FIG. 2.
Interaction of BglJ with RcsB and YjjQ. (A) In the LexA-based two-hybrid system, the sulA promoter-lacZ fusion with the wild-type LexA operator was used to analyze homodimerization, and the sulA promoter-lacZ reporter fusion with a hybrid lexA408/+ operator served as a reporter for heterodimerization. For analysis of homodimerization, a fusion of the respective protein (X) to the wild-type LexA DNA-binding domain (lexA1-87/X) was expressed from a plasmid under the control of the IPTG-inducible lacUV5 promoter (PUV5). For heterodimerization analysis, fusions of protein X to the wild-type LexA DNA-binding domain (lexA1-87/X) and of protein Y to the LexA408 mutant DNA-binding domain (lexA4081-87/Y) were coexpressed from compatible plasmids. (B) Analysis of homodimer formation in RcsB, RcsA, BglJ, and YjjQ was performed with transformants of strain S3434 with plasmids pKEMK17 (lexA1-87-rcsB), pKES192 (lexA1-87-rcsA), pKEAP30 (lexA1-87-bglJ), pKEAP27 (lexA1-87-yjjQ), and pMS604 (lexA1-87-fos) as controls. Cultures were grown in LB tetracycline medium to an OD600 of 0.5. IPTG was added to 1 mM final concentration where indicated (+). The β-galactosidase activity was determined to monitor repression of the sulA promoter by the LexA1-87-X fusion protein. The fold repression (indicated by the bars), as a measure for dimerization, was calculated as the ratio of the β-galactosidase activities measured without and with induction of the LexA fusion proteins. (C) Analysis of heterodimer formation was performed with strain S3442, which was cotransformed with plasmids coding for LexA1-87-X and LexA4081-87-Y fusions, respectively. The cultures were grown in LB with antibiotics, and IPTG was added where indicated. The fold repression of the sulA promoter-lacZ fusion with the hybrid lexA operator (lexA-op408/+) is a measure of heterodimerization (indicated by the bars). The following plasmids were used: LexA1-87-RcsB (pKEMK17), LexA1-87-RcsA (pKES192), LexA1-87-BglJ (pKEAP30), LexA1-87-YjjQ (pKEAP27), and LexA1-87-Fos (pMS604) (12), as well as LexA4081-87-RcsB (pKEAP28), LexA4081-87-RcsBD56E (pKES150), LexA4081-87-RcsBD56N (pKES151), LexA4081-87-BglJ (pKEAP29), and LexA4081-87-Jun (pDP804) (12) as controls.