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. 2015 Dec 3;291(5):2357–2370. doi: 10.1074/jbc.M115.696815

FIGURE 1.

FIGURE 1.

Homo- and heterodimer formation by RcsB, BglJ, RcsA, MatA, and DctR. The LexA two-hybrid system exploits repression of the sulA promoter by dimeric LexA (40). Expression is repressed in cases when a fusion of a protein (×) to the LexA-DNA-binding domain forms homodimers (A). For analysis of heterodimerization, a sulA promoter variant carrying a hybrid lexA 408/+ operator is used, which is repressed by heterodimers of proteins X and Y that are fused to the LexA(1–87)WT and LexA(1–87)408 DNA-binding domains, respectively (B). The LexA fusion proteins were expressed from compatible plasmids under the control of the IPTG-inducible lacUV5 promoter. C, heterodimer formation by RcsB, BglJ, RcsA, MatA, and DctR. The -fold repression of the lexAop408/+ sulA promoter lacZ fusion, as a measure of heterodimerization, is calculated as the ratio of expression values (given in smaller type) directed by the PsulA lacZ reporter when bacteria are grown without and with induction, respectively, of LexA fusion protein expression. Strain S3440 (ΔrcsB) or S3442 (ΔrcsB Δ(yjjP-yjjQ-bglJ) were co-transformed with plasmids encoding for LexA(1–87)-X and LexA(1–87)408-Y fusions, respectively. The following plasmids were used: pKEMK17 (LexA(1–87)-RcsB), pKEAP30 (LexA(1–87)-BglJ), pKES192 (LexA(1–87)-RcsA), pKEMK4 (LexA(1–87)-MatA), and pKEMK1 (LexA(1–87)-DctR) as well as pKEAP28 (LexA(1–87)408-RcsB), pKEAP29 (LexA(1–87)408-BglJ), and pKEDP59 (LexA(1–87)408-MatA). The cultures were grown to A600 of 0.5 in LB medium supplemented with ampicillin and tetracyclin. IPTG was added where indicated. Values for RcsB, BglJ, and RcsA homo- and heterodimer analysis are taken from Ref. 9. D, homodimer formation of RcsB, BglJ, RcsA, MatA, and DctR. The -fold repression, as a measure for dimerization, was calculated as the ratio of the β-galactosidase activities determined of cultures grown without and with induction of the LexA fusion proteins. Strain S3434 (ΔrcsB Δ(yjjP-yjjQ-bglJ)) was transformed with plasmids pKEMK17 (LexA(1–87)-RcsB), pKEAP30 (LexA(1–87)-BglJ), and pKES192 (LexA(1–87)-RcsA), respectively. Strain S3432 (ΔrcsB) was transformed with plasmid pKEMK4 (LexA(1–87)-MatA) and pKEMK1 (LexA(1–87)-DctR). Cultures were grown in LB tetracycline medium to A600 of 0.5 without and with 1 mm IPTG.