Pseudomonas aeruginosa was grown in LB at 37 °C for 12 h with shaking at 200 r.p.m. Cells were removed by centrifugation, and supernatants were filtered through a 0.45-μm Durapore filter (Millipore). Filtrates were ultracentrifuged (15 000 g, 1 h, 4 °C), and pellets were eluted with 10 mM HEPES (pH 6.8) containing 0.85% NaCl (HEPES-NaCl). Vesicle quantification was performed

by the phospholipid concentration assay using a previously described method (Stewart, Pirfenidone 1980; Tashiro et al., 2009). Briefly, 10 μL of vesicle sample, 100 μL of ammonium ferrothiocyanate solution (27.03 g L−1 ferric chloride hexahydrade and 30.4 g L−1 ammonium thiocyanate) and 100 μL chloroform were mixed and vortexed. After 10 min at room temperature, the absorbance of the lower layer was measured at 488 nm. l-α-Phosphatidylethanolamine CX-5461 was used as a reference standard. PQS was collected from the supernatant

of 12-h cultures and detected by thin-layer chromatography (TLC) following the method described previously (Toyofuku et al., 2008). Pyocyanin was extracted from culture supernatants and measured using the method reported by Essar et al. (1990). Briefly, 300 μL of chloroform and 500 μL of culture supernatants were vortexed. After centrifugation, the chloroform layer was transferred to a fresh tube and mixed with 100 μL of 0.2 N HCl. The absorbance of the top layer was measured at 520 nm. Using transcriptional fusions with a reporter gene xylE, the expressions of pqsABCDE and pqsH were analyzed. Insertion of the xylE gene cassette downstream of the chromosomal pqsE gene was performed as follows: two 0.8-kb DNA fragments Dimethyl sulfoxide were amplified from PAO1 chromosomal DNA with pqsA-ExylF1 (5′-CGGGATCCGGTCAACTGGATGATGATGACCTGTGCC-3′, the added restriction site is underlined)

and pqsA-ExylR1 (5′-CCTCTAGAATGTCCCGTCTCAGTCCAGAGGC-3′), or with pqsA-ExylF2 (5′-CCTCTAGAGACTGAGACGGGACATCCATTGCG-3′) and pqsA-ExylR2 (5′-CCCAAGCTTGCGACGGTACGATCTGGAACACG-3′), digested with BamHI/XbaI or XbaI/HindIII, respectively, and ligated into the BamHI–HindIII site of pG19II. The xylE fragment, digested from pX1918 with XbaI, was then inserted into the XbaI site of the constructed plasmid to yield pG19-pqsEX in which xylE was downstream of pqsE. Insertion of the xylE gene cassette downstream of the chromosomal pqsH gene was performed in the same way. Two 0.8-kb DNA fragments were amplified from PAO1 chromosomal DNA with pqsHX1 (5′-GGAATTCCTGGATGAGTCGCGCATTCGGG-3′) and pqsHX2 (5′-GCTCTAGACTACTGTGCGGCCATCTCACCG-3′), or with pqsHX3 (5′-GCTCTAGATGCCAGTGGCGTCTTGGTCGCC-3′) and pqsHX4 (5′-CCCAAGCTTGATCTCGCGCTCGGACAGCG-3′), digested with EcoRI/XbaI or XbaI/HindIII, respectively, and ligated into the EcoRI–HindIII site of pG19II.