X gal 5 bromo 4 chloro 3 indolyl β d galactopyranoside

Table 1 lists bacterial strains and plasmids used in this study. B. glumae and E. coli were grown in LB medium (1% NaCl, 1% tryptone, 0.5% yeast extract). E. coli RHO3 was grown in LB containing 200 μg/mL 2,6‐diaminopimelic acid (DAP; VWR). Some experiments used LB medium buffered with 70 mM Tris (pH 7.0). The AHL biosensor Agrobacterium tumefaciens KYC5 was grown in AT medium at 28°C (Tempé et al., 1977 (link)). Antibiotics were purchased from either Sigma‐Aldrich or VWR and used at the following concentrations: nitrofurantoin (Nit) 100 μg/mL (for selection of B. glumae), kanamycin (Kan) 30 μg/mL (E. coli) and 100 μg/mL (B. glumae), and trimethoprim (Tmp) 100 μg/mL (B. glumae and E. coli). Tetracycline (4.5 μg/mL), spectinomycin (50 μg/mL), and gentamycin (100 μg/mL) were used for A. tumefaciens KYC5. X‐gal (5‐bromo‐4‐chloro‐3‐indolyl β‐d‐galactopyranoside) was purchased from VWR. Tris base and bis‐Tris propene were purchased from J.T. Baker and Sigma‐Aldrich, respectively. N‐octanoyl homoserine lactone (C8‐HSL) and toxoflavin were purchased from Sigma‐Aldrich and Cayman Chemical, respectively. Table S1 lists oligonucleotide primers used in this study (purchased from Sigma‐Aldrich).

The plasmids and strains used in this study are listed in Tables S1 and S2 and their construction is described in the supplemental material. S. aureus strains were grown in tryptic soy broth (TSB, Difco) with aeration at 37°C or on tryptic soy agar (TSA, Difco) at 30°C or 37°C. For microscopy experiments, overnight cultures of S. aureus strains were diluted 1:200 in TSB medium and allowed to grow at 37°C until an optical density at 600 nm (OD₆₀₀) of approximately 0.5. Cells were then harvested and resuspended in the same medium. Escherichia coli strains were grown in Luria-Bertani broth (LB, Difco) with aeration, or on LB agar (LA, Difco) at 30°C. The culture medium was supplemented with the appropriate antibiotics (100 μg · mL⁻¹ ampicillin, 10 μg · mL⁻¹ erythromycin, 10 μg · mL⁻¹ chloramphenicol, or 50 μg · mL⁻¹ of both kanamycin and neomycin; Sigma-Aldrich), with 100 μg · mL⁻¹ 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; VWR) or cadmium chloride (0.1 μM, Sigma-Aldrich), when required. Cell wall-targeting antibiotics were used at their MICs: 2 μg · mL⁻¹ for vancomycin, 800 μg · mL⁻¹ for oxacillin, 1.5 μg · mL⁻¹ for CDFI [2-(2-chlorophenyl)-3-[1-(2,3-dimethylbenzyl)piperidin-4-yl]-5-fluoro-1H-indole], 40 μg · mL⁻¹ for bacitracin, 125 μg · mL⁻¹ for d-cycloserine, 300 μg · mL⁻¹ for fosfomycin, and 1 μg · mL⁻¹ for PC190273.