genotype/variation: wild type treatment: 36 uM Fe, OD 4-5
Growth protocol
Corynebacterium glutamicum ATCC13032 strain served as the wild type in the present study. Cultivation was performed in CGXII minimal medium containing 4% or 2% (wt/vol) glucose as main carbon and energy source (Keilhauer et al.) and 195 uM protocatechuic acid (PCA, Frunzke et al., 2008). To create iron limitation conditions in C. glutamicum, the amount of ferrous iron in the medium was reduced to 1 uM (compared to a standard concentration of 36 uM), similar as published by us previously (Küberl et al., 2016). Briefly, a growth experiment was started with a first pre-culture (5 ml Brain-Heart-Infusion medium) shaken for 8 h at 170 rpm. The second pre-culture (20 ml CGXII minimal medium) was inoculated using 500 ul of the first pre-culture and shaken overnight (16 h). The second pre-culture was used to inoculate the main culture (50 ml CGXII medium) to an OD600 of 1 and growth was monitored using a spectrophotometer. For cultivation under iron limitation, the cells were washed in 0.9% (wt/vol) NaCl solution before inoculating the next cultures.
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 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. (2004) Mol. Microbiol. 54: 420-438.
genotype/variation: wild type treatment: 1 uM Fe, OD 4-5
Growth protocol
Corynebacterium glutamicum ATCC13032 strain served as the wild type in the present study. Cultivation was performed in CGXII minimal medium containing 4% or 2% (wt/vol) glucose as main carbon and energy source (Keilhauer et al.) and 195 uM protocatechuic acid (PCA, Frunzke et al., 2008). To create iron limitation conditions in C. glutamicum, the amount of ferrous iron in the medium was reduced to 1 uM (compared to a standard concentration of 36 uM), similar as published by us previously (Küberl et al., 2016). Briefly, a growth experiment was started with a first pre-culture (5 ml Brain-Heart-Infusion medium) shaken for 8 h at 170 rpm. The second pre-culture (20 ml CGXII minimal medium) was inoculated using 500 ul of the first pre-culture and shaken overnight (16 h). The second pre-culture was used to inoculate the main culture (50 ml CGXII medium) to an OD600 of 1 and growth was monitored using a spectrophotometer. For cultivation under iron limitation, the cells were washed in 0.9% (wt/vol) NaCl solution before inoculating the next cultures.
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 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. (2004) Mol. Microbiol. 54: 420-438.
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 and normalization of ratios reflecting the relative mRNA levels (Ratio of Medians, GenePix Pro), GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
