Computational protocol: Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans

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Protocol publication

[…] Total RNA was isolated from 50 ml aliquots of bacteria-growing NMMP cultures in Erlenmeyer flasks at 30°C with continuous shaking at 250 rpm at the late exponential phase of growth (approx. 24 h of growth) using the RNeasy midi Kit (Qiagen). Cell lysates were extracted twice with phenol-chloroform before being loaded onto RNeasy midi columns for RNA purification.Fluorescently labelled cDNA for microarray hybridisation was obtained using the SuperScript Indirect cDNA Labelling System (Invitrogen), following the supplier’s instructions. Twenty micrograms of RNA were transformed to cDNA with Superscript III reverse transcriptase using random hexamers as primers, adding aminoallyl-modified nucleotides to the reaction mixture. After cDNA purification, the Cy3 or Cy5 fluorescent dyes (Invitrogen) were coupled to the amino-modified first-strand cDNA. Labelling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies). Prior to the hybridisation process, Streptomyces coelicolor genome-wide DNA microarrays (Eurogentec, Belgium) were blocked by immersion into a 50 ml Falcon tube containing 5xSSC, 0.1% (w/v) SDS and 1% (w/v) bovine serum albumin, and preheated to 42°C. After 45 min at 42°C, the microarrays were washed by being briefly immersed in a Falcon tube containing sterile water at room temperature, followed, when necessary, by another immersion in isopropanol, before being allowed to dry.Equal amounts of Cy3- or Cy5-labelled cDNAs (about 50 pmoles each), one sample corresponding to the control and the other to the problem under analysis, were mixed and dried in a Speed-Vac. Each sample was dissolved in 45 ml of a solution containing 50% (v/v) deionised formamide, 5 x Denhardt’s solution, 6 x SSC, 0.5% (w/v) SDS, 5% (w/v) dextran sulphate, pre-filtered and pre-heated at 42°C. After 2 min at 90°C to denature the DNA, the solution was applied to the microarray slide and covered with a 24 x 60 mm cover glass. The slide was introduced into a hybridisation chamber and incubated for 18 h away from the light, following the microarray supplier’s instructions. The microarray was then transferred to a Falcon tube containing 0.5 x SSPE (1 x SSPE contains 150 mM NaCl, 1 mM EDTA, 11.5 mM NaH2PO4, PH 7.4), 0.5% (w/v) SDA and pre-heated to 37°C. After removing the cover glass, the microarray was washed by gentle shaking for 5 min. The slide was subsequently transferred to a new tube containing 0.5 x SSPE and 0.5% (w/v) SDS, and washed again by gentle shaking for 5 min at room temperature. Similar washes with 0.5 x SSPE were conducted three more times, followed by a final wash with 0.1 x SSPE at room temperature. The microarray was allowed to dry and scanned in a microarray scanner with green and red lasers operating at 532 and 635 nm, respectively, to excite the Cy3 and Cy5. Images were taken at 10 mm resolution and spot intensity was determined using the Genepix Pro 5.0 (Axon) software package.Hybridisation data were statistically analysed using LIMMA [] software. Three independent RNA extractions were conducted out for each experiment, the corresponding microarray analyses were performed and the information was provided by three biological replicas combined in each case. The results for each replica (median intensity for each channel) were normalised and statistically analysed using the LIMMA software package []. Background subtraction was performed using a method implemented in LIMA designed to yield positive corrected intensities (i.e. to avoid negative intensity values). A convolution of normal and exponential distributions was fitted to the foreground intensities using the background intensities as covariate. This resulted in a smooth monotonic transformation of the background-subtracted intensities in such a way that all the corrected ones were positive. Differential hybridisation was calculated using linear models and empirical Bayes moderated t-statistics [, ]. The resulting log-ratios were normalised for each array through print-tip loess [] and differential hybridisation values were scaled to achieve consistency among arrays. Each probe was tested for changes in differential hybridisation over replicates by using moderated t-statistics []. The p-values were adjusted for multiple testing, as described [], to control the false discovery rate. The output file provided the fold-change and p-values for each spot, among other data. Comparisons were performed using the Venn algorithm (http://bioinfogp.cnb.csic.es/tools/venny/index.html.) (http://www.pangloss.com/seidel/Protocols/venn.cgi). Operon prediction was carried out using the Microbesonline website (http://microbesonline.org). […]

Pipeline specifications

Software tools GenePix Pro, limma, VENNY
Databases MicrobesOnline
Application Gene expression microarray analysis
Organisms Streptomyces lividans