strains: HG001 wild type and HG001 asp23 mutant growth phase: exponential growth and stationary phase
Treatment protocol
An overnight culture (HG001 wild type or isogenic asp23 mutant) was used to inoculate 120 ml medium to an OD500 of 0.075. Samples were taken during exponential growth phase (OD500=0.5) and one hour after entering stationary growth phase.
Growth protocol
S. aureus was cultured in a defined synthetic medium. Synthetic medium was used as described by Gertz et al. (1999) but without MOPS and glycine and with a final glucose concentration of 0.75 µM.
Extracted molecule
total RNA
Extraction protocol
For total RNA isolation, S. aureus cells were cultivated in synthetic medium. For Northern blot analyses, RNA was isolated as described previously using the acid-phenol method (Majumdar et al., 1991) with modifications as described by Fuchs et al. (2007). For DNA microarray analyses the RNA was further purified to eliminate traces of remaining DNA (Reiß et al., 2012). For every strain RNA was extracted from three independent cultivations.
Label
Cy3
Label protocol
Total RNA was prepared from three independent experiments. Synthesis and purification of fluorescently labeled cDNA were carried out according to Charbonnier et al. (2005) with minor modifications. In detail, 10 µg of total RNA were mixed with random primers (Promega, Madison, WI, USA) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies, Santa Clara, CA, USA). The RNA/primer mixture was incubated at 70°C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen, Karlsruhe, Germany), 5 µl of 0.1 M DTT (Invitrogen, Karlsruhe, Germany), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare, Little Chalfont, UK) and 2 µl of SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). The reaction mixture was incubated at 42°C for 60 min and then heated to 70°C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen, Karlsruhe, Germany) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare, Little Chalfont, UK). The individual samples were labeled with Cy5, whereas the reference pool was labeled with Cy3.
strain: HG001 wild type growth phase: exponential growth
Treatment protocol
An overnight culture (HG001 wild type or isogenic asp23 mutant) was used to inoculate 120 ml medium to an OD500 of 0.075. Samples were taken during exponential growth phase (OD500=0.5) and one hour after entering stationary growth phase.
Growth protocol
S. aureus was cultured in a defined synthetic medium. Synthetic medium was used as described by Gertz et al. (1999) but without MOPS and glycine and with a final glucose concentration of 0.75 µM.
Extracted molecule
total RNA
Extraction protocol
For total RNA isolation, S. aureus cells were cultivated in synthetic medium. For Northern blot analyses, RNA was isolated as described previously using the acid-phenol method (Majumdar et al., 1991) with modifications as described by Fuchs et al. (2007). For DNA microarray analyses the RNA was further purified to eliminate traces of remaining DNA (Reiß et al., 2012). For every strain RNA was extracted from three independent cultivations.
Label
Cy5
Label protocol
Total RNA was prepared from three independent experiments. Synthesis and purification of fluorescently labeled cDNA were carried out according to Charbonnier et al. (2005) with minor modifications. In detail, 10 µg of total RNA were mixed with random primers (Promega, Madison, WI, USA) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies, Santa Clara, CA, USA). The RNA/primer mixture was incubated at 70°C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen, Karlsruhe, Germany), 5 µl of 0.1 M DTT (Invitrogen, Karlsruhe, Germany), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare, Little Chalfont, UK) and 2 µl of SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). The reaction mixture was incubated at 42°C for 60 min and then heated to 70°C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen, Karlsruhe, Germany) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare, Little Chalfont, UK). The individual samples were labeled with Cy5, whereas the reference pool was labeled with Cy3.
Hybridization protocol
300 ng of Cy5-labeled cDNA and 300 ng of Cy3-labeled cDNA were hybridized together to the microarray following Agilent's hybridization, washing, and scanning protocol (Two-Color Microarray-Based Gene Expression Analysis, version 5.5)
Scan protocol
Intensity values were extracted and processed (background subtraction and LOWESS normalization) by means of the Feature Extraction software (version 10.5, Agilent Technologies, Santa Clara, CA, USA).
Data processing
Data analysis was performed using the Cyber-T program (Baldi and Long, 2001). The parameters for the Bayesian standard deviation estimation were: sliding-window size of 301 and a confidence value for the Bayesian variance estimate of 10. Genes with a Benjamini & Hochberg corrected P value of <0.05 and at least a 2-fold change in the mean fluorescence value of the three hybridization experiments were considered biologically significant expression changes.
