When applicable, cultures were pretreated with 50 mM of the catalase inhibitor 3-amino-1,2,4-triazole (AT) for 60 min. Bacteria were aerobically grown in 50 mL of LB broth using 250-mL Erlenmeyer flasks on an orbital shaker (200 r.p.m.) at Dasatinib solubility dmso 30 °C. When cultures reached an OD600 nm of 0.4, aliquots of 15 mL were exposed to UVB radiation in disposable covered Petri plates (2.0–3.0 W m−2 for 30–60 min). Radiation intensity was measured under the plastic lid using the UVB/UVA radiometer described above. After exposure, culture aliquots were subjected to serial dilutions in four replicates and spread onto LB agar plates for later counting

of CFU. Cells grown to exponential phase (OD600 nm∼0.8), were disrupted by sonic oscillation (30 s, Branson Sonifier 250) in 20 mM Tris-HCl containing 5 mM EDTA, 100 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and 14 mM β-mercaptoethanol. Lysates were cleared by centrifugation (10 000 g, 15 min) and protein concentration was estimated in the supernatant by a dye-binding assay (Bradford, 1976) using bovine serum albumin as standard. SOD activity was visualized in situ after electrophoresis of the corresponding cellular lysates in nondenaturing polyacrylamide gels as described previously (Beauchamp & Fridovich, 1971), using inhibition by H2O2 and KCN to determine the metal identity in the enzyme

(Donahue et al., 1997). SOD activity was also determined spectrophotometrically by inhibition of the xanthine/xanthine oxidase-induced reduction of cytochrome c at PAK6 pH 7.8 (McCord & Fridovich, 1969). Catalase www.selleckchem.com/products/MLN-2238.html activity was visualized

in situ after electrophoresis in nondenaturing polyacrylamide gels, as described previously (Scandalios, 1968). Spectrophotometric measurements were carried out by following the decay of H2O2 at 240 nm (Aebi, 1984). To evaluate the effect of pro-oxidants and UV radiation on the antioxidant activities in the studied strains, cell-free soluble extracts were obtained using the same protocol described above after the challenge was performed. Fragments of 800 bp of the 16S rRNA genes from the HAAW isolates Ver3, Ver5, Ver7 and N40 were subjected to sequence alignment using the clustal x 2.0.9 program (Larkin et al., 2007) including 30 available Acinetobacter NCBI entries (base 7 to base 821 of A. baumannii DSM 30007, accession number X81660.1). Figure 1 shows the resulting unrooted tree after applying the NJ algorithm (Saitou & Nei, 1987). The Ver5 and N40 isolates clustered together including seven A. lwoffii strains. When compared pairwise, Ver5 and N40 strains showed 99.26% of DNA sequence identity between them, and 99.37% and 99.27% with A. lwoffii DSM 2403 DNA, respectively (not shown). Although this similarity does not confirm Ver5 and N40 species identity with A. lwoffii, it indicates a close phylogenetic relationship among them (Achtman & Wagner, 2008). Ver3 and Ver7 strains presented 98.02% and 97.76% of DNA sequence identity with A.