M9 medium

Unless stated otherwise, all experiments used E. coli MG1655 strain (a gift from Professor Yuichi Wakamoto at Tokyo University) as wild-type (WT). We used ligation-independent cloning PCR^{37 (link)} to insert the QUEEN-2m construct into the ptqk5 plasmid vector, a low-copy modified version of the original pRSetB–QUEEN-2m plasmid¹¹ obtained from the Bacillus PS3 strain and encoding a truncated protein $ϵ$ of FoF1 ATPase, to generate the resulting ptqk5–QUEEN-2m plasmid. QUEEN-2m protein purification was performed by using the E. coli JM109 strain.
M9 medium (1x M9 salts, 2 mM MgSO4, 0.1 mM CaCl2; Sigma-Aldrich) supplemented with 1% thiamine and 0.1 mM glucose was used for glucose-deprived conditions. M9 medium supplemented with 1% thiamine and 22.2 mM glucose was used for the glucose control condition. Luria broth medium (LB medium; Merck) was used for protein purification protocols. For experiments assessing specific agents, such as 10 mM ATP (TOYO B-Net) and 10 mM glycerol (Wako), the substance of interest was combined with the various components of the glucose control medium such that the described concentrations were maintained. All mediums included ampicillin as an antibacterial agent.

Phosphate-buffered saline (PBS) (1×, pH 7.4, Gibco) was used as purchased. Lysogeny broth Miller (LB-Miller) medium was prepared by adding 10 g of tryptone (BD Difco^TM), 5 g of yeast extract (BD Difco^TM) and 10 g of sodium chloride (Fischer) to 1 L of Milli-Q^® water and autoclaving. 5× M9 medium (Sigma-Aldrich) without a carbon source (12.8 g L⁻¹ Na₂HPO₄.7H₂O, 3 g L⁻¹ KH₂PO₄, 0.5 g L⁻¹ NaCl, 1 g L⁻¹ NH₄Cl, 2 mM MgSO₄ and 0.1 mM CaCl₂) was dissolved in Milli-Q^® water, autoclaved and filtered through 0.22-μm polyethersulfone membrane (Millex-GP Syringe Filter Unit). LB-Miller agar solutions (molten) were prepared by combining LB-Miller medium with agar (final concentration of 1.5% w/v) (BD Difco^TM) and autoclaving. 2.0% w/v of ultra-low-gelling-temperature (ULGT) agarose (Sigma-Aldrich) was prepared by dissolving 100 mg in 5 mL of LB-Miller medium and autoclaving. The hydrogel was kept molten by keeping the solution in a static incubator at 37 °C. Molten solutions were only used for a week. Antibiotics were dissolved in Milli-Q^® water, filter-sterilised (0.22-μm polyethersulfone membrane) and frozen (−20 °C) as stock solutions at 100 mg mL⁻¹ for ampicillin and 30 mg mL⁻¹ for kanamycin (Sigma-Aldrich).

The bacterial strains used in this study are described in Table 1. Bacteria were cultured in Luria-Bertani (LB) broth or minimal essential medium (MEM)-HEPES (Sigma-Aldrich) supplemented with 0.1% glucose and 250 nM Fe(NO₃)₃. Caco-2 cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 15 mM l-glutamine, and 1% penicillin-streptomycin (Sigma-Aldrich). When measuring the promoter activity of sulA::gfp, strains were cultured in M9 medium (Sigma-Aldrich) supplemented with 0.2% glucose, 2 mM MgSO₄, and 0.1% acid-hydrolyzed Casamino Acids. When required, antibiotics were added to the media at the following final concentrations: 1 μg/ml for mitomycin C (MMC), 0.03 and 0.10 μM for chloramphenicol, 1.10, 1.48, and 1.85 μM for telithromycin; and 0.25, 0.50, 0.75, 1.00, 3.00, and 5.00 μM for solithromycin (Cempra Pharmaceuticals). In order to assess the impact of solithromycin on the viability of bacteria expressing or not expressing a T3SS, ZAP193 and its ΔLEE2 derivative (Table 1) were cultured overnight in LB and then inoculated into MEM-HEPES to an optical density at 600 nm (OD₆₀₀) of 0.5, at which point 3 μM solithromycin was added to the cultures and their optical densities were measured at 30-min intervals for 150 min.