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Reliable analysis of the catabolic gene landscape and transcriptome for pollutant degradation using a custom open source microarray with internal calibration

ABSTRACT: Despite various efforts to develop tools to detect and compare the activity and catabolic potential and activity for pollutant degradation of microorganisms in environmental samples, an open-source, curated and reliable method is still required. Here we report on a customize normalization system that can be applied to any microarray design, allowing the assessment of reliability of signals and enabling cross-experiment comparisons. Probes for the underlying catabolic gene array were designed based on manually curated databases for catabolic key genes. Signals were assigned to the respective catabolic protein subfamily that allows to inferring the functions and substrate specificity. The placement of probes for critical degradation nodes and subsequent metabolic steps allows the retrieval of information on the metabolic net. Information on gene localization from genome surveys was correlated to signals of putative hosts to generate phylogenetic community information. Hence, this novel array system was validated using genomic DNA of genome sequenced bacterial strains hosting biodegradation functions and applied to genomic DNA and RNA extracted from environmental samples under aromatic pollution pressure.

ORGANISM(S): Cupriavidus pinatubonensis JMP134 Paraburkholderia xenovorans LB400 Rhizorhabdus wittichii RW1 unidentified microorganism Pseudomonas putida F1

PROVIDER: GSE30303 | GEO | 2012/12/31

SECONDARY ACCESSION(S): PRJNA143805

REPOSITORIES: GEO

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Project description:Despite various efforts to develop tools to detect and compare the activity and catabolic potential and activity for pollutant degradation of microorganisms in environmental samples, an open-source, curated and reliable method is still required. Here we report on a customize normalization system that can be applied to any microarray design, allowing the assessment of reliability of signals and enabling cross-experiment comparisons. Probes for the underlying catabolic gene array were designed based on manually curated databases for catabolic key genes. Signals were assigned to the respective catabolic protein subfamily that allows to inferring the functions and substrate specificity. The placement of probes for critical degradation nodes and subsequent metabolic steps allows the retrieval of information on the metabolic net. Information on gene localization from genome surveys was correlated to signals of putative hosts to generate phylogenetic community information. Hence, this novel array system was validated using genomic DNA of genome sequenced bacterial strains hosting biodegradation functions and applied to genomic DNA and RNA extracted from environmental samples under aromatic pollution pressure. Catabolic microarray was optimized for detection and expression of catabolic genes involved in the degradation of pollutant compounds using genome genomic DNA of type strains: Burkholderia xenovorans LB400, Cupriavidus necator JMP134, Pseudomonas putida F1 and Sphingomonas wittichii RW1. Afterwards, this microarray was applied for environmental samples. For microcosm experiments, 200 ml of each groundwater were incubated in closed 1 l Erlenmeyer flasks at 20 degree C and 50 rpm. Second sets of 200 ml microcosms were supplemented with naphthalene (100 mg), which had above been identified as a contaminant present in both groundwaters in similar amounts. Naphthalene was supplied via the vapor phase and incubated under identical conditions as the control microcosms. After 4 days of incubations, 50 ml of each of the 4 microcosms were subjected to genomic DNA extraction and another 50 ml to RNA extraction.

Project description:A custom 8x60 k expression microarray for larvae of European fire salamander (Salamandra salamandra) was designed based on transcriptome sequencing. It is known the fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution) for more information. Labeled cRNA was prepared from Salamander larvae kept at 9°C and 17°C. A cRNA calibration pool was prepared with equimolar amounts of cRNA prepared from (a) a larvae (temperature: 9°C: source: pond KOE), (b) a larvae (temperature: 17°C: source: pond KOE), (c) a larvae (temperature: 9°C: source: stream KoGB (Klufterbach) and (d) a larvae (temperature: 17°C: source: stream KoGB (Klufterbach). See Steinfartz et al. (2007) (doi: 10.1111/j.1365-294X.2007.03490.x) for information of the source populations. Increasing amounts of labeled cRNA (75 ng, 150 ng, 300 ng, 600 ng, 1000 ng, 1400 ng, 1800 ng, 2200 ng), corresponding to (1/8, 1/4, 1/2, 1, 1 2/3, 2 1/3, 3 and 3 3/3 times the recommended amount of 600 ng) were hybridized to 8 microarrays (one microarray per dilution). The change in observed signal intensity in relation to the change in amount of labeled cRNA was used to infer the target-binding behavior of the individual probes. This information was extracted, to be used for a normalization procedure in another experiment with the same microarray (see Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution). The current study provides only raw data for a calibration experiment, to validate the binding behavior of the different probes on a newly designed microarray for a non model organism (European Fire salamander). This calibration is based only on raw data. More information on targeted genes is provided in a different GEO dataset (currently submitted), where biological meaningful analysis are performed with data which are normalized based on this calibration.

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Reliable analysis of the catabolic gene landscape and transcriptome for pollutant degradation using a custom open source microarray with internal calibration

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