Lb agar plates

Pseudomonas aeruginosa PAO1 (PAO1) was cultured on LB-agar plates (Roth, Karlsruhe, Germany) overnight. 5 mL LB-medium was inoculated with a single colony and cultured overnight (300 rpm, 37 °C). On the next day the 25 mL fresh medium was inoculated with bacteria from the overnight culture to yield an OD₆₀₀ ≤ 0.3. The cells were cultured under agitation until they reached an OD₆₀₀ of 1. For infection experiments with viable bacteria, PAO1 were diluted 1:10000 and applied in 15 μL to the apical surface of the air lifted cultures. For heat inactivation the undiluted solution was incubated for 5 min at 95 °C, stored in aliquots at − 20 °C and used for experiments in a dilution of 1:50 (approximately 53.4 × 10 ⁶ CFU/well).

All bacterial strains and plasmids used in this work are listed in Table 2. Escherichia coli cells were cultivated at 37°C in lysogeny broth (LB) (49 (link)) or terrific broth (TB) (12 g L⁻¹ tryptone, 24 g L⁻¹ yeast extract, 4 mL glycerol, 12.54 g L⁻¹ K₂HPO₄, 2.31 g L⁻¹ KH₂PO₄; pH 7.0) or on LB agar plates (Carl Roth, Karlsruhe, Germany). C. glutamicum strains were cultivated at 30°C in brain heart infusion medium (BHI; Difco Laboratories, Detroit, USA), in CGXII medium with 4% (wt/vol) glucose (50 (link)) containing 30 mg L⁻¹ 3,4-dihydroxybenzoate as the iron chelator, or in modified CGXII medium lacking glucose, ammonium sulfate, and urea and containing 100 mM l-glutamine as the nitrogen and carbon source. These media were also used for the preparation of solid media by addition of 15 g L⁻¹ agar. For induction of glutamate secretion, the CGXII medium with 4% (wt/vol) glucose was supplemented with 500 mg L⁻¹ ethambutol and 20 μM IPTG for strains carrying plasmids with an IPTG-inducible promoter. The glutamate concentration in the culture supernatant was measured after 24 h. To maintain plasmid stability, kanamycin was added at concentrations of 25 mg L⁻¹ (C. glutamicum) or 50 mg L⁻¹ (E. coli).

All bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli cells were cultivated at 37°C in lysogeny broth (LB) (50 (link)) or on LB agar plates (Carl Roth, Karlsruhe, Germany). C. glutamicum strains were cultivated at 30°C in brain-heart infusion medium (BHI; Difco Laboratories, Detroit, USA) or in CGXII medium with 2% (wt/vol) glucose (51 (link)) containing 30 mg L⁻¹ 3,4-dihydroxybenzoate as iron chelator. A 15 g L⁻¹ agar was added to prepare the respective solid media. Kanamycin was added at concentrations of 25 mg L⁻¹ (C. glutamicum) or 50 mg L⁻¹ (E. coli) to maintain plasmid stability. In case of the C. glutamicum ∆aceE strain, the culture medium was supplemented with 2 g L⁻¹ acetate.