Mem hepes

Expression from reporter plasmids was performed as described previously (Roe et al., 2004 (link)) using plasmids pAJR70 (promoterless control), pAJR71-75 (LEE1-5 fused to gfp), pAJR199 (fliC::gfp) and pAJR145 (rpsM::gfp). Transformants were grown overnight in LB media with appropriate antibiotics then the next morning diluted to an OD₆₀₀ of 0.08 in MEM-HEPES (Sigma). Cultures were shaken at 200 rpm in Erlenmeyer flasks incubated at 37°C. At intervals, 1ml of culture was removed from the flask and 200 μl aliquots were analysed in triplicate with a fluorescent plate reader (Fluorstar Optima; BMG) at 37°C. Fluorescence was plotted against OD₆₀₀ using Microsoft Excel software.

Bacterial strains and plasmids used in this study are listed in Table S1. Bacteria were cultured in Lysogeny Broth (LB) or M9 minimal media (Sigma-Aldrich) supplemented with 0.2% glucose, 2 mM MgSO₄ and 0.1 mM CaCl₂. For TTSS expression bacteria were cultured overnight in LB and then inoculated into minimal essential medium (MEM)-HEPES (Sigma-Aldrich) supplemented with 0.1% glucose and 250 nM Fe(NO₃)₃. Antibiotics were used at the following concentrations when required: Chloramphenicol (50 µg ml⁻¹), Mitomycin C (2 µg ml⁻¹), Nalidixic acid (50 µg ml⁻¹).

The bacterial strains, plasmids and oligonucleotides used in this study are listed in Table S4 and Table S5. Bacteria were routinely grown at 37°C in Luria broth (LB [Miller's recipe]) before diluting 1/100 into the appropriate medium for experiments or growth analysis. Chloramphenicol was used when appropriate at a concentration of 25 µg/ml. All preparations of M9 minimal medium (Sigma Aldrich; cat# M6030) were supplemented with 0.4% (w/v) glucose unless otherwise stated. For HeLa cell infection experiments, bacteria grown overnight were inoculated in prewarmed MEM-HEPES (Sigma Aldrich; cat# M7278) -/+ 1 mM D-Serine and incubated at 37°C, 200 RPM for 4.5 h. All growth media, antibiotics and chemicals were purchased from Sigma Aldrich unless stated otherwise.

The bacterial strains, plasmids and oligonucleotide primers used in this study along with relevant accompanying information are listed in S3, S4 and S5 Tables, respectively. Single bacterial colonies were inoculated into 5 ml LB broth containing the appropriate antibiotics and cultured overnight at 37°C, 200 rpm. Overnight cultures were used to inoculate pre-warmed MEM-HEPES (Sigma, St Louis, MO, USA; cat # m7278) and samples were cultured at 37°C, 200 rpm. D-serine was purchased from Sigma. Motility was assessed by inoculating the center of on 0.25% Tryptone agar plate with 5 μl of bacterial culture at OD⁶⁰⁰ 0.6 and diameter of the population swim was measured after 8 hours at 31°C.

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.

The strains used in this study are detailed in Table S1 in the supplemental material. The plasmids used in this study are described in Table S2. To induce T3SS expression, EHEC TUV93-0 was grown in minimal essential medium (MEM)-HEPES (Sigma, St. Louis, MO) with 5.62 g/liter of l-glutamine and EPEC was grown in Glutamax-Dulbecco modified Eagle medium (DMEM) (Sigma) at 37°C at 180 rpm. C. rodentium was also grown in Glutamax-DMEM (Sigma) and was cultured statically at 37°C with 5% CO₂. Growth and cell viability assays were carried out in lysogeny broth. For selection of plasmids, chloramphenicol or ampicillin was included at 20 or 50 μg/ml, respectively.