Nalidixic acid

Cycloheximide, nocodazole, sodium azide, chloramphenicol, rifampin, nalidixic acid, and a cocktail of protease inhibitors cocktail were purchased from Wako. Cytochalasin D was purchased from Focus Biomolecules (Plymouth Meeting, PA) and staurosporine from Cayman Chemical Company (Ann Arbor, MI). The solvents used and final concentrations were as follows: Cycloheximide, 100 μg/ml in dimethyl sulfoxide (DMSO); nocodazole, 10 μg/ml in DMSO; sodium azide, 50 mM in PBS; chloramphenicol, 5 μg/ml in ethanol; rifampin, 0.25 mg/ml in DMSO; nalidixic acid, 5 μg/ml in 1N NaOH; protease inhibitor cocktail containing aminoethyl benzylsulfonyl fluoride (AEBSF), 100 mM in aprotinin, 80 μM in DMSO; E-64, 1.5 mM in DMSO, leupeptin, 2 mM, bestatin, 5 mM, pepstatin, 1 mM; Cytochalasin D, 1 μg/ml in DMSO; and staurosporine, 1 μM in ethyl acetate. All chemicals and solvents were tested at a concentration used for possible adverse effects in Ca9-22 cells, as compared with cells without the inhibitor, by examining the morphology of the cells and cell viability with MTT assay (S2 Fig). Moreover, all chemicals and solvents were tested at the appropriate concentrations and found to produce no reduction in P. gulae growth (S3 Fig).

The following compounds, 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS; Pseudomonas quinolone signal), 4-hydroxy-2-heptylquinolone (HHQ), and 2-amino-6-chlorobenzoic acid (CABA), which is a pqs QS inhibitor, were purchased from Sigma-Aldrich Japan (Tokyo, Japan), and 2-heptyl-4-quinolinol-1-oxide (HQNO) was purchased from Cayman Chemical Company (Michigan, United States). As one of the quinolone analogs, nalidixic acid was also purchased from FUJIFILM Wako Pure Chemical Corporation, Ltd. (Osaka, Japan). PQS and HHQ were stocked in dimethyl sulfoxide (DMSO) to make an 80-mM standard solution. HQNO, nalidixic acid, and CABA were stocked in DMSO to be 8-mM, 16-mM, and 1.5-M stock solutions, respectively.

Bacterial growth characteristics of wecB::Cm mutant were monitored and compared with wild-type strain by measuring the optical density (OD_{600 nm}) of bacterial cultures at 37°C with shaking (150 rpm) at 2 h intervals. At the indicated time points, 100 μl of each culture was serially diluted in LB broth and 100 μl of each dilution was spread on an LB agar plate. After overnight incubation at 37°C, colonies on the plates were counted as CFUs. For the resistance assays, bacteria were grown in LB broth for 14 h and diluted to 2.0 × 10⁷ CFU/ml, and 50 μl diluted culture was mixed with 50 μl of LB broth that contained various concentrations of bile acid (Sigma-Aldrich; final concentration was 0.01 or 0.02 mM), hydrogen peroxide (H₂O₂) (Wako, Osaka, Japan; final concentration was 0.5 or 1.0 mM), or nalidixic acid (Wako; final concentration was 1.25 or 2.5 mg/ml) in 96-well flat-bottom plates (Greiner Bio-One, Kremsmünster, Austria). The plates were incubated at 37°C without shaking. Bacterial growth at the indicated time points was determined by monitoring the optical density (OD_{595 nm}) of bacterial cultures using a 96-well plate reader (Bio-Rad, Model 680 microplate reader, Hercules, CA, USA).