Listeria monocytogenes 10403S grown at 37°C to early stationary phase.
Treatment protocol
Cells were grown in BHI broth at 37°C with shaking (250 rpm; Series 25 Incubator Shaker, New Brunswick Scientific) unless otherwise indicated. All optical density measurements (OD) were made with a model DU640 spectrophotometer (Beckman); dilutions were made as appropriate in order to obtain OD readings in the linear range of the spectrophotometer. L. monocytogenes 10403S was streaked onto brain heart infusion (BHI) agar from –80°C glycerol stock cultures followed by incubation at 37°C for 24 h. A single colony was subsequently inoculated into a tube containing 5 ml BHI broth and grown overnight. 50 μl of this overnight culture was then added to a tube of 5 ml fresh, pre-warmed BHI broth and grown to logarithmic phase (OD600=0.4), then diluted 1:100 in fresh, pre-warmed BHI broth (500 μl into 50 ml broth in a 300ml side-arm flask). Cells were grown to stationary phase, which is defined as growth to an OD600=1.0 followed by incubation for an additional 3 h. A total of 10 ml of this stationary phase culture was added to 20 ml (2X volume) of RNAProtect Bacteria Reagent (Qiagen) to stabilize RNA, then vortexed, and incubated at room temperature for 5 min. Cells were then pelleted by centrifugation at 5000 rpm for 5 min (Avanti J-25 Centrifuge, Beckman). Supernatant was removed and cell pellets were stored at -80°C until RNA isolation.
Growth protocol
Cells were grown in BHI broth at 37°C with shaking (250 rpm) unless otherwise indicated. L. monocytogenes 10403S was streaked onto brain heart infusion (BHI) agar from –80°C glycerol stock cultures followed by incubation at 37°C for 24 hr. A single colony was subsequently inoculated into BHI broth and grown overnight. An aliquot of this overnight culture was then diluted 1:100 in fresh pre-warmed BHI and grown to OD600 = 0.4, then diluted 1:100 in fresh pre-warmed BHI. After growth to early stationary phase (OD600=1.0+3hr), cells were harvested for RNA isolation.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from frozen bacterial pellets with the Qiagen RNeasy Midi kit according to the manufacturer’s protocol for enzymatic and mechanical disruption, except that cells treated with lysozyme (20mg/ml) followed by sonication on ice for three 30 s bursts at 18–21 W (30 s pauses between bursts). Following RNA isolation, RQ1 DNase treatment (40 U; Promega) of total RNA in the presence of RNasin Plus RNA inhibitor (400 U; Promega). DNase treatment was incubated at 37°C for 1 h, followed by subsequent phenol:chloroform (2x) and 100% chloroform (1x) extractions. RNA was ethanol-precipitated overnight, centrifuged and washed with 70% ethanol, centrifuged and washed with 100% ethanol, before resuspenson of the final RNA pellet in RNase-free water. Total nucleic acid concentration and purity were estimated using absorbance readings (260/280 nm, 260/230 nm) on a NanoDrop ND-1000 spectrophotometer. RNA integrity was checked by agarose gel electrophoresis.
Label
Alexa Fluor 635
Label protocol
cDNA was synthesized from purified total RNA and labeled with Alexa Fluor dyes using the SuperScript Plus Indirect cDNA Labeling System according to the manufacturer's protocol with following modifications. 10 μg total RNA was combined with 1 μl of random primers included in the kit, incubated at 70°C for 5 min. Following a 5 min on ice, 6 μl 5X First-Strand buffer, 1.5 μl 0.1M dithiothreitol (DTT), 1.5 μl dNTP mix containing aminoallyl-dATP and aminohexyl-dTTP, 1 μl RNaseOUT and 2 μl SuperScript III Reverse Transcriptase were added to the reaction tube. The reverse transcription reaction was carried out at 42ºC overnight. Remaining RNA was hydrolyzed by addition of 15 μl 1N NaOH (70ºC for 10 min) and then neutralized with 15 μl 1N HCl. Each cDNA sample was purified with the QIAquick PCR purification kit (Qiagen) using RNase-free water for the final elution step. cDNA samples were dried in a SpeedVac until reduced to ~3 μl. Each cDNA sample was combined with 2X Coupling Buffer and then added to a vial of DMSO-suspended Alexa Fluor® Reactive Dye and the dye coupling reaction was carried out at room temperature overnight. Labeled cDNA samples were purified with the QIAquick PCR purification kit with an additional 35% Guanidine-HCl wash step and a final elution step in RNase-free water. Purified, labeled cDNA was quantified with UV spectrophotometer readings at wavelengths appropriate to determine the frequency of incorporation (FOI), which is defined as the number of labeled nucleotides incorporated per 1,000 nucleotides of cDNA. cDNA samples with FOIs between 20-50 were used for microarray hybridization.
Listeria monocytogenes FSL A1-254 (sigB null mutant) grown at 37°C to early stationary phase.
Growth protocol
Cells were grown in BHI broth at 37°C with shaking (250 rpm; Series 25 Incubator Shaker, New Brunswick Scientific) unless otherwise indicated. All optical density measurements (OD) were made with a model DU640 spectrophotometer (Beckman); dilutions were made as appropriate in order to obtain OD readings in the linear range of the spectrophotometer. L. monocytogenes FSL A1-254 was streaked onto brain heart infusion (BHI) agar from –80°C glycerol stock cultures followed by incubation at 37°C for 24 h. A single colony was subsequently inoculated into a tube containing 5 ml BHI broth and grown overnight. 50 μl of this overnight culture was then added to a tube of 5 ml fresh, pre-warmed BHI broth and grown to logarithmic phase (OD600=0.4), then diluted 1:100 in fresh, pre-warmed BHI broth (500 μl into 50 ml broth in a 300ml side-arm flask). Cells were grown to stationary phase, which is defined as growth to an OD600=1.0 followed by incubation for an additional 3 h. A total of 10 ml of this stationary phase culture was added to 20 ml (2X volume) of RNAProtect Bacteria Reagent (Qiagen) to stabilize RNA, then vortexed, and incubated at room temperature for 5 min. Cells were then pelleted by centrifugation at 5000 rpm for 5 min (Avanti J-25 Centrifuge, Beckman). Supernatant was removed and cell pellets were stored at -80°C until RNA isolation.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from frozen bacterial pellets with the Qiagen RNeasy Midi kit according to the manufacturer’s protocol for enzymatic and mechanical disruption, except that cells treated with lysozyme (20mg/ml) followed by sonication on ice for three 30 s bursts at 18–21 W (30 s pauses between bursts). Following RNA isolation, RQ1 DNase treatment (40 U; Promega) of total RNA in the presence of RNasin Plus RNA inhibitor (400 U; Promega). DNase treatment was incubated at 37°C for 1 h, followed by subsequent phenol:chloroform (2x) and 100% chloroform (1x) extractions. RNA was ethanol-precipitated overnight, centrifuged and washed with 70% ethanol, centrifuged and washed with 100% ethanol, before resuspenson of the final RNA pellet in RNase-free water. Total nucleic acid concentration and purity were estimated using absorbance readings (260/280 nm, 260/230 nm) on a NanoDrop ND-1000 spectrophotometer. RNA integrity was checked by agarose gel electrophoresis.
Label
Alexa Fluor 532
Label protocol
cDNA was synthesized from purified total RNA and labeled with Alexa Fluor dyes using the SuperScript Plus Indirect cDNA Labeling System according to the manufacturer's protocol with following modifications. 10 μg total RNA was combined with 1 μl of random primers included in the kit, incubated at 70°C for 5 min. Following a 5 min on ice, 6 μl 5X First-Strand buffer, 1.5 μl 0.1M dithiothreitol (DTT), 1.5 μl dNTP mix containing aminoallyl-dATP and aminohexyl-dTTP, 1 μl RNaseOUT and 2 μl SuperScript III Reverse Transcriptase were added to the reaction tube. The reverse transcription reaction was carried out at 42ºC overnight. Remaining RNA was hydrolyzed by addition of 15 μl 1N NaOH (70ºC for 10 min) and then neutralized with 15 μl 1N HCl. Each cDNA sample was purified with the QIAquick PCR purification kit (Qiagen) using RNase-free water for the final elution step. cDNA samples were dried in a SpeedVac until reduced to ~3 μl. Each cDNA sample was combined with 2X Coupling Buffer and then added to a vial of DMSO-suspended Alexa Fluor® Reactive Dye and the dye coupling reaction was carried out at room temperature overnight. Labeled cDNA samples were purified with the QIAquick PCR purification kit with an additional 35% Guanidine-HCl wash step and a final elution step in RNase-free water. Purified, labeled cDNA was quantified with UV spectrophotometer readings at wavelengths appropriate to determine the frequency of incorporation (FOI), which is defined as the number of labeled nucleotides incorporated per 1,000 nucleotides of cDNA. cDNA samples with FOIs between 20-50 were used for microarray hybridization.
Hybridization protocol
Microarray slide blocking was performed prior to hybridization. Slides were incubated for 10 min in a solution containing 0.176M succinic anhydride and 44mM sodium borate in methyl pyrrilidone, followed by 1-min washes in fresh methyl pyrrilidone (1x), deionized water (1x), 0.1% SDS (3x), deionized water (5x), and 95% EtOH (1x). The slides were dried by centrifugation and immersed in blocking solution (0.1% BSA, 5X SSC, 0.1% SDS) at 42°C. After 1 h, the slides were washed for 1 min each in deionized water (5x) and 95% EtOH (2x) and then dried by centrifugation. For the strains to be compared (parent and ΔsigB), labeled cDNAs were combined and dried, then resuspended in 50μl of hybridization buffer (5X SSC, 0.1% SDS, 0.1mM dithiothreitol (DTT), 0.5X formamide, 0.06 μg/μl salmon sperm DNA in DEPC-treated water). Samples were vortexed for 1 min and denatured at 95°C for 5 min (repeated twice), followed by a brief centrifugation, then applied to the slides using mSeries LifterSlips™ (Erie Scientific). Following overnight hybridization at 42°C, the slides were washed in 2X SSC+0.1% SDS (5 min, 42˚C), with subsequent 5-min washes at room temperature in 2X SSC+0.1% SDS, 2X SSC, and 0.2X SSC, followed by final rinse in deionized water. Slides were dried by centrifugation.
Scan protocol
Images were scanned using a GenePix 4000B scanner (Molecular Devices Corp.) using the following general scanning parameters: pixel size=10, focus position=20, lines to average=1, laser power=100%. The auto-PMT function was used to scan the slides with an acceptable PMT range of 400-900. Raw TIFF images were automatically gridded and analyzed using GenePix Pro 6.0 software.
Description
Expression profiles were measured from three independent RNA extractions, resulting in six replicate spots per gene (array contains duplicate spots).
Data processing
Analyses of microarray data were performed in R with Bioconductor using LIMMA software (Smyth, 2005). Background correction was performed using the “normexp” method. For normalization within arrays, the data were weighted for the genomic DNA controls and normalized using print-tip loess method (Smyth & Speed, 2003). The data were then scale normalized between arrays. The correlation between duplicate spots was calculated (Smyth et al., 2005) and a linear model was fitted to the normalized log ratios and empirical Bayes statistics were applied for differential expression analysis.
