mirabilis ATCC 29906 were submitted to GenBank and assigned the accession numbers AF397169 (gyrA), AF503506 (gyrB) and AF363611 (parC). CIP uptake was assayed by the method of Giraud et al. (1999) with some modifications. Bacteria suspended in PBS to OD600 nm ~1.2 were equilibrated for 10 min at 37 °C. After the addition of CIP to a final concentration of 10 μg mL−1, 0.5 mL samples were removed at different time intervals. Five minutes after this addition,
the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) 100 μM was added to the reaction mixture. The samples were diluted in 1 mL of ice-cold PBS and centrifuged for 5 min at 5600 g. The pellet was washed once with 1 mL of ice-cold PBS and
resuspended in 1 mL of 0.1 M glycine hydrochloride (pH 3.0) for 1 h at room temperature. The samples were then centrifuged at 5600 g for 10 min and the fluorescence beta-catenin tumor of the supernatant was measured with a YASCO FP-777 spectrofluorimeter at excitation and emission wavelengths of 278.5 and 448.5 nm, respectively. The concentration of CIP in the supernatant was calculated by comparison with a standard curve for CIP in 0.1 M glycine hydrochloride. The results were expressed as nanograms of CIP incorporated mg−1 of protein. The FRAP assay (Benzie & Strain, 1999) was adapted to measure the antioxidant capacity of P. mirabilis. A volume of 100 μL of bacterial suspensions (OD600 nm ~1) was incubated with 125 μL of 3.1 mg mL−1 of 2,4,6-tripyridyl-1,3,5-triazine (TPTZ) in 40 mM HCl, 125 μL of FeCl3·6H2O 5.4 mg mL−1 and 1.25 mL of 300 mM acetate buffer (pH 3.6). Absorbances Selleckchem Pexidartinib were
determined at 593 nm and expressed as μM of FeSO4 mg−1 protein. The concentration of proteins in bacterial suspension was determined by Folin–Ciocalteau assay (Stauffer, 1975). Bacterial suspensions of 1 mL were incubated with CIP or using PBS (control). The incubations were stopped Methocarbamol at 2 h with 1 mL of TCA 35% (p/v) in the absence of light. After 20 min, 1 mL of 0.5% (p/v) thiobarbituric acid was added and the samples were heated to 80 °C for 30 min. An ice bath was used to cool the samples, which were centrifuged at 1500 g and the absorbance of the supernatant was determined at 535 nm. A calibration curve of malondialdehyde (MDA) solutions was applied to estimate lipid oxidation. MDA levels were expressed as nmol MDA mg−1 protein. Bacterial suspensions of 3 mL were incubated with 0.5 mL of CIP or PBS (control) for 2 h. After that, 1 mL of the samples was treated with 1 mL of 0.1% 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl for 1 h. The proteins were precipitated in 5% trichloroacetic acid (TCA), centrifuged 20 min at 10 000 g, and the supernatant discarded. Samples were extracted three times with 1 mL ethanol/ethylacetate (1 : 1, v/v) to remove any remaining residual of DNPH. The precipitate was dissolved in 6 M guanidine hydrochloride solution in PBS and incubated for 30 min at 37 °C.