Outer membrane proteins used in proteoliposomes were purified as described by Calderón et al. (2011). E. coli Top10 cells carrying pBAD-ompA or pBAD-ompW were grown in 500 ml to OD600 ~ 0.6 at 37°C and overexpression was performed for 5 h in the presence of 1 mM arabinose. His-tagged porins were purified by affinity chromatography using HisTrap HP columns (Amersham) according to the manufacturer’s instructions. Plasmid pBAD-ompW was generated amplifying the coding region of S. Typhimurium ompW by PCR using primers 5′ ATGAAAAAATTTACAGTGGC 3′ (pBAD-ompWF) and 5′ GAAACGATAGCCTGCCGAG 3′ (pBAD-ompWR) and cloned into Smoothened inhibitor plasmid pBAD-TOPO TA® (Invitrogen) according to the manufacturer’s instructions.

Insertion was verified by DNA sequencing. RNA isolation and ompW mRNA detection Overnight cultures were diluted (1:100) and cells CX-5461 chemical structure were grown to OD600 ~ 0.4. Genetically complemented cells (∆arcA/pBAD-arcA and ∆arcB/pBAD-arcB) were grown in the presence of arabinose (1 mM) and ampicillin (100 μg ml-1).

At this point, H2O2 (1.5 mM) or NaOCl (530 μM) was added and cells were grown for 20 min. Control cells received no treatment. After exposure to the toxic compounds, 4 ml were withdrawn from the culture and used to extract total RNA using GenElute Total RNA purification Kit® (Sigma). Total RNA treatment with DNase I and cDNA synthesis was performed as previously described [19]. Relative quantification of ompW mRNA was performed using Brilliant

II SYBR Green QPCR Master Reagent Kit and the Mx3000P detection system (Stratagene). 16S rRNA Protein kinase N1 was used for normalization. Specific primers were 5′ ATGAAAAAATTTACAGTGG 3′ (RTompWF) and 5′ GAAACGATAGCCTGCCGA 3′ (RTompWR) for the ompW gene; 5′ GTAGAATTCCAGGTGTAGCG 3′ (16SF) and 5′ TTATCACTGGCAGTCTCCTT 3′ (16SR) for 16S rRNA gene (16S). The reaction mixture was carried out in a final volume of 20 μl containing 1 μl of diluted cDNA (1:1000), 0.24 μl of each primer (120 nM), 10 μl of 10 x Master Mix, 0.14 μl of diluted ROX (1:200) and 8.38 μl of H2O. The reaction was performed under the following conditions: 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 53°C and 45 s at 72°C. Finally a melting cycle from 53 to 95°C was performed to check for HSP cancer amplification specificity. Amplification efficiency was calculated from a standard curve constructed by amplifying serial dilutions of RT-PCR products for each gene. These values were used to obtain the fold change in expression for the gene of interest normalized with 16S levels according to [47]. Experiments were performed in three biological and technical replicates. DNA binding assays Non-radioactive EMSAs were performed as described [48]. Briefly, increasing amounts of purified ArcA (phosphorylated and unphosphorylated) were incubated with 20 or 60 ng of PCR product(s) in binding buffer (100 mM Tris-Cl [pH 7.4], 100 mM KCl, 10 mM MgCl2, 10% glycerol, and 2 mM dithiothreitol) for 20 min at 30°C.