1 M Decitabine phosphate buffer pH 7.0 (PB). The pellet was resuspended in 2 ml PB with addition of 100 μg/ml lysozyme and 1 mM EDTA pH 8.0 and incubated at room temperature for 10 minutes. Cells were disintegrated using a French Press and centrifuged as above to remove unbroken cells. The low-speed centrifugation supernatant was then centrifuged at 30,000 × g for 30 minutes at 4°C to separate the cytoplasm (supernatant) and the membrane fraction (pellet). The pellet was resuspended in 1 ml of PB. Protein concentrations were determined and 25 μg of total
proteins was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Bands of interest were excised from the gel and the corresponding proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of the peptide generated by in-gel trypsin digestion ([35]; performed by CEINGE, University of Naples, Italy http://www.ceinge.unina.it/). Measurement of gene expression by Real Time-PCR Gene expression determination was performed using Real Time-PCR as previously described [29]. RNA was extracted from bacterial cultures grown as for membrane protein extraction. Production
of cDNAs was obtained by reverse transcription using 1 μg total RNA, along with negative control samples incubated without reverse transcriptase. Primer sequences for genes of interest were designed based on the available genome sequences for A. baumannii and were tested in PCR experiments on A. baumannii SMAL genomic DNA to verify the Selleckchem Rapamycin presence of the gene and the correctness of the expected products. Primer sequences were as follows: fchR_for: 5′-ACGTCAAGCGGTTGCTCCAT-3′, fchR_rev: 5′-CCTGTAATCGGGTCTGTTGG-3′, tonB_for: 5′-ATGGCAAGATACCGATGCCC-3′, tonB_rev: 3-mercaptopyruvate sulfurtransferase 5′-CCGATATCTTCGCTTGAGCG-3′, csuC_for: 5′-GCCCGCCTGTAGCCAAAATT-3′, csuC_rev: 5′-GAAGCATCTTGCTCGTTGCC-3′, csuE_for: 5′-TAGCGGGCCTGATGGCAATT-3′, csuE_rev: 5′-ACCCAGGGCTCTCAAAGAAG-3′, 16S_for: 5′-TGTCGTCAGCTCGTGTCGTGA-3′, 16S_rev: 5′-TGATGACTTGACGTCGTCCCC-3′.
Each Real Time PCR experiment was performed in triplicate and included negative control samples, which never showed significant threshold cycles. The relative transcript amounts were determined using 16S rRNA as the reference gene ([CtGene of interest-Ct16S] = ΔCt value). The results are the average of at least three independent experiments showing standard deviations ≤10%. Other methods Resistance to desiccation was performed as described in [29]. Sensitivity to oxidative stress was determined by treatment with hydrogen peroxide (H2O2), as described previously [50]. Transmission electron microscopy analysis was performed as described [51]. Acknowledgements We would like to thank M. Spalla for her excellent technical collaboration and L. Dolzani for providing A. baumannii strains RUH134 and RUH875.