Metagenomic Clones Research Articles

Biochemical characterization of a SusD-like protein involved in β-1,3-glucan utilization by an uncultured cow rumen Bacteroides.

In ruminants, the rumen is a specialized stomach that is adapted to the breakdown of plant-derived complex polysaccharides through the coordinated activities of a diverse microbial community. Bacteroidota is a major phylum in this bovine rumen microbiota. They contain several clusters of genes called polysaccharide utilization loci (PULs) that encode proteins working in concert to capture, degrade, and transport polysaccharides. Despite the critical role of SusD-like proteins for efficient substrate transport, they remain largely unexplored. Here, we present the biochemical characterization of a SusD-like protein encoded by a β-glucan utilization locus from an Escherichia coli metagenomic clone previously isolated by functional screening of the bovine rumen microbiome. In this study, we show that clone 41O1 can grow on laminaritriose, cellotriose, and a mixture of cellobiosyl-cellobiose and glucosyl-cellotriose as sole carbon sources. Based on this, we used various in vitro analyses to investigate the binding ability of 41O1_SusD-like towards these oligosaccharides and the corresponding polysaccharides. We observed a clear binding affinity for β-1,6 branched β-1,3-glucans (laminarins, yeast β-glucan) and laminaritriose. Comparison of the AlphaFold2 model of 41O1_SusD-like with its closest structural homologs highlights a similar pattern of substrate recognition. In particular, three tryptophan residues are shown to be crucial for laminarin recognition. In the context of the cow rumen, we discuss the possible substrates targeted by the 41O1_PUL, such as the (1,3;1,4)-β-d-glucans present in cereal grains or the β-1,3- and (1,3;1,6)-β-d-glucans that are components of the cell wall of ruminal yeasts.IMPORTANCEThe rumen microbiota can majorly impact overall animal health, feed efficiency, and release of harmful substances into the environment. This microbiota is involved in the fermentation of organic matter to provide the host with valuable and assimilable nutrients. Bacteroidota efficiently captures, breaks down, and imports complex polysaccharides through the concerted action of proteins encoded by polysaccharide utilization loci (PULs). Within this system, SusD-like protein has proven necessary for the active internalization of the substrate. Nevertheless, the vast majority of SusD-like proteins characterized to date originate from cultured bacteria. With regard to the diversity and importance of uncultured bacteria in the rumen, further studies are required to better understand the role of polysaccharide utilization loci in ruminal polysaccharide degradation. Our detailed characterization of the 41O1_SusD-like therefore contributes to a better understanding of the carbohydrate metabolism of an uncultured Bacteroides from the cow rumen.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

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.

Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes.

Here, we outline how to identify hydrogenase enzymes from metagenomic fosmid libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis MR-1 (ΔhyaB) via triparental mating. If a fosmid clone exhibits hydrogen-uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. The screen enables screening of 48 metagenomic fosmid clones in parallel.

448 Isolation and identification of bioprospects capable of metabolizing 17-beta-estradiol and 17-alpha-ethinylestradiol using metagenomics and culture-dependent techniques in Puerto Rico

OBJECTIVES/GOALS: This research project aims to isolate and identify bioprospects capable of metabolizing estrogen using culture-dependent, culture independent methods and the identification of the gene/genes responsible for the metabolization of estrogen by the bioprospects. METHODS/STUDY POPULATION: For the culture dependent technique, samples were collected from the water treatment plant in Mayagüez, cultivated on TSA medium and selected specific and diverse colonies were patched on M-9NC (no carbon sources), M-9-glucose (M9G) and M-9-hormone mixture (M9H: 17-beta-Estradiol and 17-alpha-Ethynylestradiol). After the 48hrs incubation at 25 and 37 Celsius, growth was scored on the different media, to choose those potential bioprospects that use the hormones as the sole carbon source. For the culture independent approach, metagenomic clones from libraries generated from the Guajatacas water reservoir and Cabo Rojos Saltern microbial mats were cultivated on the appropriate M9NC, M-9G and M-9H, supplemented with Leucine and Thiamine for 48 hours at 37 Celsius. RESULTS/ANTICIPATED RESULTS: The culture-dependent approach showed a total of 6 potential bioprospects capable of growing on hormones as a sole carbon source on M9H at 25 and 37 Celsius and no-growth on the M-9 media used. Currently, we are in the process of determining the identities of the cultivable isolates genetically, any preference on hormone metabolization, monitoring a larger number of metagenomic clones and optimizing the selection conditions. DISCUSSION/SIGNIFICANCE: The identification of bioprospects capable of metabolizing hormones could potentially lead to the generation of new water treatments that could help remove unwanted chemicals, such as high concentrations of estrogens.

The complete genome sequence of a novel chrysovirus from the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana, an entomopathogenic fungus, is used for arthropod pest control worldwide. Here, we report the discovery and characterization of a novel double-stranded RNA (dsRNA) mycovirus, Beauveria bassiana chrysovirus 2 (BbCV-2), isolated from a Chinese B. bassiana strain. The genome sequence of the virus was determined by metagenomic sequencing, RT-PCR, and RACE cloning and was found to consist of four dsRNA segments that are 3441bp, 2779bp, 2925bp, and 2688bp long, respectively. Each dsRNA segment contains a single ORF. The ORF of dsRNA1 encodes a 1114-amino-acid (aa) protein (123.4kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif, the sequence of which showed the highest identity of only 16.13% to that of Beauveria bassiana chrysovirus-1 (BbCV-1). The ORF of dsRNA2 encodes an 805-aa coat protein (CP) (84.7kDa). The ORFs of dsRNAs 3 and 4 encodes proteins of undetermined function. The virus is a new member of the family Chrysoviridae from B. bassiana.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.

Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity.

A New High-Throughput Screening Method to Detect Antimicrobial Volatiles from Metagenomic Clone Libraries.

The ever-growing spread of resistance in medicine and agriculture highlights the need to identify new antimicrobials. Microbial volatile organic compounds (VOCs) are one of the most promising groups of chemicals to meet this need. These rarely exploited molecules exhibit antimicrobial activity and their high vapour pressure makes them ideal for application in surface sterilisation, and in particular, in biofumigation. Therefore, we adapted the previously developed Two Clamp VOCs Assay (TCVA) to a new high-throughput screening for the detection of novel antifungal VOCs from metagenomic clone libraries. As a proof of concept, we tested the new high-throughput TCVA (htTCVA) by sourcing a moss metagenomic library against Fusarium culmorum. This led to the identification of five clones that inhibited the growth of mycelium and spores in vitro by up to 8% and 30% and subsequently, to the identification of VOCs that are potentially, and in part responsible for the clones’ antifungal activity. For these VOCs, the in vitro effect of the pure compounds was as high as 100%. These results demonstrate the robustness and feasibility of the htTCVA, which provides access to completely new and unexplored biosynthetic pathways and their secondary metabolites.

Distribution and diversity of endophytic fungi in Gentiana rigescens and cytotoxic activities

ObjectiveIn the present study, Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts. Meanwhile, the identification and activity assays of the strains were also conducted. MethodsBy culture-dependent (endophytic fungi isolations from plant sections) and culture-independent (metagenomic library and cloning from plant sections) techniques, fungi communities were studied. The metagenomic library was generated using direct DNA isolation of whole plants, plant radixes, plant stems, plant leaves, plant flowers and soils around the plant. Meanwhile, endophytes were isolated from all parts of G. rigescens plants. After fermentation of the fungi isolations, all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines (HCT116, BEL7404, A549, MDA-MB-231). ResultsEventually, 200 strains were isolated and 103 strains were further identified through the internal transcribed spacer (ITS, ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4. A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units (OTU) were identified by 454 pyrosequencing. Through 454 pyrosequencing, the main fungal genera were Sebacina, Botrytis, Mycosphaerella, Boletus and Gibberella, and the major fungal genera which were directly isolated were Aspergillus, Fusarium, Penicillium and Alternaria. Activity assays showed strains 5–26 (Aspergillus sp.) and 6–2 (Fusarium avenaceum) had the outstanding cytotoxicity to all the tested cell lines with IC50 values <5 μg/mL. ConclusionThis study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G. rigescens by high-throughput 454 sequencing technology and fungi isolation methods. Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.

Biotechnological potential of mangrove sediments: Identification and functional attributes of thermostable and salinity-tolerant β-glucanase

Microorganisms native to mangroves are expected to contain enzymes capable of hydrolyzing different carbon sources. However, most of these microorganisms aren't cultivable; hence, alternative techniques as metagenomics are tools for studying and obtaining some of the natural genomes, genes and enzymes of biotechnological interest. The β-glucanase was produced using a metagenomic clone of mangrove sediments and detected by functional screening on carboxymethylcellulose substrate. The enzyme was purified by cation exchange chromatography. The peptides detected by mass spectrometry showed 20% identity with the polypeptide deduced from the genomic fragment sequenced. The ORF identified as BglfosD9 possessed 729 bp and the encoded protein showed predicted MW and pI of 28kD and 6.8, respectively. The enzyme was active in a wide range of pH (5–10) with optimum pH at 8, had relative activity greater than 50% at all temperatures tested (5–90 °C), was stable at temperatures of 5, 50 and 90 °C and showed excellent relative activity at high NaCl concentrations. This β-glucanase also showed high relative activity in the presence of SDS and it could hydrolyze β-glucan, CMC and Avicel as substrates. These findings support the idea of a new thermostable and active enzyme at basic pH from metagenomic library of mangrove sediment.

Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China.

In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.

Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments.

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

“Delineating bacterial community structure of rhizosphere metagenome from Kansal Forest”

Rhizosphere bacteria play crucial role in numerous biochemical and ecological processes ranging from biomass disintegration, mineralization, biological nitrogen fixation and de nitrification. The present investigation was carried out to delineate the microbial diversity of rhizosphere soil using metagenomic approach. Extraction of metagenomic DNA, PCR amplification and cloning of 16S rRNA genes resulted in library consisting of ~2000 clones. Sequencing of randomly selected 400 clones resulted in 352 unique sequences of 16S rRNA. Analysis of recovered sequences using BlastN (NCBI) revealed that ~47% of these sequences had uncultured representatives, uncultured Proteobacteria constituted ~8% of the total sequence diversity, uncultured acidobacteria comprised 5% of the total diversity among recovered clones. Other minor groups represent 2–5% of the total sequence diversity and include Bacillus and Pseudomonas . In addition, 1–2% of the total sequences also include uncultured Commensalibacter sp., Streptomyces sp., uncultured Gemmatimonadales , Halomonas (Gram-negative), uncultured Burkholderia sp., uncultured Pelomonas and other uncultured species. Phylogeny tree revealed that all sequence can be clustered into twelve main clusters indicating their diverse origin. Further clustering of these clones using QIIME resulted in 300 unique OTUs that constitutes 83.57% of the total sequence diversity, 52 clones were clustered as single OTU making a total 14.21%, while one sequence remain unclassified. At the Genus level, Bacillus, Pseudomonas and Staphylococcus demonstrated abundant presence in this soil. At species level Flavisolibacter tropicus of phylum Bacteriodetes showed maximum abundance followed by Bacillus cereus of phylum Firmicutes and Betaproteobacteria bacterium GR16-43 of phylum Proteobacteria.

Complete nucleotide sequence of a novel mycovirus from Trichoderma harzianum in China

A new mycovirus was identified in Trichoderma harzianum strain 137 isolated in Xinjiang province, China. The whole genome sequence of the mycovirus was determined by metagenomic sequencing, RT-PCR, and RACE cloning. The mycovirus contained two genomic segments. The first segment was 2088 bp long and contained a single ORF (ORF1) encoding the RNA-dependent RNA polymerase (RdRP) (72.26 kDa). The second segment was 1634 bp long and also contained a single ORF (ORF2) encoding a hypothetical protein of 37.472 kDa. We named this novel mycovirus “Trichoderma harzianum bipartite mycovirus 1” (ThBMV1). Phylogenetic analysis showed that ThBMV1 clusters with other unclassified dsRNA mycoviruses.

Abstract B066: Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics

Abstract The immune system, which is profoundly affected by the microbiota composition and activity, plays an anti-tumorigenic role by surveying for and killing cancer cells. Indeed, recent studies found the microbiota to be essential for the success of cancer immunotherapy treatments in mice, while the bacterial effectors remain unknown. We developed an innovative high throughput screening system to isolate microbiota-derived effectors that modulate the ability of immune cells to kill cancer cells. We utilize the mouse CT26 colon carcinoma cell line as targeT-cells and the GSW11 specific CCD2Z T-cell hybridoma cell line as the effector cells. Bacterial DNA extracted from fecal contents of mice bearing colorectal tumors served for generation of metagenomics libraries. Supernatnants of the metagenomic clones were added to the CT26-CCD2Z co-culture and CCD2Z activation was measured using a LacZ reporter, regulated by NFAT. We identified about 40 clones that activate CCD2Z cells. We performed secondary screens and tested the activation of CD8+ T-cells sorted from mouse spleens. We sequenced the metagenomic DNA of several clones and analyzed the coding sequences. Currently, we are sub-cloning the clones to find the microbial genes responsible for CD8+ T-cell activation and design strategies for isolation of active molecules from these bacterial supernatnats. Finally, we hope that our future results could be taken to the clinic and serve for diagnosis, prognosis and therapeutics. Citation Format: Lior Lobel, Wendy S. Garrett. Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B066.

Identification and characterization of a novel KG42 xylanase (GH10 family) isolated from the black goat rumen-derived metagenomic library

This study was conducted to isolate and functionally characterize a novel xylan-degrading enzyme from the microbial metagenomes of black goat rumens. A novel gene, KG42, was isolated from one of the 17 xylan-degrading metagenomic fosmid clones obtained from black goat rumens. The KG42 gene, comprising a 1107 bp open reading frame, encodes a protein composed of 368 amino acids (41 kDa) with a glycosyl hydrolase family 10 (GH10) domain, consisting of a “salad-bowl” shaped tertiary structure (a typical 8-fold α/β barrel (α/β)8) and two catalytic residues. KG42 xylanase protein has at best 40% sequence identity with other homologous GH10 xylanase proteins. The enzyme displayed its optimum activity at pH 5.0 and 50 °C. The enzyme was thermally stable at pH and temperature ranges of 5.0–10.0 and 20–60 °C, respectively. Substrate specificity and hydrolytic patterns implied that the KG42 xylanase functions as an endo-β-1,4-xylanase (EC 3.2.1.8). The KG42 xylanase was also used for the preparation of bifidogenic xylan hydrolysates, demonstrating its potential applications toward preparing prebiotic xylooligosaccharides.

Enzymatic characterization of a soluble aggregate induced by N-terminal extension to a lipolytic enzyme

A self-assembling peptide (27PEP) was isolated from an open reading frame (ORF). The ORF consisted of an unknown functional domain and a catalytic (lipolytic and phospholipolytic) domain (MPlaG) on metagenomic fosmid clone. This extension of 27 amino acids prior to the N-terminus of the catalytic domain (27PEP-MPlaG), starting at Met261, produced an aggregate of high molecular weight (> 700 kDa). Compared with MPlaG, 27PEP-MPlaG showed the same temperature and pH effect for maximum activity but was stable in the presence of inhibitors such as EDTA and PMSF. The 27PEP-MPlaG exhibited lower specific activity than that of MPlaG, but when pre-incubated for 30 min at temperatures between 4 and 100 °C, its activity increased at temperatures greater than 40 °C under alkaline conditions and eventually reached the specific activity level of MPlaG at 60 °C. We experimentally determined that the aggregate caused by 27PEP was dissociated at elevated temperatures resulting in an active catalytic monomer. The 27PEP-indued aggregation may be attractive as application tool for improving or engineering of biocatalysts and biomaterials.

Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community.

Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5–10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83) is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments.

The gut bacterium and pathobiont Bacteroides vulgatus activates NF-κB in a human gut epithelial cell line in a strain and growth phase dependent manner

The gut microbiota is increasingly implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) although the identity of the bacteria that underpin these diseases has remained elusive. The pathobiont Bacteroides vulgatus has been associated with both diseases although relatively little is known about how its growth and functional activity might drive the host inflammatory response. We identified an ATP Binding Cassette (ABC) export system and lipoprotein in B. vulgatus ATCC 8482 and B. vulgatus PC510 that displayed significant sequence similarity to an NF-κB immunomodulatory regulon previously identified on a CD-derived metagenomic fosmid clone. Interestingly, the ABC export system was specifically enriched in CD subjects suggesting that it may be important for colonization and persistence in the CD gut environment. Both B. vulgatus ATCC 8482 and PC510 activated NF-κB in a strain and growth phase specific manner in a HT-29/kb-seap-25 enterocyte like cell line. B. vulgatus ATCC 8482 also activated NF-κB in a Caco-2-NF-κBluc enterocyte like and an LS174T-NF-κBluc goblet cell like cell lines, and induced NF-κB-p65 subunit nuclear translocation and IL-6, IL-8, CXCL-10 and MCP-1 gene expression. Despite this, NF-κB activation was not coincident with maximal expression of the ABC exporter or lipoprotein in B. vulgatus PC510 suggesting that the regulon may be necessary but not sufficient for the immunomodulatory effects.

Synthesis and Physical Properties of Polyhydroxyalkanoate Polymers with Different Monomer Compositions by Recombinant Pseudomonas putida LS46 Expressing a Novel PHA SYNTHASE (PhaC116) Enzyme

A recombinant of Pseudomonas putida LS461 (deletion of the phaC1phaZphaC2 genes) was constructed by introducing cosmid JC123 carrying a novel phaC116 gene from a metagenomic clone. The resulting strain, P. putida LS46123, was able to synthesize polyhydroxyalkanoate (PHA) polymers with novel monomer compositions when cultured on glucose or free fatty acids, and accumulated PHAs from 9.24% to 27.09% of cell dry weight. The PHAs synthesized by P. putida LS46123 contained up to 50 mol % short chain length subunits (3-hydroxybutyrate and 3-hydroxyvalerate), with the remaining monomers consisting of various medium chain length subunits. The PhaC116 protein expressed by P. putida LS46123 had an amino acid sequence similarity of 45% with the PhaC1 protein of the parent strain, P. putida LS46. Predicted 3D structures of the PhaC116 proteins from P. putida LS46123 and P. putida LS46 revealed several differences in the numbers and locations of protein secondary structures. The physical and thermal properties of the novel polymers synthesized by P. putida LS46123 cultured with glucose or free fatty acids differed significantly from those produced by P. putida LS46 grown on the same substrates. PHA polymers with different subunit compositions, and hence different physical and thermal properties, can be tailor-made using novel PHA synthase for specific applications.