In this study the wild type Gluconobacter oxydans 621H strain DSM 2343 from the German Collection of Microorganisms and Cell Cultures (DSMZ) was used. The cells were cultivated in mannitol medium containing 220 mM (4% w/v) mannitol, 5 g/L yeast extract, 1 g/L KH2PO4, 1 g/L (NH4)2SO4, 2.5 g/L MgSO4 x 7 H2O, and 50 µg/mL cefoxitin as antibiotic. Cells were grown in 500 mL shaking flasks with three baffles containing 50 mL of the mannitol medium (30°C, 140 rpm). Cell growth in liquid culture was followed by measuring the optical density at 600 nm (OD600) using a spectrophotometer. If rifampicin was applied, it was added to a cell culture at OD600 of 0.6 to 0.8 from a stock (50 mg/mL in methanol) to obtain the final concentration of 150 µg/mL.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004), Mol Microbiol 54: 420-438.)
Label
Cy5
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm (2003) Appl. Environm. Microbiol. 69:2521-2532, yet for pairwise comparisons the A mix and the B mix of the Agilent Spike-In Kit (Agilent Technologies) was used to spike t0 and tx RNA samples.
In this study the wild type Gluconobacter oxydans 621H strain DSM 2343 from the German Collection of Microorganisms and Cell Cultures (DSMZ) was used. The cells were cultivated in mannitol medium containing 220 mM (4% w/v) mannitol, 5 g/L yeast extract, 1 g/L KH2PO4, 1 g/L (NH4)2SO4, 2.5 g/L MgSO4 x 7 H2O, and 50 µg/mL cefoxitin as antibiotic. Cells were grown in 500 mL shaking flasks with three baffles containing 50 mL of the mannitol medium (30°C, 140 rpm). Cell growth in liquid culture was followed by measuring the optical density at 600 nm (OD600) using a spectrophotometer. If rifampicin was applied, it was added to a cell culture at OD600 of 0.6 to 0.8 from a stock (50 mg/mL in methanol) to obtain the final concentration of 150 µg/mL.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004), Mol Microbiol 54: 420-438.)
Label
Cy3
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm (2003) Appl. Environm. Microbiol. 69:2521-2532, yet for pairwise comparisons the A mix and the B mix of the Agilent Spike-In Kit (Agilent Technologies) was used to spike t0 and tx RNA samples.
Hybridization protocol
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
Scan protocol
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
Data processing
For calculation of mRNA half-lives, first the ratio of median values (GenePix Pro 6.0) reflecting the relative mRNA level changes were normalized for each array hybridization separately using a normalization factor. This factor was calculated for each hybridization based on the log base 2 of the non-normalized ratio of median values of the Spike-In (+)E1A_r60_1 and (+)E1A_r60_a20 RNAs as internal controls, each having 32 spots randomly scattered over the array. Both RNAs are present in A mix and B mix at an expected ratio amount (A/B) of 1:1 (Agilent Technologies). Accordingly, the factor was calculated that the log base 2 of the factor-normalized ratio of median values of the 1:1 control RNAs is 0 on average. Subsequently, the ratio of median value for each gene was normalized with the calculated factor. A normalized ratio value was included in the calculation of the average of the triplicates for each time point if the following quality filter was fulfilled by the spot data (GenePix Pro 6.0): i) Flags ≥0 and ii) signal/noise ≥3 for Cy5 (F635Median / B635Median) or Cy3 (F532Median / B532Median). The resulting data matrix with the average values of the four time points was used to calculate the mRNA half-lives and R2 in Excel (Microsoft) by linear fit.
