Labeled cDNAs were combined, mixed with Agilent hybridization buffer, and competitively hybridized to custom-designed Agilent microarrays according to the manufacturer’s

instructions (Agilent). Data extraction and normalization was QNZ ic50 performed using Agilent Feature Extraction Software 9.5.3.1 (Agilent). The custom-designed arrays contain 9–11 probes covering a region around the translational start site (−300 to +200 relative to the translational start site +1) of each gene. Only those probes downstream of the translational start site were considered for estimating the fold change in gene expression. Ratios obtained for probes corresponding to the same gene were averaged and genes showing a ratio log2 (mutant/parental) < −1 https://www.selleckchem.com/products/epoxomicin-bu-4061t.html or log2 (mutant/parental) > 1 in all three biological replicates were considered as differentially expressed between the strains analyzed. Complete microarray dataset was deposited in GEO (GSE 32406). Cell fractionation and Western blot analysis Protein extracts were obtained from cultures of parental strain NA1000 and a CC3252 mutant with both C131 and C181 replaced for serine before and after treatment with 55 μM dichromate for 30 min. Cells were cultured until OD600 0.5, harvest by centrifugation and washed once with 0.2 M Tris–HCl pH 8.0.

Cells were then resuspended in 1 ml 60 mM Tris–HCl pH 8.0, 0.2 M sucrose, 0.2 mM EDTA, 200 μg ml-1 of lysozyme and incubated for 10 min at room temperature. After brief

Silibinin sonication (three 10 s pulses), cell debris were removed and the supernatant was centrifuged at 150,000 x g for 1 h. The pellet was washed once with 60 mM Tris–HCl pH 8.0 and resuspended in 1 ml 60 mM Tris–HCl pH 8.0, 0.2 M sucrose, 0.2 mM EDTA. Equal amounts of total protein (20 μg) were resolved through SDS-PAGE and transferred to nitrocelulose membrane, as previously described [45]. Membranes were incubated overnight at 4°C with anti-σF (1:500) [16] or anti-FtsH (1:2000) (kindly provided by T. Ogura, Kumamoto University, Japan) antibody in 10 mM Tris–HCl pH 8.0 containing 150 mM NaCl, 0.02% Tween 20, and 0.03% Triton X-100. The blots were developed using fluorescent CF680 Goat Anti-Rabbit IgG (1:10000- Uniscience) and imaged using Odyssey Imager- LI-COR (Biosciences). Promoter activity assay β-galactosidase assays were carried out with cells carrying a CC3255-lacZ transcription fusion (pCKlac54-1 or pCKlac54-2) or a sigF-lacZ transcription fusion (pCKlac53-1 or pCKlac53-2). For that, cells were cultured to exponential phase, harvested and used for the enzymatic assay. The empty ARN-509 plasmid placZ290 [46] was used as the control in the experiments. β-galactosidase activity was measured as previously described [41]. All experiments were performed in duplicates and repeated on three different occasions. Stress sensitivity tests Exponentially growing cells were exposed to 55 μM dichromate or kept under unstressed conditions.