Functional Metagenomic Approach Research Articles

Cat and dog feces as reservoirs of diverse novel antibiotic resistance genes

Companion animals have the potential to greatly enhance the physical and mental health of humans, thus leading to an increased focus on the interactions between humans and pets. Currently, the inappropriate and excessive utilization of antimicrobial agents has become prevalent in veterinary clinical practice for pets. This antibiotic contamination phenomenon has a profound impact on the enrichment of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in pets. However, the pet-associated resistome, especially the novel ARGs in pets, represents a relatively neglected area. In this study, we successfully constructed a total of 12 libraries using the functional metagenomics approach to assess the diversity of ARGs in pet cats and dogs from four pet hospitals. Through the integration of functional screening and high-throughput sequencing, a total of 122 antibiotic resistance determinants were identified, of which 15 were classified as putative novel ARGs originating from five classes. Functional assessment demonstrated that 6 novel ARGs including one β-lactam, two macrolides, two aminoglycosides, and one rifamycin (RIF), namely blaPF, ermPF, msrPF, aac(6′)PF, aph(3′)PF, and arrPF, exhibited functionally activity in conferring bacterial phenotypic resistance by increasing the minimum inhibitory concentrations (MICs) with a 4- to 128-fold. Genetic context analysis demonstrated that, with the exception of aac(6′)PF and arrPF, the remaining four novel ARGs were found adjacent to mobile genetic elements (MGEs) including IS elements or transposases, which provided a prerequisite for horizontal transfer of these novel ARGs, thereby offering an explanation for their detection in diverse samples collected from various sampling sites. The current study has unveiled the significant role of cat and dog feces as one source of reservoirs of diverse novel ARGs, while also highlighting the potential adverse consequences of their further spread to medically significant pathogens and human commensal organisms.

Functional Metagenomics Approach for Discovery of Novel Cold-Active Protease from the Antarctic Region

Functional Metagenomics Approach for Discovery of Novel Cold-Active Protease from the Antarctic Region

Potential for functional divergence in ectomycorrhizal fungal communities across a precipitation gradient.

Functional traits influence the assembly of microbial communities, but identifying these traits in the environment has remained challenging. We studied ectomycorrhizal fungal (EMF) communities inhabiting Populus trichocarpa roots distributed across a precipitation gradient in the Pacific Northwest, USA. We profiled these communities using taxonomic (meta-barcoding) and functional (metagenomic) approaches. We hypothesized that genes involved in fungal drought-stress tolerance and fungal mediated plant water uptake would be most abundant in drier soils. We were unable to detect support for this hypothesis; instead, the abundance of genes involved in melanin synthesis, hydrophobins, aquaporins, trehalose-synthases, and other gene families exhibited no significant shifts across the gradient. Finally, we studied variation in sequence homology for certain genes, finding that fungal communities in dry soils are composed of distinct aquaporin and hydrophobin gene sequences. Altogether, our results suggest that while EMF communities exhibit significant compositional shifts across this gradient, coupled functional turnover, at least as inferred using community metagenomics is limited. Accordingly, the consequences of these distinct EMF communities on plant water uptake remain critically unknown, and future studies targeting the expression of genes involved in drought stress tolerance are required.

Identification of Cutinolytic Esterase from Microplastic-Associated Microbiota Using Functional Metagenomics and Its Plastic Degrading Potential.

Plastic pollution has threatened biodiversity and human health by shrinking habitats, reducing food quality, and limiting the activities of organisms. Therefore, global interest in discovering novel enzymes capable of degrading plastics has increased considerably. Within this context, the functional metagenomic approach, which allows for unlocking the functional potential of uncultivable microbial biodiversity, was used to discover a plastic-degrading enzyme. First, metagenomic libraries derived from microplastic-associated microbiota were screened for esterases capable of degrading both tributyrin and polycaprolactone. Clone KAD01 produced esterase highly active against p-nitrophenyl esters (C2-C16). The gene corresponding to the enzyme activity showed moderate identity (≤ 55.94%) to any known esterases/cutinases. The gene was extracellularly expressed with a 6× histidine tag in E. coli BL21(DE3), extracellularly. Titer of the enzyme (CEstKAD01) was raised from 21.32 to 35.17U/mL by the statistical optimization of expression conditions and media components. CEstKAD01 was most active at pH 7.0 and 30°C. It was noteworthy stable over a wide pH (6.0-10.0) and temperature (20-50°C). The enzyme was active and stable in elevated NaCl concentrations up to 12% (w/v). Pre-incubation of CEstKAD01 with Mg2+, Mn2+, and Ca2+ increased the enzyme activity. CEstKAD01 displayed an excellent tolerance against various chemicals and solvents. It was determined that 1mg of the enzyme caused the release of 5.39 ± 0.18mM fatty acids from 1g apple cutin in 120min. Km and Vmax values of CEstKAD01 against p-nitrophenyl butyrate were calculated to be 1.48mM and 20.37µmol/min, respectively. The enzyme caused 6.94 ± 0.55, 8.71 ± 0.56, 7.47 ± 0.47, and 9.22 ± 0.18% of weight loss in polystyrene, high-density polyethylene, low-density polyethylene, and polyvinyl chloride after 30-day incubation. The scanning electron microscopy (SEM) analysis indicated the formation of holes and pits on the plastic surfaces supporting the degradation. In addition, the change in chemical structure in plastics treated with the enzyme was determined by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Finally, the degradation products were found to have no genotoxic potential. To our knowledge, no cutinolytic esterase with the potential to degrade polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyvinyl chloride (PVC) has been identified from metagenomes derived from microplastic-associated microbiota.

Functional metagenomics reveals wildlife as natural reservoirs of novel β-lactamases

The antibiotic resistances in bacteria are believed to rapidly evolve over time in the anthropogenic environments which enriched with selection pressures. However, the knowledge regarding the development of antibiotic resistance in wildlife and their habitats is scarce. It is, therefore, of great interest and significance to unveil the yet-unknown antibiotic resistances in wildlife in accordance with One Health concept. To this end, we analyzed the samples taken from wildlife and surrounding environments using a functional metagenomics approach. By functional screening in combination with Illumina sequencing, a total of 32 candidate genes which encoding putative novel β-lactamase were identified. These putative β-lactamase were taxonomically assigned into bacteria of 23 genera from 7 phyla, where Proteobacteria, Actinobacteria and Firmicutes were dominant. The following functional assessment demonstrated that 4 novel β-lactamases, namely blaSSA, blaSSB1, blaSSB2 and blaSSD, were functionally active to confer the phenotypical resistance to bacteria by increasing MICs up to 128-fold. Further analysis indicated that the novel β-lactamases identified in the current study were able to hydrolyze a broad spectrum of β-lactams including cephalosporins, and they were genetically unique comparing with known β-lactamases. The plausible transmission of some novel β-lactamase genes was supported by our results as the same gene was detected in different samples from different sites. This study shed the light on the active role of wildlife and associated environments as natural reservoirs of novel β-lactamases, implying that the antibiotic resistances might evolve in absence of selection pressure and threaten public health once spread into clinically important pathogens.

Functional metagenomic libraries generated from anthropogenically impacted environments reveal importance of metabolic genes in biocide and antibiotic resistance

Anthropogenic activities result in the release of antimicrobial resistant bacteria and a cocktail of antimicrobial compounds into the environment that may directly select or indirectly co-select for antimicrobial resistance (AMR). Many studies use metagenome sequencing or qPCR-based approaches to study the environmental resistome but these methods are limited by a priori knowledge. In this study, a functional metagenomic approach was used to explore biocide resistance mechanisms in two contaminated environments and a pristine site, and to identify whether potentially novel genes conferring biocide resistance also conferred resistance or reduced susceptibility to antibiotics. Resistance was predominately mediated through novel mechanisms exclusive of the well-known qac efflux genes. UDP-galactose 4-epimerase (galE) –like genes were identified in both contaminated environments and were shown to confer cross-resistance to biocides and clinically important antibiotics for the first time (to our knowledge), compared to knockout mutants. GalE -like genes were also co-located with transposons, suggesting mobilisation potential. These results show that housekeeping genes may play a significant yet underappreciated role in AMR in environmental microbiomes.

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics.

Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining high-throughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion.

Functional Metagenomics Approach for the Discovery of Novel Genes Encoding Phosphatase Activity.

Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.

Functional metagenomic screening identifies an unexpected β-glucuronidase.

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added β-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently 'unpredictable' by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

ObjectiveMany sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice.Materials and methodsAs a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed.ResultsScreening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively.ConclusionMultiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field.

Ascomycetes versus Spent Mushroom Substrate in Mycoremediation of Dredged Sediments Contaminated by Total Petroleum Hydrocarbons: The Involvement of the Bacterial Metabolism

Two mycoremediation approaches for the depletion of the total petroleum hydrocarbons in dredged sediments were compared: co-composting with spent mushroom substrate (SMS) from Pleurotus ostreatus and bioaugmentation with Lambertella sp. MUT 5852, an ascomycetes autochthonous to the sediment, capable of utilizing diesel oil its sole carbon source. After 28 days of incubation, 99% depletion was observed in presence of Lambertella sp. MUT 5852. No total petroleum hydrocarbon depletion was observed in sediment co-composting with the SMS after 60 days of incubation. 16S rDNA metabarcoding of the bacterial community was performed to evaluate the potential synergism between fungi and bacteria in the bioremediation process. The functional metagenomic prediction approach indicated that the biodiversity of the bacterial genera potentially involved in the degradation of TPH was higher in sediment bioaugmented with Lambertella sp. MUT 5852, which resulted in being mandatory for TPH depletion. Mechanisms of co-substrate inhibition of the hydrocarburoclastic bacterial species, due to the bioavailable organic matter of the SMS, are suggested to be involved in the observed kinetics of TPH depletion, failing in the case of SMS and successful in the case of Lambertella sp. MUT 5852.

Shape-function of a novel metapyrocatechase, RW4-MPC: Metagenomics to SAXS data based insight into deciphering regulators of function

The oxygenases have attracted considerable attention in enzyme-mediated bioremediation of xenobiotic compounds due to their high specificity, cost-effectiveness, and targeted field applications. Here, we performed a functional metagenomics approach to cope with culturability limitations to isolate a novel extradiol dioxygenase. Fosmid clone harboring dioxygenase gene was sequenced and analyzed by bioinformatics tools. One ring-cleaving dioxygenase RW4-MPC (metapyrocatechase) was purified and characterized to examine its degradation efficiency. The RW4-MPC was significantly active in the temperature and pH range of 5 to 40 °C, and 7–10, respectively, with an optimum temperature of 25 °C and pH 8. To gain insight into observed differential activity, Small-Angle X-ray Scattering (SAXS) data of the protein samples were analyzed, which brought forth that the RW4-MPC molecules form tight globular tetramers in solution. This native association was stable till 35 °C, and protein started to associate at higher temperatures, explaining heat-induced loss of function. Similarly, RW4-MPC aggregated or lost globular profile below pH 7 or at pH 10, respectively. The kinetic parameters showed the six folds high catalytic efficiency of RW4-MPC towards 2,3-dihydroxy biphenyl than catechol and its derivatives. RW4-MPC molecules showed remarkable retention of functionality in hypersaline conditions with more than 70% activity in a buffer having 3 M NaCl concentration. In concordance, SAXS data analysis showed retention of functional shape profile in hypersaline conditions. The halotolerant and oxygen insensitive nature of this enzyme makes it a potential candidate for bioremediation.

Mining for Perchlorate Resistance Genes in Microorganisms From Sediments of a Hypersaline Pond in Atacama Desert, Chile.

Perchlorate is an oxidative pollutant toxic to most of terrestrial life by promoting denaturation of macromolecules, oxidative stress, and DNA damage. However, several microorganisms, especially hyperhalophiles, are able to tolerate high levels of this compound. Furthermore, relatively high quantities of perchlorate salts were detected on the Martian surface, and due to its strong hygroscopicity and its ability to substantially decrease the freezing point of water, perchlorate is thought to increase the availability of liquid brine water in hyper-arid and cold environments, such as the Martian regolith. Therefore, perchlorate has been proposed as a compound worth studying to better understanding the habitability of the Martian surface. In the present work, to study the molecular mechanisms of perchlorate resistance, a functional metagenomic approach was used, and for that, a small-insert library was constructed with DNA isolated from microorganisms exposed to perchlorate in sediments of a hypersaline pond in the Atacama Desert, Chile (Salar de Maricunga), one of the regions with the highest levels of perchlorate on Earth. The metagenomic library was hosted in Escherichia coli DH10B strain and exposed to sodium perchlorate. This technique allowed the identification of nine perchlorate-resistant clones and their environmental DNA fragments were sequenced. A total of seventeen ORFs were predicted, individually cloned, and nine of them increased perchlorate resistance when expressed in E. coli DH10B cells. These genes encoded hypothetical conserved proteins of unknown functions and proteins similar to other not previously reported to be involved in perchlorate resistance that were related to different cellular processes such as RNA processing, tRNA modification, DNA protection and repair, metabolism, and protein degradation. Furthermore, these genes also conferred resistance to UV-radiation, 4-nitroquinoline-N-oxide (4-NQO) and/or hydrogen peroxide (H2O2), other stress conditions that induce oxidative stress, and damage in proteins and nucleic acids. Therefore, the novel genes identified will help us to better understand the molecular strategies of microorganisms to survive in the presence of perchlorate and may be used in Mars exploration for creating perchlorate-resistance strains interesting for developing Bioregenerative Life Support Systems (BLSS) based on in situ resource utilization (ISRU).

Perspectives on Expanding the Repertoire of Novel Microbial Chitinases for Biological Control

Interest in chitin-degrading enzymes has grown over the years, and microbial chitinases are the most attractive and promising candidates for the control of plant pests (fungi and insects). Currently, there are many studies on chitinases produced by cultivable microorganisms; however, almost none of them have achieved acceptable applicability as a biopesticide in the field. Approximately 99% of the microorganisms from soil cannot be isolated by conventional culture-dependent methods, thus having an enormous biotechnological/genetic potential to be explored. On the basis of this, the present paper aims to provide a brief overview of the metagenomic opportunities that have been emerging and allowing access to the biochemical potential of uncultivable microorganisms through the direct mining of DNA sequences recovered from the environment. This work also shortly discussed the future perspectives of functional and sequence-based metagenomic approaches for the identification of new chitinase-coding genes with potential for applications in several agricultural and biotechnological industries, especially in biological control.

Phenanthrene degradation in soil using biochar hybrid modified bio-microcapsules: Determining the mechanism of action via comparative metagenomic analysis

A strategy involving biochar (BC) hybrid modification was developed to promote the bioremediation effect of degrading bacteria immobilized in layer-by-layer assembly (LBL) microcapsules for the treatment of phenanthrene (PHE) polluted soil. A taxonomic and functional metagenomic approach was used to investigate changes in the microbial community structures and functional gene compositions in the PHE-polluted soil during the bioremediation process. Biofortification with an initial PHE concentration of 100 mg kg−1 dry soil in soils using the BC (3%) hybrid LBL bio-microcapsule (BC-LBL, 2.0 g kg−1 dry soil, 107 colony forming unite cell g−1 dry soil) was faster; further, a higher PHE degradation efficiency (80.5% after 25 d) was achieved when compared with that by the LBL agent (66.2% after 25 d) used. Sphingomonas, Streptomyces, Gemmatirosa, Ramlibacter, Flavisolibacter, Phycicoccus, Micromonospora, Acidobacter, Mycobacterium and Gemmatimonas were more abundant in BC-LBL treatment than those in LBL one. Functional gene annotation results showed that more gene number with BC-LBL treatment than those with LBL one. More abundant functions in the former were primarily related to the growth, reproduction, metabolism, and transportation of bacteria. BC hybridization promoting PHE degradation by microencapsulated bacteria may be due to the strong adsorption property of BC, which results in the enrichment of the nutrients that needed for bacterial growth and reproduction, as well as enhancing the mass transfer performance of PHE to BC-LBL; Meanwhile, BC could also stimulate and improve the metabolism and membrane transportation of the degrading bacteria, and finally improving the degradation function.

Duck wastes as a potential reservoir of novel antibiotic resistance genes

Overuse of antibiotics in animal husbandry has led to an increase of antibiotic resistance microorganisms as well as antibiotic-resistance genes (ARGs). Duck farming in China is practiced on a large and diverse scale and the overuse of antibiotics in this field is gaining attention recently. We evaluated the diversity of ARGs from five duck farms using a functional metagenomic approach and constructed five libraries. A total of seventy-six resistant determinants were identified, of which sixty-one were gene variants or novel genes. The novel genes contained five β-lactamase-encoding genes designated as blaDWA1, blaDWA2, blaDWA3, blaDWA4 and blaDWB1, respectively, and two genes conferring resistance to fosfomycin designated as fosA-like1 and fosA-like2. Three of the five β-lactamase-encoding genes were further identified as extended-spectrum β-lactamases (ESBL) that can hydrolyze both penicillins and cephalosporins. Besides, two of the five β-lactamase-encoding genes were associated with mobile genetic elements, indicating a high potential for transfer of the genes to other bacterial hosts. The two novel fosA-like genes were able to increase the MICs of the test Escherichia coli strain from 2 μg/mL to as high as 256 μg/mL(up to 128-fold increase). Our study provides a reference for ARGs prevalence in duck farm wastes and implies that they are an important resistome reservoir, especially for novel ARGs with high spread potential.

Role of metagenomics in prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review.

As the fossil fuel reserves are depleting rapidly, there is a need for alternate fuels to meet the day to day mounting energy demands. As fossil fuel started depleting, a quest for alternate forms of fuel was initiated and biofuel is one of its promising outcomes. First-generation biofuels are made from edible sources like vegetable oils, starch, and sugars. Second-generation biofuels (SGB) are derived from lignocellulosic crops and the third-generation involves algae for biofuel production. Technical challenges in the production of SGB are hampering its commercialization. Advanced molecular technologies like metagenomics can help in the discovery of novel lignocellulosic biomass-degrading enzymes for commercialization and industrial production of SGB. This review discusses the metagenomic outcomes to enlighten the importance of unexplored habitats for novel cellulolytic gene mining. It also emphasizes the potential of different metagenomic approaches to explore the uncultivable cellulose-degrading microbiome as well as cellulolytic enzymes associated with them. This review also includes effective pre-treatment technology and consolidated bioprocessing for efficient biofuel production.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

Functional metagenomics of the thioredoxin superfamily

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

The Antibacterial Agent Identified from Acidocella spp. in the Fluid of Nepenthes gracilis Against Multidrug-Resistant Klebsiella pneumoniae: A Functional Metagenomic Approach

Aims: The fluid of Nepenthes gracilis harbors diverse bacterial taxa that could serve as a gene pool for the discovery of the new genre of antimicrobial agents against multidrug-resistant Klebsiella pneumoniae. The aim of this study was to explore the presence of antibacterial genes in the fluids of N. gracilis growing in the wild. Methods: Using functional metagenomic approach, fosmid clones were isolated and screened for antibacterial activity against three strains of K. pneumoniae. A clone that exhibited the most potent antibacterial activity was sent for sequencing to identify the genes responsible for the observed activity. The secondary metabolites secreted by the selected clone was sequentially extracted using hexane, chloroform, and ethyl acetate. The chemical profiles of a clone (C6) hexane extract were determined by gas chromatography/mass spectrometry (GC-MS). Results: Fosmid clone C6 from the fluid of pitcher plant that exhibited antibacterial activity against three strains of K. pneumoniae was isolated using functional metagenome approach. A majority of the open reading frames detected from C6 were affiliated with the largely understudied Acidocella genus. Among them, the gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway could be involved in the observed antibacterial activity. Based on the GC-MS analysis, the identities of the putative bioactive compounds were 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane. Conclusions: The gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway as well as the secondary metabolites, namely 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane could be the potential antibacterial molecules responsible for the antibacterial activity of C6.