Activation of the bgl operon by BglJ requires RcsB. (A) Schematic of a transposon mutagenesis screen for mutants in which activation (relief of H-NS-mediated repression) of bgl by BglJ is abrogated. Strain S2822 carries the bgl operon and a bgl promoter-lacZ fusion as dual reporters for bgl expression. In addition, the strain carries allele yjjQ/bglJ-Y6::miniTn10-cat (bglJ_c) for constitutive expression of bglJ. A miniTn10-tet (mTn10) transposon mutagenesis screen yielded Bgl- and Lac-negative mutants, one of which carried a transposon insertion in rcsB (assigned to strain S2828 [Table 1]). (B) RcsB is required for derepression of bgl by BglJ. The Bgl phenotypes of the E. coli K-12 wild type (wt) (strain S524) and its isogenic derivatives, which constitutively express bglJ (bglJ_C; strain S2176) or carry a deletion of rcsB (ΔrcsB; strain S3918), as well as the double mutant bglJ_C ΔrcsB (strain S3919), was determined on BTB salicin indicator plates. Shown is complementation of the ΔrcsB mutants with plasmids encoding wild-type RcsB (pKETS6) or the RcsB mutants D56E (pKETS7), D56N (pKETS8), and D56A (pKES235).

Coimmunoprecipitation of BglJ-FLAG with RcsB-HA. (A) Coimmunoprecipitation of BglJ-FLAG with RcsB-HA was performed for lysates prepared from strain S3377 [ΔrcsB Δ(yjjP-yjjQ-bglJ)] (−) and for transformants of strain S3377 with plasmids pKEAP38 (RcsB-HA) (RcsB), pKERV10 (BglJ-FLAG) (BglJ), or both plasmids. Immunoprecipitation (IP) was performed with rabbit anti-HA IgG. The lysates and the coimmunoprecipitates were separated on SDS-PAGE and analyzed by Western blotting. For simultaneous detection of BglJ-FLAG and RcsB-HA, the Western blot was developed with rat anti-HA (α-HA) and mouse anti-FLAG as primary antibodies and fluorescence labeled anti-mouse and anti-rat secondary antibodies.

Sequence of the bgl promoter and upstream regulatory region. Indicated are the −35, −10, and transcription start sites of the promoter; the CRP binding site (boxed) (48); and the Fis binding sites (dotted lines) (4), as well as the LeuO (dashed lines) and BglJ-RcsB (solid lines) binding sites characterized here. H-NS binds to the upstream regulatory element and the promoter, but the H-NS nucleation sites have so far not been mapped. The stop codon of the phoU gene located upstream of bgl is underlined, and the inverted arrows indicate an inverted repeat that may represent the phoU transcriptional terminator.

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 lexA_408/+ 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 (lexA_1-87/X) was expressed from a plasmid under the control of the IPTG-inducible lacUV5 promoter (P_UV5). For heterodimerization analysis, fusions of protein X to the wild-type LexA DNA-binding domain (lexA_1-87/X) and of protein Y to the LexA408 mutant DNA-binding domain (lexA408_1-87/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 (lexA_1-87-rcsB), pKES192 (lexA_1-87-rcsA), pKEAP30 (lexA_1-87-bglJ), pKEAP27 (lexA_1-87-yjjQ), and pMS604 (lexA_1-87-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 LexA_1-87-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 LexA_1-87-X and LexA408_1-87-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: LexA_1-87-RcsB (pKEMK17), LexA_1-87-RcsA (pKES192), LexA_1-87-BglJ (pKEAP30), LexA_1-87-YjjQ (pKEAP27), and LexA_1-87-Fos (pMS604) (12), as well as LexA408_1-87-RcsB (pKEAP28), LexA408_1-87-RcsB_D56E (pKES150), LexA408_1-87-RcsB_D56N (pKES151), LexA408_1-87-BglJ (pKEAP29), and LexA408_1-87-Jun (pDP804) (12) as controls.

Effect of mutation of the BglJ-RcsB binding site on derepression of bgl by BglJ-RcsB. The expression levels directed by bgl promoter-lacZ fusions (shown schematically in panel B) with wild-type and mutant BglJ-RcsB binding sites (A) were determined for exponential cultures grown in LB (with appropriate antibiotics and 1 mM IPTG) (C). (A) The BglJ-RcsB binding site is underlined, and mutations are indicated in lowercase boldface letters. (B) The bgl-lacZ fusions were integrated at the phage λ attB site (the strains are listed in Table 1). The bgl promoter (P_bgl) is indicated by a flagged arrowhead, the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) binding site is shown as a black box, and the mutation of terminator t1 (t1_RAT) is indicated by a crossed hairpin loop. (C) Expression levels were determined in strain T314 [Δ(yjjP-yjjQ-bglJ) ΔleuO] (−) transformed with the empty vector pKESK22, with plasmid pKETS1 for expression of BglJ in trans (+BglJ), or with plasmid pKEDR13 for expression of LeuO (+LeuO). The β-galactosidase activities with LeuO provided in trans were determined 6 times independently, as the standard deviation was up to 40%. In addition, the expression levels of the bgl-lacZ fusions were analyzed in transformants of Δhns, ΔrcsB, and Δhns stpA mutant derivatives, as indicated.

Model illustrating regulation of the bgl promoter and regulatory interactions of the transcriptional regulators LeuO and BglJ-RcsB (for details, see Discussion). Pointed arrowheads indicate activation, and blunt arrowheads indicate repression. Promoters (P) are indicated by pointed flags. In addition to being controlled by H-NS, StpA, LeuO, and BglJ-RcsB, the bgl promoter is CRP dependent and is repressed by FIS (4, 48, 52).