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DETAILED MATERIAL AND METHODS FOR WECKE ET AL. 2008
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Daptomycin versus Friulimicin B: In-depth Profiling
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of the Bacillus subtilis Cell Envelope Stress Responses
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Strains, media and growth conditions. B. subtilis strains were routinely grown in LB medium
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at 37C with aeration, except where stated otherwise. Erythromycin (1 g/ml) plus lincomycin (25
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g/ml) for MLS resistance were used for the selection of strain BFS2469, tetracycline (10 µg/ml) was
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used for selection of strain TMB389. FRI was obtained from MerLion Pharmaceuticals GmbH, and
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other drugs from their respective manufacturers.
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RNA preparation. B. subtilis 168 wild type strain was grown aerobically at 37°C in LB medium
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to mid-log phase. The culture was split and induced with FRI or DAP (1 µg/ml each) with one sample
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remaining as the uninduced control. After 10 min of induction 30 ml of each sample were mixed with
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15 ml cold killing buffer (20 mM Tris-HCl pH 7.0, 5 mM MgCl2, 20 mM NaN3), harvested by
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centrifugation and frozen in liquid nitrogen. For cell disruption, the pellet was resuspended in 200 µl
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killing buffer, immediately dropped into the Teflon vessel (filled and pre-cooled with liquid nitrogen),
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and then disrupted with a Mikro-Dismembrator U (Sartorius). The resulting cell powder was
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resuspended in 3 ml of lysis solution (4 M guanidine-thiocyanate, 0.025 M Na-acetat pH 5.2, 0.5% N-
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lauroylsarcosinate) and the RNA was extracted twice by phenol/chloroform/isoamylalcohol 25/24/1
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followed by chloroform/isoamylalcohol 24/1 extraction and ethanol precipitation. RNA samples were
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DNase-treated with the RNase-free DNase kit (Qiagen) according to the manufacturer’s instructions
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and purified using RNeasy mini columns (Qiagen). The quality control of the RNA preparations was
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performed with the RNA 6000 Nano LabChip Kit (Agilent Technologies) on the Agilent 2100
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Bioanalyzer according to the manufacturer’s instructions.
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DNA microarray analysis. The RNA samples obtained from three independent cultivations
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were used for independent cDNA synthesis and DNA array hybridization. Generation of the Cy3/Cy5-
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labeled cDNAs and hybridization to B. subtilis whole-genome DNA microarrays (Eurogentec) were
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performed as described (6). The slides were scanned with a ScanArray Express scanner
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(PerkinElmer). Quantitation of the signal and background intensities was carried out using the
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ScanArray Express image analysis software.
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Transcriptome data analysis. Data was analyzed using the GeneSpring software (Agilent
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Technologies). Raw signal intensities were first transformed by intensity dependent LOWESS
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normalization. The normalized array data were subjected to a statistical analysis using Cyber-T, a
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program based on a t-test combined with a Bayesian statistical framework (2). The software is
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accessible through a web interface at http://cybert.microarray.ics.uci.edu. The mRNA abundance was
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considered to be significantly different between the untreated control samples and the samples
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obtained after treatment with the respective antibiotic if (i) the Cyber-T Bayesian P value was < 0.001
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and (ii) the average fold change was at least 3 in three independent experiments. The potential and
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known
functions
of
the
encoded
proteins
1
were
initially
inferred
from
the
SubtiList
1
(http://genolist.pasteur.fr/SubtiList/) or BSORF databases (http://bacillus.genome. ad.jp/). An in-depth
2
analysis of the identified marker genes (as listed in Table 2) was performed using the SMART (7, 9)
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and
4
http://www.microbesonline.org/, respectively.
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MicrobesOnline
(1)
databases,
at
http://smart.embl-heidelberg.de/
and
Quantitative real time RT-PCR. Measurement of transcript abundance was performed by
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quantitative real-time RT-PCR using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad)
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according to the manufacturer’s procedure with minor modifications: In brief, 100 ng of DNA-free total
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RNA was used in a total reaction volume of 20 µl with 0.3 µM of each primer (see Table 1). The
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amplification reaction was carried out in an MyiQ Cycler (BioRad) using the following program: reverse
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transcription at 50°C for 10 min, followed by a 95°C denaturing/activation step for 5 min, followed by
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45 cycles (95°C for 10 sec), (60°C for 30 sec). After a subsequent denaturation (95°C for 1 min) and
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annealing (55°C for 1 min) the setpoint temperature was increased in 80 cycles (10 sec each) by
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0.5°C/cycle, starting from 55°C, to determine the melting temperatures of the PCR products.
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Expression of rpsJ and rpsE was monitored as constitutive reference. These genes were chosen due
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to their stable expression behaviour under various growth and stress conditions in B. subtilis (data not
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shown). Expression of liaI and genes encoding ECF  factors was calculated as fold changes using
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the formula: Fold change = 2-Ct; with -Ct = (Ct(gene x)-Ct(constitutive gene))condition I – (Ct(gene x)-Ct(constitutive
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gene))condition II (11).
Concentration-dependent killing curve/β-galactosidase assays. These experiments were
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performed as described (8). In brief, strain BFS2470 was grown in LB medium with MLS selection to
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OD600 ~ 0.5 and DAP/FRI were added to a final concentration ranging from 0.01 to 50 g/ml. An
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uninduced culture was used as a negative control. The cultures were incubated with aeration at 37°C.
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A sample was taken after 30 min for -galactosidase assay and the turbidity of the remaining culture
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was measured for at least 5 hours to monitor the concentration-dependent effects of the antibiotics on
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growth.
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L-[35S]methionine labelling of proteins and 2D-PAGE analysis. B. subtilis 168 wild type
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strain was grown aerobically at 37°C in Belitsky minimal medium (10) to mid-log phase. 10 and 30
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minutes after addition of DAP or FRI (1,5 and 1,0 µg/ml, respectively) the cells as well as untreated
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control cells were labelled with 15 µCi/ml [35S]-methionine. After 5 minutes incorporation of radioactive
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methionine, the reaction was stopped by adding an excess of nonradioactive methionine (1mM) and
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chloramphenicol (100µg/ml) to stop translation. Samples were taken and the cytoplasmic protein
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fraction isolated as described earlier (3). 2D-PAGE using Immobiline dry strips (IPG, Amersham
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Biosciences) (pH 4-7) loaded with 80 µg protein extract as well as visualization of radiolabelled
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proteins and dual channel imaging using Delta2D software (Decodon) were carried out as described
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(4, 5).
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