To investigate the effects of colicin M on the whole genome expression profile, an overnight culture of the

E. coli strain MG1655 (F-lambda-ilvG-rfb-50 rph-1) was grown as described above. One part was treated with colicin M (30 ng/ml), while the untreated part served as the control. For gene expression analysis by microarray and qPCR, total RNA was isolated from 2-ml samples removed from each flask following 30 min and 60 min incubations at 37°C. The experiments were repeated at least two times. For quantification of colanic acid, the growth conditions and the application of subinhibitory concentrations of colicin M were as described above. learn more Colanic acid was purified from 50 ml cultures treated with colicin M for 60 min, 90 min and 120 min at 37°C, with aeration. The experiment was repeated at least two times. RNA isolation Total RNA was extracted using the RNAProtect Bacteria Reagent (Qiagen) and RNeasy Mini kits (Qiagen), according to the manufacturer instructions. To remove residual DNA, on-column DNase digestion was performed during the RNA purification using the RNase-Free DNase Set (Qiagen). A Nanodrop ND 1000 spectrophotometer (Thermo Scientific) was used to confirm

total RNA concentrations, while an Agilent 2100 Bioanalyser (Agilent Technologies, CA, NVP-BGJ398 ic50 USA) was used to evaluate the RNA quality. The isolated RNA was stored at −80°C until use. Microarray procedures Gene expression analysis was performed using Affymetrix GeneChip® E. coli Genome 2.0 arrays. Target preparation, hybridization, washing, staining and scanning were performed as recommended by the Affymetrix GeneChip® Expression Analysis Technical Manual. The experiment was repeated at least two times. The acquisition of array images and the data quality assessment were performed using an Affymetrix

GeneChip Command Console. The GeneChip data was processed using several different R/Bioconductor packages. The Affymetrix raw data were normalized using the RMA algorithm from the XPS package. The data have been deposited in the NCBI Gene Expression Omnibus database (GEO, http://​www.​ncbi.​nlm.​nih.​gov/​geo) under GEO series accession number GSE37026. Annotation of the genes and the data representation was managed using the G protein-coupled receptor kinase ANNAFFY and AFFYCORETOOLS packages. The normalized data, converted to log2 values, were first limited to the ENTREZ-annotated probes from strain K12 (10208 probes). The remaining data were tested for differential expression, which was performed using the LIMMA package for the 30-min treated versus the 30-min untreated control and for the 60-min treated versus the 60-min untreated control bacterial culture. Differential expression was assessed using the 2-way factorial ANOVA model constructed using LIMMA package. Differential expression was assessed using the false discovery rate multiple test correction [82] and controlling type I error at α = 0.05.