Gel purified pcr products

The genes encoding selected F. tularensis proteins were amplified by PCR with specific primers (Supplementary Table S2), and the gel-purified PCR products (Qiagen) were inserted into pET28b using either the NcoI and XhoI restriction sites (pepSY, rho, pepB, gene of YCII-related domain protein) or HindIII and XhoI restriction sites (trxA). The resulting plasmids encoded the aforementioned proteins with a C-terminal histidine tag. The final plasmid constructs were verified by direct DNA sequencing. For protein expression, the plasmids were transformed into E. coli NiCo21 expression strains (New England BioLabs, Ipswich, AM, United States). The cells were grown in Terrific Broth medium at 28°C overnight. Bacteria were lysed in a French pressure cell and the lysates were applied to a TALON^® Metal Affinity Resin purification system (Clontech, Mountain View, CA, United States) for purification of His-tagged proteins. Finally, the elution buffer was changed to T₁₀₀N₁₅₀ (100 mM Tris-HCl, 150 mM NaCl, [pH 7.6]) using an Amicon^® Ultra-15 centrifugal filter (Sigma–Aldrich). Eluted proteins were verified by SDS-PAGE followed by Coomassie staining (data not shown), and the protein concentrations were determined using Coomassie Plus Bradford assay reagent (Thermo Fisher).

To validate selected GapA interaction partners identified by SILAC-Pull-Down, the gapA gene was amplified from F. tularensis FSC200 genomic DNA (forward primer: 5′-GGACTCAGATCTCGAGAGTTGCAATTAATGGTTTCGGTAG-3′, reverse primer: 5′-CCGCGGTACCGTCGACTTATAGAGCTCCGAAGTACTCTAC-3′). The gel-purified PCR products (Qiagen) were inserted into pEGFP-C2 expression vector (Clontech–Takara Holding, Kyoto, Japan) between XhoI and SalI restriction sites using In-Fusion HD Cloning Kit (Takara), resulting in the plasmid encoding GapA fused with the green fluorescent protein (GFP) at its N-terminus (pEGFP-C2::gapA). The plasmid construct was verified by DNA sequencing.