Rapid identification of genes encoding DNA polymerases by function-based screening of metagenomic libraries derived from glacial ice.
Ontology highlight
ABSTRACT:
SUBMITTER: Simon C
PROVIDER: S-EPMC2681694 | biostudies-literature | 2009 May
REPOSITORIES: biostudies-literature
Publications
Rapid identification of genes encoding DNA polymerases by function-based screening of metagenomic libraries derived from glacial ice.
Simon Carola C Herath Judith J Rockstroh Stephanie S Daniel Rolf R
Applied and environmental microbiology 20090306 9
Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered. ...[more]
Similar Datasets
Project description:Glacial retreat creates new habitat which is colonized and developed by plants and animals during the process of primary succession. While there has been much debate about the relative role of deterministic and stochastic processes during terrestrial succession, evidence from freshwater ecosystems remains minimal and a general consensus is lacking. Using a unique 27 years record of community assembly following glacial recession in southeast Alaska, we demonstrate significant change in the trait composition of stream invertebrate communities as catchment glacial cover decreased from ?70% to zero. Functional diversity increased significantly as glacier cover decreased and taxonomic richness increased. Null modelling approaches led to a key finding that niche filtering processes were dominant when glacial cover was extensive, reflecting water temperature and dispersal constraints. Thereafter the community shifted towards co-occurrence of stochastic and deterministic assembly processes. A further novel discovery was that intrinsic functional redundancy developed throughout the study, particularly because new colonizers possessed similar traits to taxa already present. Rapid glacial retreat is occurring in Arctic and alpine environments worldwide and the assembly processes observed in this study provide new fundamental insights into how glacially influenced stream ecosystems will respond. The findings support tolerance as a key primary successional mechanism in this system, and have broader value for developing our understanding of how biological communities in river ecosystems assemble or restructure in response to environmental change.
| S-EPMC3664027 | biostudies-literature
Project description:Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
| S-EPMC5341072 | biostudies-literature
Project description:To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.
| S-EPMC161467 | biostudies-literature
Project description:Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5-10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.
| S-EPMC6481063 | biostudies-literature
Project description:BackgroundIt is generally believed that there are many natural sources of as yet unknown bioactive compounds with a high biotechnological potential. However, the common method based on the use of cell extracts in the preliminary screening for particular molecules or activities is problematic as amounts of obtained compounds may be low, and such experiments are hardly reproducible. Therefore, the aim of this work was to test whether a novel strategy to search for previously unknown biological activities can be efficient. This strategy is based on construction of metagenomic libraries and employment of Escherichia coli strains as cell factories producing compounds of properties potentially useful in biotechnology.ResultsThree cyanobacterial metagenomic libraries were constructed in the fosmid system. The libraries were screened for various biological activities. Extracts from selected E. coli clones bearing constructs with fragments of cyanobacterial genomes revealed antimicrobial or anticancer activities. Interestingly, stimulation of growth of host bacteria bearing particular plasmids with certain cyanobacterial genes was detected, suggesting a potential possibility for improvement of E. coli cultivation during biotechnological production. The most interesting plasmids were sequenced, and putative mechanisms of biological effects caused by cyanobacterial gene products are discussed.ConclusionsThe strategy of exploring cyanobacteria as sources of bioactive compounds, based on E. coli cell factories producing compounds due to expression of genes from metagenomic libraries, appears to be effective.
| S-EPMC4347599 | biostudies-literature
Project description:Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes, and metabolites that have a variety of potential clinical and industrial uses. Here, we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700 000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase, and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities.
| S-EPMC4028719 | biostudies-literature
Project description: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.
| S-EPMC7690600 | biostudies-literature
Project description:A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.
| S-EPMC4432631 | biostudies-literature
Project description:Blooms of microalgae on glaciers and ice sheets are amplifying surface ice melting rates, which are already affected by climate change. Most studies on glacial microorganisms (including snow and glacier ice algae) have so far focused on the spring and summer melt season, leading to a temporal bias, and a knowledge gap in our understanding of the variations in microbial diversity, productivity, and physiology on glacier surfaces year-round. Here, we investigated the microbial communities from Icelandic glacier surface snow and bare ice habitats, with sampling spanning two consecutive years and carried out in both winter and two summer seasons. We evaluated the seasonal differences in microbial community composition using Illumina sequencing of the 16S rRNA, 18S rRNA, and ITS marker genes and correlating them with geochemical signals in the snow and ice. During summer, Chloromonas, Chlainomonas, Raphidonema, and Hydrurus dominated surface snow algal communities, while Ancylonema and Mesotaenium dominated the surface bare ice habitats. In winter, algae could not be detected, and the community composition was dominated by bacteria and fungi. The dominant bacterial taxa found in both winter and summer samples were Bacteriodetes, Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria. The winter bacterial communities showed high similarities to airborne and fresh snow bacteria reported in other studies. This points toward the importance of dry and wet deposition as a wintertime source of microorganisms to the glacier surface. Winter samples were also richer in nutrients than summer samples, except for dissolved organic carbon-which was highest in summer snow and ice samples with blooming microalgae, suggesting that nutrients are accumulated during winter but primarily used by the microbial communities in the summer. Overall, our study shows that glacial snow and ice microbial communities are highly variable on a seasonal basis.
| S-EPMC9149292 | biostudies-literature