.IMPORTANCELarge-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes.

It has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans.

BACKGROUND: Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. RESULTS: Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. CONCLUSION: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action.

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.

Gut bacteria responding to dietary change encode sialidases that exhibit preference for red meat-associated carbohydrates.

Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes and cancer1. In Western diets, red meat is a frequently eaten food2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can use free sialic acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13.

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156).

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.

Complete Genome Sequence of Kluyveromyces lactis Strain GG799, a Common Yeast Host for Heterologous Protein Expression.

We report the genome sequence of the dairy yeast Kluyveromyces lactis strain GG799 obtained using the Pacific Biosciences RS II platform. K. lactis strain GG799 is a common host for the expression of proteins at both laboratory and industrial scales.