, 2008). Plates were incubated either with or without ferric chloride (1 mM) and 2, 2′-dipyridyl (0.5 mM) at 30 °C. Pseudomonas aeruginosa cells were grown in LB to exponential phase. The cells were incubated at 30 °C for 24 h without agitation. Subsequently, the aggregation percentage was obtained according to a previous report GDC941 (Liu et al., 2008). For the

swimming and swarming assay, 2 μL of cells grown overnight were inoculated in plates with modified M9 medium, [20 mM NH4Cl; 12 mM Na2HPO4; 22 mM KH2PO4; 8.6 mM NaCl; 1 mM MgSO4; 1 mM CaCl2 2H2O; 10 mM glucose; 0.5% casamino acids (Difco)] solidified with Bacto-agar (Difco; swimming 0.2%; swarming 0.5%) for 24 h at 30 °C. The pyocyanin assay is based on the absorbance of pyocyanin at 520 nm in acidic solution (Essar et al., 1990). A 5-mL sample of culture grown in LB was extracted with 3 mL

of chloroform and then re-extracted into 1 mL of 0.2 N HCl to give a pink to deep red solution. The absorbance of this solution was measured at 520 nm. To measure pyoverdine production, bacteria were grown in LB to stationary phase and the absorbance of the culture supernatants was measured. Pyoverdine has a characteristic absorbance spectrum with a peak at 403 nm (Hohnadel et al., 1986). Spectral analysis of CFS was performed using an Optizen 2120 UV/VIS spectrophotometer (Mecasys, Korea). Cultures grown in LB underwent NMR analysis. After allowing Osimertinib ic50 the cultures to grow in 50 mL of LB medium (37 °C, 16 h, with agitation), the cells were harvested with centrifugation (4 °C, 1 h, 2800 g). Supernatants were lyophilized until they could be analyzed. All 1H NMR spectra were acquired on a Varian Inova 600-MHz NMR spectrometer (Varian) at ambient temperature. The NMR spectral data were reduced to 0.001 p.p.m. Endonuclease spectral buckets and the region corresponding to water (4.6–4.8 p.p.m.) was removed (Jung et al., 2012). The PM assay of the mioC mutant was performed with chemicals using

the Biolog system (Fig. 1a). Sensitivity to the antibiotics, metals and chelator was detected. Although some antibiotics and metals did not significantly affect the PM results, a mutant strain has resistance or sensitivity under various antibiotics and metals. Therefore, our data suggested that the mioC gene might be involved in antibiotic resistance and metabolism of metals in P. aeruginosa. Sensitivity tests were performed with the wild-type, mioC mutant and mioC over-expressed complementation strains using antibiotics, metals and chelator (Fig. 1b and c). Our laboratory experimental data were consistent with those of the PM assay. The mutant strain was resistant against oxidative stresses, including superoxide [paraquat (PQ)] and peroxide (H2O2 and CHP) stresses (Fig. 1b). The mutant strain was also resist to Amp and Gm antibiotics (Fig. 1b).