Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.     

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.     

A low-cost,high-throughput microfluidic nano-culture platform for functional metagenomics

Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries.     

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Biomining active cellulases from a mining bioremediation system

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.     

Construction, Analysis, and β-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for β-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with β-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Identification and characterization of metagenomic fragments from tidal flat sediment

Phylogenetic surveys based on cultivation-independent methods have revealed that tidal flat sediments are environments with extensive microbial diversity. Since most of prokaryotes in nature cannot be easily cultivated under general laboratory conditions, our knowledge on prokaryotic dwellers in tidal flat sediment is mainly based on the analysis of metagenomes. Microbial community analysis based on the 16S rRNA gene and other phylogenetic markers has been widely used to provide important information on the role of microorganisms, but it is basically an indirect means, compared with direct sequencing of metagenomic DNAs. In this study, we applied a sequence-based metagenomic approach to characterize uncultivated prokaryotes from tidal flat sediment. Two large-insert genomic libraries based on fosmid were constructed from tidal flat metagenomic DNA. A survey based on end-sequencing of selected fosmid clones resulted in the identification of clones containing 274 bacterial and 16 archaeal homologs in which majority were of proteobacterial origins. Two fosmid clones containing large metagenomic DNAs were completely sequenced using the shotgun method. Both DNA inserts contained more than 20 genes encoding putative proteins which implied their ecological roles in tidal flat sediment. Phylogenetic analyses of evolutionary conserved proteins indicate that these clones are not closely related to known prokaryotes whose genome sequence is known, and genes in tidal flat may be subjected to extensive lateral gene transfer, notably between domains Bacteria and Archaea. This is the first report demonstrating that direct sequencing of metagenomic gene library is useful in underpinning the genetic makeup and functional roles of prokaryotes in tidal flat sediments.     

Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences

The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [β-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the blast analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.     

Phylogenetic Analysis of Polyketide Synthase I Domains from Soil Metagenomic Libraries Allows Selection of Promising Clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Functional screening of a metagenomic library for genes involved in microbial degradation of aromatic compounds

A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive clones. Based on their substrate specificity for various catecholic compounds, 38 clones were subjected to sequence analysis. Each insert contained at least one EDO gene, and a total of 43 EDO genes were identified. More than half of these belonged to new EDO subfamilies: I.1.C (2 clones), I.2.G (20 clones), I.3.M (2 clones) and I.3.N (1 clone). The fact that novel I.2.G family genes were over-represented in these clones suggested that these genes play a specific role in environmental aromatic degradation. The I.2.G clones were further classified into six groups based on single-nucleotide polymorphisms (SNPs). Based on the combination of the SNPs, the evolutionary lineage of the genes was reconstructed; further, taking the activities of the clones into account, potential adaptive mutations were identified. The metagenomic approach was thus used to retrieve novel EDO genes as well as to gain insights into the gene evolution of EDOs.     

Metagenomic Guilt by Association: An Operonic Perspective

Next-generation sequencing projects continue to drive a vast accumulation of metagenomic sequence data. Given the growth rate of this data, automated approaches to functional annotation are indispensable and a cornerstone heuristic of many computational protocols is the concept of guilt by association. The guilt by association paradigm has been heavily exploited by genomic context methods that offer functional predictions that are complementary to homology-based annotations, thereby offering a means to extend functional annotation. In particular, operon methods that exploit co-directional intergenic distances can provide homology-free functional annotation through the transfer of functions among co-operonic genes, under the assumption that guilt by association is indeed applicable. Although guilt by association is a well-accepted annotative device, its applicability to metagenomic functional annotation has not been definitively demonstrated. Here a large-scale assessment of metagenomic guilt by association is undertaken where functional associations are predicted on the basis of co-directional intergenic distances. Specifically, functional annotations are compared within pairs of adjacent co-directional genes, as well as operons of various lengths (i.e. number of member genes), in order to reveal new information about annotative cohesion versus operon length. The results suggests that co-directional gene pairs offer reduced confidence for metagenomic guilt by association due to difficulty in resolving the existence of functional associations when intergenic distance is the sole predictor of pairwise gene interactions. However, metagenomic operons, particularly those with substantial lengths, appear to be capable of providing a superior basis for metagenomic guilt by association due to increased annotative stability. The need for improved recognition of metagenomic operons is discussed, as well as the limitations of the present work.     

Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach

Studies of the prevalence and identity of genes encoding resistance to antibiotics in a microbial community are usually carried out on only the cultivable members of the community. However, it is possible to include the as-yet-uncultivable organisms present by adopting a metagenomic approach to such studies. In this investigation, four metagenomic libraries of the oral microbiota were prepared from three groups of 20 adult humans and screened for antibiotic-resistant clones. Clones resistant to tetracycline and amoxycillin were present in all four libraries while gentamicin-resistant clones were found in three of the libraries. The genes encoding tetracycline resistance in the clones were identified and found to be tet(M), tet(O), tet(Q), tet(W), tet37 and tet(A). However, only the first three of these were detected in all three groups of individuals investigated.     

Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut

Background/Aim

The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota.

Methods

A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-κB binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn''s disease patients was performed to identify NF-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition.

Results

The two proinflammatory cytokines, TNF-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB.

Conclusions

We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.