Abstract
BackgroundMicroorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes.ResultsFirst we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently.ConclusionsWe successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to study any group of genes. The Metabolic Design software is freely available from the authors and can be downloaded and modified under general public license.
Highlights
Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions
Based on Metabolic Design defined probes, targeting eight genes coding for enzymes involved in the degradation of various polycyclic aromatic hydrocarbons (PAHs) by strain EPA505, we demonstrate that our design strategy is useful for most of the determined probes
This study evaluates the efficiency of a new probe design software tool, Metabolic Design, dedicated to DNA functional microarrays
Summary
Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. Microorganisms display vast diversity, each one having its own set of genes, cell components and these sequence data were used to identify different species in environmental or clinical samples with DNA microarrays [5]. These data should improve our knowledge of genome organization and genome evolution and of biological processes and biological activities. Spurious functional assignments are usually caused by species homology-based transfer of information from existing database entries to new target sequences [8,9]. Unlike TrEMBL, the Swiss-Prot database contains curated datasets of high quality [12]
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