Chloramphenicol

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).

Amikacin, ampicillin, carbenicillin, chloramphenicol, ciprofloxacin, norfloxacin, and tetracycline were purchased from Wako Pure Chemicals Industries, Ltd (Osaka, Japan). Moxifloxacin and levofloxacin were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Imipenem/cilastatin was purchased from Sandoz K.K. (Tokyo, Japan).

The E. coli strains and their mutants used in this study are listed in Table 1. Whole-genome sequences of the 442 O121:H19 strains (Data set S1) previously used in our phylogenetic analysis (13 ) were used for the analysis of IS insertion into the lacZ and iee genes.
Bacteria were grown in the following media: LB (1% [wt/vol] Bacto Tryptone, Gibco; 0.5% [wt/vol] Bacto Yeast Extract, Becton, Dickinson [BD]; 1% [wt/vol] sodium chloride, nacalai tesque), LB agar (LB containing 1.5% [wt/vol] Bacto Agar, BD), MAC (Difco MacConkey agar base, BD; 1% [wt/vol] lactose monohydrate, Wako), MAC not supplemented with lactose (Difco MacConkey agar base), Pearlcore MAC (Pearlcore MacConkey agar, Eiken Chemical Co.), MM (Difco M9 Minimal Salts, BD; 2 mM magnesium sulfate heptahydrate, Wako; 0.1% D-[+]-glucose, nacalai tesque), and MM agar (MM containing 1.5% [wt/vol] Bacto Agar). The growth media were supplemented with regents and antibiotics when necessary at the following concentrations: L(+)-arabinose (Wako), 1 mM; IPTG (Wako), 0.3 mM or 30 mM; X-gal (TaKaRa), 40 μg/mL; sucrose (nacalai tesque), 10% (wt/vol); D-(+)-glucose, 0.1%, 0.2%, or 0.4% (wt/vol); lactose monohydrate, 1.0% (wt/vol); D(+)-maltose monohydrate (Wako), 1.0% (wt/vol); chloramphenicol (Wako), 20 μg/mL; ampicillin (Sigma), 50 μg/mL; tetracycline (nacalai tesque), 10 μg/mL.