Bacteriomatch 2 two hybrid system

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.

A C. glutamicum genomic library was prepared as previously described [29] (link). The BacterioMatch II Two-Hybrid system (Agilent Technology) was used according to manufacturer instructions. Briefly, the two plasmids, pBT and pTRG, containing the “bait” and “target” genes, respectively, were used to transform E. coli simultaneously. Protein–protein interactions were screened based on the expression of his3 and aadA, which confer histidine prototrophy (His⁺) and streptomycin resistance (Str⁺), respectively. For screening, 50 ng of each pSL500 (i.e., pBT-glxR) and target library DNA were introduced into reporter cells and spread onto selective media (His⁻ and Str⁺). Colonies were isolated and the plasmids in the growing cells were analyzed.

C. glutamicum AS019E12 [27] (link) was used to construct HL1385, which harbored a ΔsprA mutation. C. glutamicum HL1516 and HL1389 harbored the sprA-complementing plasmid pSL535 and sprA-overexpressing plasmid pSL509, respectively. E. coli DH10B (Invitrogen) was used for the construction and propagation of plasmids. E. coli BL21-CodonPlus (DE3)-RIL (Stratagene) and E. coli DH5αF′ (Bethesda Research Laboratories) were used for the expression of histidine-tagged SprA (His₆-SprA) and maltose-binding protein (MBP)-fused GlxR (MBP-GlxR), respectively. E. coli and C. glutamicum strains were cultured in Luria-Bertani broth at 37°C and MB medium at 30°C, respectively [27] (link). MCGC minimal media for C. glutamicum were prepared as described previously [28] . Glucose and acetate were added as carbon sources to the MCGC minimal medium at 1% and 2% (w/v), respectively. Selective and nonselective broths (BacterioMatch II Two-Hybrid System, Agilent Technologies) for E. coli XL1-Blue MRF′ kan were prepared as described [29] (link). Antibiotics were added at the following concentrations (μg mL⁻¹): 50 ampicillin, 5 tetracycline, 20 chloramphenicol, and 25 kanamycin.