Methicillin-resistant S. aureus (MRSA) SA137/93A, isolated from a tracheal secretion and displays heterogeneous intermediate vancomycin resistance (hVISA strain, MIC: 8 mg/L in BHI). Source: Reipert, A., K. Ehlert, T. Kast, and G. Bierbaum. 2003. Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin. Antimicrob.Agents Chemother. 47:568-576.
Treatment protocol
S. aureus cultures for RNA preparations were diluted 200-fold from fresh BHI broth overnight cultures and further incubated until they reached an OD600 ~ 1.0. CaCl2 was supplemented to all study cultures to a final concentration of 1 mM since Ca2+-ions enhance the bactericidal effect of mersacidin.
Extracted molecule
total RNA
Extraction protocol
Exponential-phase cultures of 10 ml for total RNA preparations were grown as aforementioned and fixed by incubation with two volumes of prewarmed RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) for 5 min at 37°C. The cells were subsequently harvested by 20 min of centrifugation at 4000 rpm and 37°C, and the pellets were shock-frozen in fluid nitrogen and kept at -70°C. The cells were lysed in the presence of 400 µg/ml lysostaphin (Genmedics, Reutlingen, Germany) and total RNA was extracted using the PrestoSpin R bug kit including DNase I treatment (Molzym, Bremen, Germany) following the manufacturer’s instructions. Quality and quantity of total RNA were confirmed by agarose gel electrophoresis and measured using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, USA).
Label
Cy3
Label protocol
Fluorescence-labeled single-stranded cDNA was obtained by reverse transcription of total RNA. To this end, three different aliquots of total RNA preparations from separate cultures of the respective experiment were pooled to a total amount of 9 µg (3 µg each) and transcribed into cDNA using 100 Units (U) of BioScript reverse transcriptase (Bioline, Luckenwalde, Germany) following the manufacturer’s instructions. For direct cDNA labeling, the total reaction volume of 40 µl contained 75 µg/ml pd(N)6 random hexamers (GE Healthcare - Amersham, NJ, USA), 0.1 mM CyDye3- or CyDye5-dCTPs (GE Healthcare - Amersham) aside from 0.2 mM dCTP, 0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dGTP and 25 U/ml RNase-OUT (Invitrogen, Karlsruhe, Germany). RNA was degraded by alkaline hydrolysis at 65°C and fluorescence-labeled cDNA was purified using the MinElute PCR purification kit (Qiagen). cDNA synthesis and Cy3/Cy5 incorporation was verified using the Nanodrop spectrophotometer (Nanodrop Technologies).
Methicillin-resistant S. aureus (MRSA) SA137/93A, isolated from a tracheal secretion and displays heterogeneous intermediate vancomycin resistance (hVISA strain, MIC: 8 mg/L in BHI). Source: Reipert, A., K. Ehlert, T. Kast, and G. Bierbaum. 2003. Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin. Antimicrob.Agents Chemother. 47:568-576.
Treatment protocol
S. aureus cultures for RNA preparations were diluted 200-fold from fresh BHI broth overnight cultures and further incubated until they reached an OD600 ~ 0.5. Next, mersacidin was added in subinhibitory concentrations and the cultures were further grown to an OD600 of ~ 1.0. CaCl2 was supplemented to all mersacidin study cultures to a final concentration of 1 mM since Ca2+-ions enhance the bactericidal effect of mersacidin
Extracted molecule
total RNA
Extraction protocol
Exponential-phase cultures of 10 ml for total RNA preparations were grown as aforementioned and fixed by incubation with two volumes of prewarmed RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) for 5 min at 37°C. The cells were subsequently harvested by 20 min of centrifugation at 4000 rpm and 37°C, and the pellets were shock-frozen in fluid nitrogen and kept at -70°C. The cells were lysed in the presence of 400 µg/ml lysostaphin (Genmedics, Reutlingen, Germany) and total RNA was extracted using the PrestoSpin R bug kit including DNase I treatment (Molzym, Bremen, Germany) following the manufacturer’s instructions. Quality and quantity of total RNA were confirmed by agarose gel electrophoresis and measured using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, USA).
Label
Cy5
Label protocol
Fluorescence-labeled single-stranded cDNA was obtained by reverse transcription of total RNA. To this end, three different aliquots of total RNA preparations from separate cultures of the respective experiment were pooled to a total amount of 9 µg (3 µg each) and transcribed into cDNA using 100 Units (U) of BioScript reverse transcriptase (Bioline, Luckenwalde, Germany) following the manufacturer’s instructions. For direct cDNA labeling, the total reaction volume of 40 µl contained 75 µg/ml pd(N)6 random hexamers (GE Healthcare - Amersham, NJ, USA), 0.1 mM CyDye3- or CyDye5-dCTPs (GE Healthcare - Amersham) aside from 0.2 mM dCTP, 0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dGTP and 25 U/ml RNase-OUT (Invitrogen, Karlsruhe, Germany). RNA was degraded by alkaline hydrolysis at 65°C and fluorescence-labeled cDNA was purified using the MinElute PCR purification kit (Qiagen). cDNA synthesis and Cy3/Cy5 incorporation was verified using the Nanodrop spectrophotometer (Nanodrop Technologies).
Hybridization protocol
Microarray transcriptional profiling by competitive hybridization of fluorescence-labeled cDNA was performed using the custom PCR product full-genome chip sciTracer (Scienion, Berlin, Germany) containing 2338 unique open reading frames representing 90% of the S. aureus N315 genome (NC_002745). Additional information on the microarray platform is described elsewhere. To increase reproducibility, each experiment was performed 4 times including a dye swap resulting in four chips per competitive comparison with the incorporated dye reversed on two separate arrays. All hybridizations were done with equal amounts of fluorescence-labeled cDNA probes or picomols of dye. Fluorescence-labeled cDNA probes were mixed in hybridization buffer (Scienion) in a total volume of 55 µl and denatured at 95°C for 2 min and subsequently applied to the microarray slide following incubation at 42°C for 72 hours under humidified conditions according to the manufacturer’s instructions. Hybridized microarrays were washed at room temperature in SSC buffer with decreasing salt concentrations (1 x SSC/0.3% SDS for 5 min, 0.2 x SSC for 5 min, 0.06 x SSC for 30s).
Scan protocol
The intensity of fluorescence of the microarray was scanned with a GenePix 4000B scanner (Axon Instruments/Distribution by Biozyme Scientific GmbH, Hessisch Oldendorf, Germany). TIFF images were captured and analysed with GenePixPro4.1 software (Axon Instruments). The actual signal intensity was calculated by substracting the mean value of the local background intensity from the mean value of the signal intensity of the individual spot.
Description
no additional information provided
Data processing
The data sets were then normalized by using Acuity 3.1 software (Axon Instruments) and by applying the LOWESS algorithm. Significant changes of gene expression were determined by implementing SAM (significance analysis of microarrays; http://www-stat.stanford.edu/~tibs/SAM/; (Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc.Natl.Acad.Sci.U.S.A 98:5116-5121.)) using the one class response and a false discovery rate of < 1% with a medium number of falsely called significant genes of <1.
