Gluconobacter oxydans ATCC 621H Δupp (ATCC 621H is identical to DSM2343), which lacks the upp gene for uracil phosphoribosyltransferase, was obtained from Armin Ehrenreich (Technical University of Munich, Germany). The strain was cultivated on mannitol medium containing 220 mM (4% w/v) mannitol, 5 g l-1 yeast extract, 2.5 g l-1 MgSO4 x 7 H20, 1 g l-1 (NH4)2SO4, 1 g l-1 KH2PO4 and 10 μM thymidine. The initial pH value of the medium was 6.0. G. oxydans possesses a natural resistance towards cefoxitin; as a precaution to prevent bacterial contaminations, cefoxitin was added to the media at a concentration of 50 μg ml-1. When required, kanamycin (50 ug ml-1) was added. Precultures were grown in baffled shaking flasks at 30°C and 140 rpm. For determination of growth parameters, DNA microarray analyses and enzyme activity measurements, cells were cultivated in 250 ml of the same medium in a bioreactor system (DASGIP, Jülich, Germany) composed of four 400-ml vessels, each equipped with electrodes for measuring the dissolved oxygen concentration (DO) and the pH. The system allows to constantly controlling these two parameters. The carbon dioxide concentration in the exhaust gas was measured continuously by an infrared spectrometer. The pH was kept at pH 6.0 by automatic titration of 2 M NaOH. The oxygen availability was kept constant at 15% DO by mixing air, O2 and N2. Calibration was performed by gassing with air (100% DO) and N2 (0% DO). The agitation speed was kept constant at 900 rpm. Control and recording of all data was carried out by the software “Fedbatch Pro” (DASGIP, Jülich, Germany).
Extracted molecule
total RNA
Extraction protocol
25 ml of a G. oxydans culture was added to 25 g of crushed ice in a Falcon tube which had been pre-cooled in liquid nitrogen. The cells were sedimented by centrifugation for 3 min at 4 °C and 10,397 x g and after removal of the supernatant the tube with the cell pellet was immersed in liquid nitrogen and then stored at -70°C. For total RNA preparation, the RNeasy system (Qiagen, Hilden, Germany) was used. The cell pellet was resuspended in 700 µl of RLT buffer containing 7 µl ß-mercaptoethanol and transferred to two vials each containing 0.5 g of 0.1-mm diameter zirconium silica beads (Roth, Karlsruhe, Germany). Cells were disrupted by 4 x 30 s bead beating using a Silamat S5 (Ivoclar Vivadent, Ellwangen, Germany). The lysate was centrifuged at 8,000 x g for 2 min and the supernatant was mixed with 250 µl ethanol (-20 °C). RNA was extracted with two RNeasy mini-columns following the manufacturer’s instructions. DNA digestion was performed for 20 min on column by adding 30 Kuniz U RNase-free DNase. RNA concentration and quality were checked photometrically and on formaldehyde gels according to standard procedures (Sambrook and Russell, 2001).
Label
Cy5
Label protocol
cDNA synthesis for DNA microarray analysis was performed as described (Polen et al., 2007). 25 µg RNA was used for random hexamer-primed synthesis of fluorescently-labeled cDNA with the fluorescent nucleotide analogues Cy3-dUTP and Cy5-dUTP (GE Healthcare, Freiburg, Germany). The mixture contained 3 µl 1 mM Cy3-dUTP or Cy5-dUTP, 3 µl 0.1 M DTT, 6 µl 5x first strand buffer (Invitrogen, Karlsruhe, Germany), 0.6 µl dNTP-mix (25 mM each of dATP, dCTP, and dGTP and 10 mM dTTP) and 2 µl Superscript II reverse transcriptase (Invitrogen, Darmstadt, Germany). The mixture was incubated for 10 min at room temperature and 110 min at 42 °C, stopped by addition of 10 μl 0.1 N NaOH, incubated for 10 min at 70 °C, and then neutralized by addition of 10 μl 0.1 N HCl. The sensors were purified by washing three times with water on a Microcon column (Millipore, YM-30).
Gluconobacter oxydans ATCC 621H Δupp (ATCC 621H is identical to DSM2343), which lacks the upp gene for uracil phosphoribosyltransferase, was obtained from Armin Ehrenreich (Technical University of Munich, Germany). The strain was cultivated on mannitol medium containing 220 mM (4% w/v) mannitol, 5 g l-1 yeast extract, 2.5 g l-1 MgSO4 x 7 H20, 1 g l-1 (NH4)2SO4, 1 g l-1 KH2PO4 and 10 μM thymidine. The initial pH value of the medium was 6.0. G. oxydans possesses a natural resistance towards cefoxitin; as a precaution to prevent bacterial contaminations, cefoxitin was added to the media at a concentration of 50 μg ml-1. When required, kanamycin (50 ug ml-1) was added. Precultures were grown in baffled shaking flasks at 30°C and 140 rpm. For determination of growth parameters, DNA microarray analyses and enzyme activity measurements, cells were cultivated in 250 ml of the same medium in a bioreactor system (DASGIP, Jülich, Germany) composed of four 400-ml vessels, each equipped with electrodes for measuring the dissolved oxygen concentration (DO) and the pH. The system allows to constantly controlling these two parameters. The carbon dioxide concentration in the exhaust gas was measured continuously by an infrared spectrometer. The pH was kept at pH 6.0 by automatic titration of 2 M NaOH. The oxygen availability was kept constant at 15% DO by mixing air, O2 and N2. Calibration was performed by gassing with air (100% DO) and N2 (0% DO). The agitation speed was kept constant at 900 rpm. Control and recording of all data was carried out by the software “Fedbatch Pro” (DASGIP, Jülich, Germany).
Extracted molecule
total RNA
Extraction protocol
25 ml of a G. oxydans culture was added to 25 g of crushed ice in a Falcon tube which had been pre-cooled in liquid nitrogen. The cells were sedimented by centrifugation for 3 min at 4 °C and 10,397 x g and after removal of the supernatant the tube with the cell pellet was immersed in liquid nitrogen and then stored at -70°C. For total RNA preparation, the RNeasy system (Qiagen, Hilden, Germany) was used. The cell pellet was resuspended in 700 µl of RLT buffer containing 7 µl ß-mercaptoethanol and transferred to two vials each containing 0.5 g of 0.1-mm diameter zirconium silica beads (Roth, Karlsruhe, Germany). Cells were disrupted by 4 x 30 s bead beating using a Silamat S5 (Ivoclar Vivadent, Ellwangen, Germany). The lysate was centrifuged at 8,000 x g for 2 min and the supernatant was mixed with 250 µl ethanol (-20 °C). RNA was extracted with two RNeasy mini-columns following the manufacturer’s instructions. DNA digestion was performed for 20 min on column by adding 30 Kuniz U RNase-free DNase. RNA concentration and quality were checked photometrically and on formaldehyde gels according to standard procedures (Sambrook and Russell, 2001).
Label
Cy3
Label protocol
cDNA synthesis for DNA microarray analysis was performed as described (Polen et al., 2007). 25 µg RNA was used for random hexamer-primed synthesis of fluorescently-labeled cDNA with the fluorescent nucleotide analogues Cy3-dUTP and Cy5-dUTP (GE Healthcare, Freiburg, Germany). The mixture contained 3 µl 1 mM Cy3-dUTP or Cy5-dUTP, 3 µl 0.1 M DTT, 6 µl 5x first strand buffer (Invitrogen, Karlsruhe, Germany), 0.6 µl dNTP-mix (25 mM each of dATP, dCTP, and dGTP and 10 mM dTTP) and 2 µl Superscript II reverse transcriptase (Invitrogen, Darmstadt, Germany). The mixture was incubated for 10 min at room temperature and 110 min at 42 °C, stopped by addition of 10 μl 0.1 N NaOH, incubated for 10 min at 70 °C, and then neutralized by addition of 10 μl 0.1 N HCl. The sensors were purified by washing three times with water on a Microcon column (Millipore, YM-30).
Hybridization protocol
Preparation of the slides was performed in 50 ml Falcon tubes. All reagents were from the OpArray system from Eurofins MWG Operon. Slides were incubated at 42°C in Pre-Hyb solution for 1 h, then the slides were transferred to Wash 1 solution (1.25 ml Wash B and 48.75 ml H2O) and incubated for 5 min at 37°C. Slides were washed with H2O and dried in a centrifuge (Sigma Laboratory Centrifuge 4K15C, 5 min, 521 x g). Hybridization was carried out for 16–18 h at 42°C using a MAUI hybridization system (BioMicro Systems, Salt Lake City, USA).
Scan protocol
For post-hybridization, slides were washed at 37°C in Wash 2 solution (5 ml Wash A, 2.5 ml Wash B and 42.5 ml H2O) and Wash 3 solution (5 ml Wash A and 45 ml H2O) for 10 minutes each. Slides were rotated in Wash 4 solution (1 ml Wash A and 49 ml H2O) for 5 min at room temperature. Finally, the slides were dried by centrifugation (Sigma Laboratory Centrifuge 4K15C, 5 min, 521 x g). Fluorescence of the hybridized DNA microarrays was determined at 532 nm (Cy3-dUTP) and 635 nm (Cy5-dUTP) at 10 µm resolution with a GenePix 4000B laser scanner (Molecular Devices, Sunnyvale, CA, USA).
Data processing
The transcriptome comparisons of Δupp Δgnd zwf* vs. Δupp and of Δupp Δedd-eda vs. Δupp, and of Δupp Δgnd versus Δupp, were repeated independently three times in biological replicates. Quantitative image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0). For data normalization, GPR-files were processed using the BioConductor/R-packages limma and marray (http://www.bioconductor.org). For further analysis, the processed and loess-normalized data as well as detailed experimental information according to MIAME (Brazma et al., 2001) were stored in the in-house microarray database (Polen and Wendisch, 2004). To filter for differentially expressed genes, the following criteria had to be fulfilled: (i) flags 0 (GenePix Pro 6.0), (ii) signal/noise 5 for Cy5 (F635Median/B635Median, GenePix Pro 6.0) or Cy3 (F532Median/B532Median, GenePix Pro 6.0), (iii) mRNA ratio 2.0 (lower ones allowed in the case of operons) or 0.5 (higher ones allowed in the case of operons), (iv) in a paired Students t test, relative RNA levels had to be significantly different from all spots (p < 0.05).
Mutational analysis of the pentose phosphate pathway and the Entner-Doudoroff pathway in Gluconobacter oxydans reveals improved growth of an edd-eda deletion mutant on mannitol
