RNA was quantified by NanoDrop® spectrophotometer (NanoDrop Products, Wilmington, DE). cDNA was synthesized from the extracted RNA using the QuantiTech Reverse Transcription Kit (QIAGEN). Ipatasertib solubility dmso For qRT-PCR, a 200-ng aliquot of cDNA and 250 nM of specific primer (see Additional file 1) were mixed with SYBR Green PCR Master Mix (Life Technologies, Carlsbad, CA). Three independent biological replicates were used for RNA extraction. Additionally, each PCR reaction was set up in triplicate. The 30S ribosomal RNA gene rpsL was used as an internal standard to normalize the quantity of cDNA in different samples [58]. Gene expression analysis was done using StepOne Plus software

version 2.2.2 (Life Technologies). For RT-PCR, PCR was performed using the prepared cDNA and specific primers to amplify regions

of PA2782, and PA2782-PA2783 (see Additional file 1). As a positive control, genomic DNA extracted from PAO1 was amplified by PCR using the primers for PA2782-PA2783. PCR extension was conducted at temperatures appropriate for each primer. To exclude DNA contamination, each RNA sample was subjected to PCR without reverse transcriptase. The products were examined using 0.8% agarose gel electrophoresis. TnphoA mutagenesis This was done using the previously described method by Boquet et al.[34]. Plasmid pAB2 that carries PA2783 was transformed into E. coli strain CC102 that carries the F’ factor, F42 lacI3 zzf::TnphoA[34]. The transformants were selected on LB agar plates containing BB-94 concentration carbenicillin and kanamycin. Individual colonies were grown in LB broth, diluted and spread on LB agar plates containing carbenicillin, kanamycin (300 μg/ml), and chromogenic alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (XP) (40 μg/ml) (Sigma Aldrich). The high kanamycin concentration is essential to enrich for cells in which the TnphoA transposon has inserted in pAB2. Blue

color colonies indicative of alkaline phosphatase activity Cyclic nucleotide phosphodiesterase were VX-680 in vivo streaked on the XP plates to confirm the alkaline phosphatase production phenotype. Additionally, plasmid DNA was extracted from these colonies and transformed into the E. coli alkaline phosphatase deficient strain CC118. We confirmed the in-frame PA2783::phoA fusion by DNA sequence analysis using an appropriate primer (see Additional file 1). Cellular fractionation E. coli cells were fractionated using the cold shock osmotic procedure as described by Koshland and Botstein and Lee et al.[36, 42]. Fractionation of P. aeruginosa was conducted according to the procedure described by Cheng et al.[59]. Overexpression of rPA2783 (rMep72) and outer membrane preparation Plasmid pAB4 was transformed into the E. coli strain LMG194 and transformants were selected on LB agar with carbenicillin. Transformants were grown for 16 h at 37°C in RM minimal medium (Invitrogen) that was supplemented with 0.