155 research outputs found

    CRISPy-web:An online resource to design sgRNAs for CRISPR applications

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    CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the Streptococcus pyogenes endonuclease Cas9. To utilize Cas9, so-called single guide RNAs (sgRNAs) need to be designed for each target gene. While there are many tools available to design sgRNAs for the popular model organisms, only few tools that allow designing sgRNAs for non-model organisms exist. Here, we present CRISPy-web (http://crispy.secondarymetabolites.org/), an easy to use web tool based on CRISPy to design sgRNAs for any user-provided microbial genome. CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs. After checks for potential off-target matches, the resulting sgRNA sequences are displayed graphically and can be exported to text files. All steps and information are accessible from a web browser without the requirement to install and use command line scripts

    Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

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    Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats such as rule-based BGC detection, sequence and annotation quality and cluster boundary prediction, which all have to be considered while planning for, performing and analyzing the results of genome mining studies

    NRPSpredictor2-a web server for predicting NRPS adenylation domain specificity

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    The products of many bacterial non-ribosomal peptide synthetases (NRPS) are highly important secondary metabolites, including vancomycin and other antibiotics. The ability to predict substrate specificity of newly detected NRPS Adenylation (A-) domains by genome sequencing efforts is of great importance to identify and annotate new gene clusters that produce secondary metabolites. Prediction of A-domain specificity based on the sequence alone can be achieved through sequence signatures or, more accurately, through machine learning methods. We present an improved predictor, based on previous work (NRPSpredictor), that predicts A-domain specificity using Support Vector Machines on four hierarchical levels, ranging from gross physicochemical properties of an A-domain's substrates down to single amino acid substrates. The three more general levels are predicted with an F-measure better than 0.89 and the most detailed level with an average F-measure of 0.80. We also modeled the applicability domain of our predictor to estimate for new A-domains whether they lie in the applicability domain. Finally, since there are also NRPS that play an important role in natural products chemistry of fungi, such as peptaibols and cephalosporins, we added a predictor for fungal A-domains, which predicts gross physicochemical properties with an F-measure of 0.84. The service is available at http://nrps.informatik.uni-tuebingen.de/

    plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters

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    Plant specialized metabolites are chemically highly diverse, play key roles in host-microbe interactions, have important nutritional value in crops and are frequently applied as medicines. It has recently become clear that plant biosynthetic pathway-encoding genes are sometimes densely clustered in specific genomic loci: Biosynthetic gene clusters (BGCs). Here, we introduce plantiSMASH, a versatile online analysis platform that automates the identification of candidate plant BGCs. Moreover, it allows integration of transcriptomic data to prioritize candidate BGCs based on the coexpression patterns of predicted biosynthetic enzyme-coding genes, and facilitates comparative genomic analysis to study the evolutionary conservation of each cluster. Applied on 48 high-quality plant genomes, plantiSMASH identifies a rich diversity of candidate plant BGCs. These results will guide further experimental exploration of the nature and dynamics of gene clustering in plant metabolism. Moreover, spurred by the continuing decrease in costs of plant genome sequencing, they will allow genome mining technologies to be applied to plant natural product discovery.</p

    Dissemination of antibiotic resistance genes from antibiotic producers to pathogens

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    AbstractIt has been hypothesized that some antibiotic resistance genes (ARGs) found in pathogenic bacteria derive from antibiotic-producing actinobacteria. Here we provide bioinformatic and experimental evidence supporting this hypothesis. We identify genes in proteobacteria, including some pathogens, that appear to be closely related to actinobacterial ARGs known to confer resistance against clinically important antibiotics. Furthermore, we identify two potential examples of recent horizontal transfer of actinobacterial ARGs to proteobacterial pathogens. Based on this bioinformatic evidence, we propose and experimentally test a ‘carry-back’ mechanism for the transfer, involving conjugative transfer of a carrier sequence from proteobacteria to actinobacteria, recombination of the carrier sequence with the actinobacterial ARG, followed by natural transformation of proteobacteria with the carrier-sandwiched ARG. Our results support the existence of ancient and, possibly, recent transfers of ARGs from antibiotic-producing actinobacteria to proteobacteria, and provide evidence for a defined mechanism.</jats:p
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