Cells were first grown overnight at 37°C on a shaker table (150 rpm) in 70 mL of Luria-Bertani medium (LB) containing streptomycin (100 mg/L) to produce biomass. They were then centrifuged at room temperature for 2 min at 5,000 g and transferred to a sterile minimum mineral medium 457 containing for one liter of distilled water, 2,440 mg of Na2HPO4, 1,520 mg of KH2PO4, 500 mg of (NH4)2SO4, 200 mg of MgSO4.7H2O, 50 mg of CaCl2.2H2O, 200 mg of Tween 80 and 10 mL of SL-4 solution as described by DSMZ. Cultures were prepared as follows, 2 mL of the PAH stock solutions were evaporated in sterile 250-mL Erlenmeyer flasks. Then, 100 mL of sterile medium 457 were added and inoculated with 7.5 × 107 cells prepared as above. Such cultures were incubated at 28°C on a shaker table (150 rpm) for 27 h and bacterial growth was monitored spectrophotometrically at 620 nm. A culture was also grown with glucose (15 g/L) as sole carbon source and energy in order to define basal expression of genes implicated in PAH degradation.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA from pure culture of strain EPA505 was extracted at different times of the PAH degradation kinetics with the RNeasy Mini kit (Qiagen GmbH), treated with 1.5 U of DNase I (Invitrogen) to eliminate DNA contamination. RNA sample concentration and purity was then estimated using the Nanodrop spectrophotometer (Nanodrop). 15 µL of strain EPA505 total RNA were enriched using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Ambion) as recommended by the suppliers. Each enriched mRNA was then amplified using the MessageAmp™ II-Bacteria RNA Amplification Kit (Ambion) with a modified protocol for the in vitro transcription step. Briefly, the purified double-stranded template (~14 µL) was transcribed in vitro with 12 µL of ATP, CTP and GTP mix (25mM each) (Ambion), 3 µL of UTP (75mM) (Ambion), 3 µL of amino-allyl-UTP (50mM) (Ambion), 4 µL of 10X reaction buffer (Ambion) and 4 µL of T7 enzyme mix (Ambion) at 37°C during a 14 h incubation period. Finally, the aRNA was purified using the MessageAmp™ II-Bacteria RNA Amplification Kit (Ambion) as recommended by the manufacturer’s instructions. Total DNA was extracted from 5g of contaminated soil, named S3, following the protocol described by Zhou (Zhou, J., Bruns, M.A. and Tiedje, J.M. (1996) DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 62, 316-322.). Three extractions were realized and pooled to minimize potential biases. DNA quality was checked on a 0.8% agarose gel.
Label
Cy5
Label protocol
10 µg of purified aRNA for each sample was vacuum dried and labeled using the Amersham CyDyeTM Post-Labeling Reactive Dye Packs (GE Healthcare, Little Chalfont, United Kingdom) with Cyanine3 or Cyanine5 dyes as recommended by the supplier. Briefly, the aRNA pellet was resuspended in 20µL of 0.1M bicarbonate buffer (pH 8.7) and incubated for 90 min with 40 nM of dye compound (coupling the dye to amino-allyl-UTP) dissolved in 20 µL of DMSO in the dark at room temperature. Exceeding dye was quenched by addition of 15 μl of 4 M hydroxylamine solution incubated for 15 min in the dark at room temperature. Then, labeled aRNA was purified with NucleoSpin RNA Clean-Up kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s manual. Total DNA was amplified and labeled using the BioPrime® Total Genomic Labeling System (Invitrogen) following the manufacturer’s instructions. The quantity and quality of labeling were estimated by using the Nanodrop spectrophotometer (Nanodrop) as recommended by protocols.
Hybridization protocol
For each hybridization experiment, 3.33µg of labeled RNA for kinetics experiments (one sample in Cy3 and another in Cy5) or 12µg of labeled DNA (in Alexa Fluor® 5) for soil were mixed, vacuum dried and resuspended in 5.6 µL of water. Then, the hybridization mix (Roche NimbleGen) was realized according to the manufacturer's protocols. The arrays were hybridized on a 4-bay NimbleGen Hybridization System (Roche NimbleGen) at 42°C for 72 hr. Arrays were washed with NimbleGen wash buffers I, II and III according to vendor protocols.
Scan protocol
Samples were scanned using a Scanner Innoscan 900AL (Innopsys, Carbonne, France) at 2 μm resolution. Individual array images were acquired independently, adjusting the PMT gain (30 for aRNA and 100 for total DNA) for each image as recommended using the software Mapix® (Innopsys). Then, for each array image, raw expression data had been extracted using the NimbleScan software v2.1. (Roche NimbleGen) and feature intensities were exported as .pair files.
Description
Strain EPA505 with Fluoranthene as sole carbon and energy source. total RNA extracted after 3h of culture
Data processing
The background noise was then determined using random probes present on the microarrays (8,863 probes in our experiment) with the method described in the publication Sébastien Terrat, Eric Peyretaillade, Olivier Gonçalves, Eric Dugat-Bony, Fabrice Gravelat, and Pierre Peyret. 2010 - Studying the ‘Unkown’ with Metabolic Design, a new probe design software for explorative functional microarrays development. Nucleic Acids Research (submited). This background noise is defined by two components: the background median intensity (Bposition) and its dispersion (Bdispersion). Finally, a modified signal-to-noise ratio called SNR’ and based on the formula of Verdik (Verdick, D., Handran, S. and Pickett, S. (2002) In LLC, D. P. (ed.), In G. Kamberova (ed.), DNA array image analysis: nuts and bolts. Salem, MA., pp. p. 83-98) is calculated as follow in order to reduce-centralize our data: SNR’ = (probe signal intensity - Bposition)/ Bdispersion. However, spatial effect across array surface is a predominant within-slide experimental artifact that needs to be eliminated before any other normalization procedure (Wang, X., He, H., Li, L., Chen, R., Deng, X.W. and Li, S. (2006) NMPP: a user-customized NimbleGen microarray data processing pipeline. Bioinformatics, 22, 2955-2957). That is why, for all array images obtained in this work, surface was segmented to 16 sub-squares according to probe position (X, Y) indicated in pair report. A Perl script was developed to calculate local background noise in all sub-squares and the median SNR’ retrieved from the three replicates of each probe. Finally, another Perl script was implemented to summarize each replicate probe treated and determine the median value between the three replicates. Positive hybridization was considered significant for probes having an SNR’ > 3 (value to avoid all false positives).
Expression analysis of Sphingomonas paucimobilis sp. EPA505 during biodegradation kinetics of various PAHs and detection of gene in a contaminated soil S3
Data table header descriptions
ID_REF
VALUE
SNR'-normalized, averaged gene-level signal intensity (by median calculation).
