Cultures were harvested by centrifugation (8,000 rpm, 4°C, 10 min) and washed once with minimum medium (BT medium) without urea (7 g ammonium sulfate, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.5 g MgSO4･7H2O, 6 mg FeSO4･7H2O, 6 mg MnSO4･5H2O, 1 mg ZnSO4･7H2O, 0.2 mg CuSO4･5H2O, 0.2 mg NiCl2･6H2O, 0.2 mg biotin, 0.2 mg thiamin per liter). Samples of 3.6 g of washed cells were resuspended in 54 mL of BT medium without urea, resulting in suspensions of cells at 6% wet weight/volume in 60 mL reaction solution in a lidded 100 mL medium bottle. At the beginning of reactions, 4.32 g of glucose (400 mM) and 1.01 g NaHCO3 (200 mM) were added in reaction solution. Cell suspensions were incubated at 30 or 42.5°C for 2 h.
Growth protocol
Strains were aerobically cultivated at 30.0ºC for 16 h in 500 mL of a nutrient rich medium (A medium containing 2 g yeast extract, 7 g casamino acids, 2 g urea, 7 g ammonium sulfate, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.5 g MgSO4･7H2O, 6 mg FeSO4･7H2O, 6 mg MnSO4･5H2O, 1 mg ZnSO4･7H2O, 0.2 mg CuSO4･5H2O, 0.2 mg NiCl2･6H2O, 0.2 mg biotin, 0.2 mg thiamin per liter supplemented with 2% [w/v] glucose) in a 2 L flask.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from C. glutamicum cells using NucleoSpin RNA II kit (Macherey-Nagel, Germany) according to the manufacture's manual.
Label
Cy3
Label protocol
The cDNAs were synthesized from RNA by using reverse transcriptase from 10 μg of total RNAs and labeled with cyanine 3 (Cy3) or cyanine 5 (Cy5) using the SuperScript Indirect cDNA Labeling system (Life Technologies).
Cultures were harvested by centrifugation (8,000 rpm, 4°C, 10 min) and washed once with minimum medium (BT medium) without urea (7 g ammonium sulfate, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.5 g MgSO4･7H2O, 6 mg FeSO4･7H2O, 6 mg MnSO4･5H2O, 1 mg ZnSO4･7H2O, 0.2 mg CuSO4･5H2O, 0.2 mg NiCl2･6H2O, 0.2 mg biotin, 0.2 mg thiamin per liter). Samples of 3.6 g of washed cells were resuspended in 54 mL of BT medium without urea, resulting in suspensions of cells at 6% wet weight/volume in 60 mL reaction solution in a lidded 100 mL medium bottle. At the beginning of reactions, 4.32 g of glucose (400 mM) and 1.01 g NaHCO3 (200 mM) were added in reaction solution. Cell suspensions were incubated at 30 or 42.5°C for 2 h.
Growth protocol
Strains were aerobically cultivated at 30.0ºC for 16 h in 500 mL of a nutrient rich medium (A medium containing 2 g yeast extract, 7 g casamino acids, 2 g urea, 7 g ammonium sulfate, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.5 g MgSO4･7H2O, 6 mg FeSO4･7H2O, 6 mg MnSO4･5H2O, 1 mg ZnSO4･7H2O, 0.2 mg CuSO4･5H2O, 0.2 mg NiCl2･6H2O, 0.2 mg biotin, 0.2 mg thiamin per liter supplemented with 2% [w/v] glucose) in a 2 L flask.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from C. glutamicum cells using NucleoSpin RNA II kit (Macherey-Nagel, Germany) according to the manufacture's manual.
Label
Cy5
Label protocol
The cDNAs were synthesized from RNA by using reverse transcriptase from 10 μg of total RNAs and labeled with cyanine 3 (Cy3) or cyanine 5 (Cy5) using the SuperScript Indirect cDNA Labeling system (Life Technologies).
Hybridization protocol
Labeled cDNA (4.5 μg each) are mixed with 1 × hybridization buffer (6 × SSC, 0.2% SDS, 5 × Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA) in a total volume of 340 μl, and denatured at 95˚C for 2 min. The hybridization mixture was hybridized to a microarray in an Agilent Technologies Microarray chamber at 60˚C for 17 h in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 5 minutes with wash buffer 1 (2x SSC, 0.2% SDS) at room temperature , 5 minutes with wash buffer 2 (0.2x SSC, 0.2% SDS) at 60˚C, and 5 minutes with wash buffer 3 (0.2x SSC) at room temperature.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) at a resolution of 5 µm. PMT is set to 100% for Cy3 and Cy5 channels.
Data processing
The scanned images were analyzed quantified with Feature Extraction Software 10.5.5.1 (Agilent) using default parameters (protocol GE2-NonAT_105_Dec08). Feature extracted data were analyzed using GeneSpring GX v 13.0 software from Agilent. After taking the average of two replicate features on each array, normalization of the data was done in GeneSpring GX using the Lowess normalization algorithm without baseline transformation.
