SILAC Metabolic Labeling, Cell Lysate Preparation, Affinity Purification

For metabolic labeling, the F. tularensis wild-type strain (wt) or strain expressing Strep-tagged GapA protein (FSC200/gapA_S) were cultivated in CDM containing the heavy labeled amino acids L-arginine hydrochloride [¹³C₆ ¹⁵N₄] and L-lysine hydrochloride [¹³C₆ ¹⁵N₂] (Sigma–Aldrich, Schnelldorf, Germany) in the same concentrations as in the light medium. After three generations, the incorporation levels were checked by mass spectrometry (MS) and the labeled bacteria were stored as frozen stocks at −150°C. For the experiment, the labeled bacterial stock was cultivated in heavy medium overnight at 37°C, 200 rpm. Non-labeled bacteria were grown in the light medium. The overnight cultures were diluted with fresh media to optical density at 600 nm (OD_600nm) = 0.1 and cultivated to OD_600nm = 0.8 (approximately 3.2 × 10⁹ bacteria/ml, late exponential phase). Bacteria were then harvested by centrifugation and washed with phosphate-buffered saline (PBS, pH 7.4). At this point, the light and heavy bacteria were mixed in a 1:1 cell ratio. Three sets of biological replicates were prepared. Each set consisted of three samples. In two samples, the wt FSC200 was grown in light medium and the FSC200/gapA_S in heavy medium. For the third sample, the SILAC groups were swapped (FSC200/gapA_S was cultivated in light medium, wt FSC200 strain in heavy medium). After supplementation with protease inhibitor cocktail Complete EDTA-free (Roche Diagnostics, Mannheim, Germany) and benzonase (150 U/ml; Sigma–Aldrich), the bacterial cells were disrupted in a French press (Thermo IEC, Needham Heights, MA, United States) by two passages at 16,000 psi. Unbroken cells were removed by centrifugation at 6,000 × g for 40 min at 4°C. The clarified lysate supplemented with an appropriate amount of Avidin (11 U/mg; IBA Lifesciences, Göttingen, Germany) was applied on a column containing Strep-Tactin^® affinity resin and the GapA/S protein was purified using the Strep-tag^® purification system according to the manufacturer’s instructions (IBA Lifesciences).