Cells were grown in BHI broth at 37°C with shaking (250 rpm) unless otherwise indicated. L. monocytogenes 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
8mL of RNAProtect was added to 4mL of stationary phase culture and vortexed. After 5 min incubation at room temperature, the bacterial cell and RNA protect mixture was centrifuged for 5 min at 5000xg and stored at -80C. 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 5μg random primers, incubated at 70°C for 10 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 for 2h. 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.
Cells were grown in BHI broth at 37°C with shaking (250 rpm) unless otherwise indicated. L. monocytogenes 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
8mL of RNAProtect was added to 4mL of stationary phase culture and vortexed. After 5 min incubation at room temperature, the bacterial cell and RNA protect mixture was centrifuged for 5 min at 5000xg and stored at -80C. 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 5μg random primers, incubated at 70°C for 10 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 for 2h. 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 immersed in blocking solution (1% BSA, 5X SSC, 0.1% SDS) at 42°C. After 1 h, the slides were washed for 5 min in 0.1x SSC twice, followed by 30 sec in deionized water and then dried by centrifugation. For the strains to be compared, labeled cDNAs were combined and dried, then suspended 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
Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Description
Expression profiles were measured for 4 independent biological replicates. Array data were analyzed using a one way ANOVA model to compare differences between strains, and a two way ANOVA model to identify genes regulated by SigB and PrfA.
Data processing
Raw intensity values for all probes on each array were normalized using pin-tip LOWESS in R v.2.2.1 with the MAANOVA (v. 0.98-8) package. Signals from two replicate probes on each array were averaged and log2 transformation applied after normalization. Differences in transcript levels between strains were determined using a mixed model one way ANOVA in R/MAANOVA, where the log transformed intensity data, Y = array + dye + strain + sample (biological replicate) + E (error). Contribution of the transcriptional regulator PrfA and sigma factor SigB to changes in transcript levels was determined using a mixed model two way ANOVA, where the log transformed intensity data, Y = array + dye + prfA + sigB + prfA*sigB + sample + E. In this model, the factors prfA and sigB have two levels, determined by whether or not the protein is active or inactive. The ANOVA modeling allows for consideration of appropriate error structures for experiments with multiple sources of variation in microarray measurements. The random effects of the models were biological replicate and array effects, whereas the fixed effects were strain or prfA and sigB effects as well as dye effects. The Fs statistic, a shrinkage estimator for gene-specific variance components that makes no assumptions about the distribution of variances across genes, was estimated. Significant differences in expression between strains were determined by calculating the p-values for the Fs statistic for each gene using 1000 random permutations. The p-values were adjusted to correct for type I error with the Benjamini-Hochberg (B-H) linear step-up correction implemented in R/MAANOVA and a cutoff adjusted p-value < 0.05. Pair wise contrasts of time points were estimated by the t-test in R/MAANOVA. Contrast p-values were corrected for multiple testing by using the B-H step-up correction.
