For induction of TOL catabolic genes during transcriptomics experiments, the P. putida mt-2 strain was precultured overnight in M9 medium with succinate, and bacterial cultures were then diluted 100-fold in the same medium and grown to the exponential phase (OD600 = 0.3-0.5). Samples were then cultured further without additional substrate or exposed to vaporous m-xylene, toluene or o-xylene (1/2 dilution in dibutylphthalate, which is a non-effector for TOL genes) in a flask (2 h). For soluble substrates, benzoate (5 mM) or 3MBz (5 mM) was added to the culture medium once growth had reached OD600c~ 0.3. For the C-runout condition, the cells were cultured for 96 h in succinate-supplemented minimal medium. The antibiotics kanamycin (Km) 50 μg/ml, ampicillin (Amp) 150 μg/ml, streptomycin (Sm) 100 μg/ml, chloramphenicol (Cm) 30 μg/ml and gentamycin (Gm) 10 μg/ml were added to bacterial cultures when necessary.
Growth protocol
P. putida cultures were routinely grown at 30°C in M9 minimal medium including 6 g/l Na2HPO4, 3 g/l KH2PO4, 1.4 g/l (NH4)2SO4, 0.5 g/l NaCl, 0.2 g/l MgSO4·7H2O and 2.5 ml/l of a trace element solution, with a carbon source as described in treatment protocol.
Extracted molecule
total RNA
Extraction protocol
Cell cultures were transferred to 1/10 sample volume of ice-cold ethanol/phenol solution (5% water-saturated phenol in ethanol) to protect RNA from degradation and harvested by centrifugation (3,800 rpm, 15 min, 4°C). After supernatant aspiration, pellets were frozen in liquid nitrogen and stored at -80°C until required. Total RNA was extracted by using the miRNeasy kit (Qiagen) with some modifications. The collected pellets were resuspended into 0.3 ml Tris-HCl (pH 7.5) containing 2 mg/ml lysozyme and incubated (10 min, 37°C). Lysate (0.1 ml) was used according to manufacturer's instructions. RNase-free DNase (Qiagen) treatment was performed during the isolation procedure to eliminate residual DNA and quality was evaluated on Model 2100 Bioanalyzer (Agilent).
Label
Hy5
Label protocol
Total RNA (20 μg each) was retrotranscribed and aminoallyl-labeled using the SuperScript Indirect cDNA Labeling System (Invitrogen) and 5-(3-aminoallyl)-2'deoxyuridine-5'-triphosphate (aa-dUTP, Ambion) following manufacturers' protocols. To avoid antisense artifacts from second-strand cDNA during reverse transcription, actinomycin D was added after the denaturing step at 70°C to a final concentration of 6 μg/ml. For each sample, aminoallyl-labeled cDNA was resuspended in 0.1 M Na2CO3 (pH 9.0) and conjugated with Cy3 or Hyper 5 Mono NHS Ester (CyTMDye Post-labelling Reactive Dye Pack, Amersham), following a dye-swap strategy. Samples were purified with Megaclear (Ambion) following the manufacturer's instructions. Cy3 and Hyper 5 incorporation was measured in a NanoDrop spectrophotometer (NanoDrop Technologies). Probe preparation and hybridizations were performed as described (Two-Color Microarray-Based Prokaryote Analysis, Agilent Technologies).
For induction of TOL catabolic genes during transcriptomics experiments, the P. putida mt-2 strain was precultured overnight in M9 medium with succinate, and bacterial cultures were then diluted 100-fold in the same medium and grown to the exponential phase (OD600 = 0.3-0.5). Samples were then cultured further without additional substrate or exposed to vaporous m-xylene, toluene or o-xylene (1/2 dilution in dibutylphthalate, which is a non-effector for TOL genes) in a flask (2 h). For soluble substrates, benzoate (5 mM) or 3MBz (5 mM) was added to the culture medium once growth had reached OD600c~ 0.3. For the C-runout condition, the cells were cultured for 96 h in succinate-supplemented minimal medium. The antibiotics kanamycin (Km) 50 μg/ml, ampicillin (Amp) 150 μg/ml, streptomycin (Sm) 100 μg/ml, chloramphenicol (Cm) 30 μg/ml and gentamycin (Gm) 10 μg/ml were added to bacterial cultures when necessary.
Growth protocol
P. putida cultures were routinely grown at 30°C in M9 minimal medium including 6 g/l Na2HPO4, 3 g/l KH2PO4, 1.4 g/l (NH4)2SO4, 0.5 g/l NaCl, 0.2 g/l MgSO4·7H2O and 2.5 ml/l of a trace element solution, with a carbon source as described in treatment protocol.
Extracted molecule
total RNA
Extraction protocol
Cell cultures were transferred to 1/10 sample volume of ice-cold ethanol/phenol solution (5% water-saturated phenol in ethanol) to protect RNA from degradation and harvested by centrifugation (3,800 rpm, 15 min, 4°C). After supernatant aspiration, pellets were frozen in liquid nitrogen and stored at -80°C until required. Total RNA was extracted by using the miRNeasy kit (Qiagen) with some modifications. The collected pellets were resuspended into 0.3 ml Tris-HCl (pH 7.5) containing 2 mg/ml lysozyme and incubated (10 min, 37°C). Lysate (0.1 ml) was used according to manufacturer's instructions. RNase-free DNase (Qiagen) treatment was performed during the isolation procedure to eliminate residual DNA and quality was evaluated on Model 2100 Bioanalyzer (Agilent).
Label
Cy3
Label protocol
Total RNA (20 μg each) was retrotranscribed and aminoallyl-labeled using the SuperScript Indirect cDNA Labeling System (Invitrogen) and 5-(3-aminoallyl)-2'deoxyuridine-5'-triphosphate (aa-dUTP, Ambion) following manufacturers' protocols. To avoid antisense artifacts from second-strand cDNA during reverse transcription, actinomycin D was added after the denaturing step at 70°C to a final concentration of 6 μg/ml. For each sample, aminoallyl-labeled cDNA was resuspended in 0.1 M Na2CO3 (pH 9.0) and conjugated with Cy3 or Hyper 5 Mono NHS Ester (CyTMDye Post-labelling Reactive Dye Pack, Amersham), following a dye-swap strategy. Samples were purified with Megaclear (Ambion) following the manufacturer's instructions. Cy3 and Hyper 5 incorporation was measured in a NanoDrop spectrophotometer (NanoDrop Technologies). Probe preparation and hybridizations were performed as described (Two-Color Microarray-Based Prokaryote Analysis, Agilent Technologies).
Hybridization protocol
Samples were placed on ice and rapidly loaded onto arrays, hybridized (65ºC, 17 h), then washed once in GE Wash Buffer 1 (room temperature, 1 min) and once in GE Wash Buffer 2 (37ºC, 1 min). Arrays were drained by centrifugation (2000 rpm, 2 min).
Scan protocol
Images from Cy3 and Hyper 5 channels were equilibrated and captured with a GenePix 4000B (Axon) and spots were quantified using GenePix software (Axon) for tiling array data sets (m-xylene VS succinate). For other tiling array data sets that are reference condition-based comparisons, we used the Agilent DNA Microarray Scanner and quantified images using Agilent Feature Extraction software.
Raw intensities were background-corrected by the normexp method with an offset of 50. Background-corrected intensities were converted to log2 scale and normalized by adjusting the quantiles of all replicates. After normalization, differential expression for each probe was calculated as log2Ratios = log2Intensity (experimental condition) - log2Intensity (control condition).
