Temporal transcriptome analysis of Bacillus subtilis NDmed in the submerged biofilm model

[Lien vers la fiche source] Investigation of the kinetics of whole genome gene expression level changes in Bacillus subtilis NDmed strain during formation of submerged biofilm and pellicle. The Bacillus subtilis NDmed strain analyzed in this study is able to form thick and highly structurated submerged biofilms as described in Bridier et al., (2011) The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging. PLoS ONE 6(1):e16177. A seven chip study using total RNA recovered from static cultures of Bacillus subtilis NDmed growing in TSB medium in 96-well microplates for different times after adhesion to the bottom of the wells. Each chip measures the expression level of 5,737 transcripts from Bacillus subtilis 168. 200 µL of an overnight culture of Bacillus subtilis NDmed in TSB (adjusted to an OD 600nm of 0.02) were added in each well of 96-well microtiter plates, incubated at 30°C for 90 min to allow the bacteria to adhere to the bottom of the wells. Wells were then rinsed with TSB to eliminate non-adherent bacteria and refilled with 200 µL of sterile TSB. For each time point (1h, 3h, 4h, 5h, 7h, 24h and 48h) 96-well plates were prepared. Total RNA was extracted as described by Nicolas et al., (2012) Science 335, 1103–1106 (PMID:22383849). RNA concentration was measured using a NanoDrop spectrophotometer; RNA quality was checked by analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies). For cDNA synthesis, 10 µg of total RNA were mixed with random primers (FairPlay III Microarray Labeling Kit) and spike-ins (One-Color RNA Spike-In Kit, Agilent Technologies) and incubated at 70°C for 10 min followed by 5 min incubation on ice. Then, first-strand Master Mix, Actinomycin D (final conc. 40 µg/ml) and AffinityScript HC Reverse Transcriptase were added. The reaction was incubated for 60 min at room temperature and for 60 min at 42°C. After hydrolyzing the RNA, cDNA was precipitated overnight at -20°C. NHS-ester dye coupling (CyDye Mono-Reactive Dye, GE Healthcare) and purification of labeled cDNA were performed according to the FairPlay III instructions. cDNA and Cy-dye concentrations were quantified by means of a NanoDrop spectrophotometer. 1200 ng of Cy3-labeled cDNA were hybridized to the tiling array following Agilent’s hybridization, washing and scanning protocol (One-Color Microarray-based Gene Expression Analysis, version 5.5). The microarray (BaSysBio Bacillus subtilis T3 array, 2x400K [Agilent-044473]) was scanned with Agilent Technologies Scanner, model G2505C. Grid: 044473_D_F_20121025. Protocol: GE1_107_Sep09. An aggregated expression value was computed for each Genbank annotated CDS and newly defined transcribed region as the median log2 expression signal intensity of probes lying entirely within the corresponding region as described in (Nicolas et al., 2012 PMID: 22383849). The expression intensity was computed from the raw intensity data using a model of signal shift and drift and correcting for probe affinity variations as described in (Nicolas et al., 2009, Bioinformatics 25, 2341-2347). Data were quantile normalized using LIMMA package.To control for possible cross-hybridization artefacts the sequence of each probe was BLAST-aligned against the whole chromosome sequence and probes with a SeqS value above the 1.5 cut-off were discarded (Wei et al., 2008 Nucl. Acids Res. 36, 2926-2938).