Rapid Protein-Protein Interaction Screening

The BacterioMatch II Two-Hybrid system (Agilent Technologies, CA, USA) was used to rapidly detect possible interactions between proteins. The B2H assay was carried out according to a procedure used in laboratory^{45 (link)}. In short, target genes containing coding regions were PCR-cloned into pBT and pTRG vectors, respectively, and these were transformed into E. coli XL1-Blue MRF ´ Kan. The vectors pBT-GacS and pTRG-GacS were used as positive controls^{45 (link)} and empty pTRG and pBT plasmids as negative controls. All co-transformed strains were spotted onto selective agar plates (selective agar, denoted as +3AT + Str^r) and cultured at 28°C for 2 to 3 days. Strains with pBT-RpfG and pTRG-WspR would be expected to grow well on the selective agar plates if there is a direct physical interaction between WspR and RpfG. Selective agar consisted of minimal medium (M9) supplemented with 30 μg/mL kanamycin, 34 μg/mL chloramphenicol, 12.5 μg/mL tetracycline, 5 mM 3-AT, and 8 μg/mL Str^{45 (link)}. LB agar is nonselective (denoted as -3AT-Str^r) and comprises 12.5 μg/mL tetracycline, 34 μg/mL chloramphenicol, and 30 μg/mL kanamycin^{45 (link)}. The purpose of LB agar is to confirm that all recombinant vectors were successfully transformed into E. coli XL1-Blue MRF ´ Kan.