Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli

Abstract

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility, and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (also known as EcpR) and with DctR. Our data suggest that the MatA-dependent transcription regulation is mediated by the MatA-RcsB heterodimer and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet of conserved residues, and of surface-exposed residues for activity of RcsB. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various auxiliary regulators and their differential dependence on phosphorylation add an additional level of control to the Rcs system that is operating at the output level.

Keywords: DNA binding protein; bacterial signal transduction; protein phosphorylation; protein-protein interaction; transcription coregulator.

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 lexAop_408/+ 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. . 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.

FIGURE 2.

MatA-RcsB activates the matACFT073 promoter and inhibits motility. A, β-galactosidase expression levels directed by the matA_CFT073 promoter lacZ reporter were determined in rcsB⁺ strain T1749, ΔrcsB strain T1747, rcsB⁺ P_L-matA strain T1986, and P_L-matA ΔrcsB strain T1987. These strains were either transformed with control plasmid pKESK22 (pCtrl) or plasmids pKETS6 (pRcsB), pKET7 (pD56E), and pKES235 (pD56A), respectively. Cultures for β-galactosidase assays were grown to A₆₀₀ of 0.5 in LB medium, supplemented with 1 mm IPTG and 25 mg/ml kanamycin. B, motility was determined of wild-type strain T1241 and ΔrcsB strain U89 and of transformants of these strains ectopically expressing MatA under the control of Ptac using plasmid pKEDP30 (pMatA). Overnight cultures were grown in LB medium, which was supplemented with 1 mm IPTG and 25 mg/ml kanamycin for growth of the transformants. Three μl of each culture was spotted on the center of a soft agar plate (0.2% agar), supplemented with 0.2 mm IPTG and 25 mg/ml kanamycin in the case of transformants, and the plates were incubated at 37 °C for 5 h. The plates were scanned, and the motility radii that are indicated by arrows were measured in mm. Error bars represent S.D. The images of the plates have been scaled to 25% of the original size.

FIGURE 3.

Validation of a PrprA-lacZ fusion as a reporter system of RcsB activity. Expression levels directed by the chromosomal PrprA lacZ fusion were determined in rcsB⁺ strain T2023, ΔgalU strain T2041, and ΔrcsB strain T1052. The strains were either untransformed or complemented with rcsB, encoded by plasmid pKETS6 (pRcsB). RcsB derivatives D56E (pD56E) and D56A (pD56A) were expressed from plasmids pKETS7 and pKES235, respectively. Cultures for β-galactosidase assays were grown in LB medium to an A₆₀₀ of 0.5, which was supplemented with 1 mm IPTG and 25 μg/ml kanamycin in the case of the transformants. Error bars represent S.D.

FIGURE 4.

Establishing Pwza-lacZ as reporter system of RcsA-RcsB activity. The expression levels of the chromosomally encoded Pwza lacZ fusion were determined in rcsB⁺ strain T2037, ΔgalU strain T2042, ΔrcsB strain T864, ΔrcsBCD strain T921, P_L-rcsA strain T2039, P_L-rcsA ΔgalU strain T2045, and P_L-rcsA ΔrcsBCD strain T963. Expression levels were determined of non-transformed strains or of strains complemented with plasmidic rcsB, expressed from plasmid pKETS6 (pRcsB), pKET7 (pD56E), and pKES235 (pD56A), respectively. Cultures for β-galactosidase assays were grown to A₆₀₀ of 0.5 in LB medium, which was supplemented with 1 mm IPTG and 25 μg/ml kanamycin in the case of transformants. Error bars represent S.D.

FIGURE 5.

Structure model of the receiver and helix-turn-helix domains of RcsB. The structure model of the RcsB receiver domain predicted by the Phyre2 server (57) on the basis of the crystal structure (PDB code 3EUL) of M. tuberculosis NarL (36). Colored amino acids were replaced by alanine. Blue, active site residues Asp-11, Thr-87, and Lys-109 and highly conserved residues Pro-60, Gly-67, and Met-88. Green, the phosphorylation site Asp-56. Pink, presumably surface-exposed residues. For structure presentation, we used the PyMOL Molecular Graphics System, version 1.7.4 (Schrödinger, LLC). A and B show the same side and top views of the RcsB receiver domain. In A, residues of the active quintet and conserved residues of RcsB are labeled, whereas in B, other surface-exposed residues that were mutated are labeled. C, structure of the RcsB helix-turn-helix domain of E. amylovora (PDB code 1P4W) (32), which is 92% identical to E. coli RcsB. α helices 8 and 9, which bind to the DNA, are shown in blue. Residue Lys-180, which was mutated, is labeled K180.

FIGURE 6.

Activation by RcsB mutants as RcsB-RcsB homodimer and as RcsA-RcsB, BglJ-RcsB, and MatA-RcsB heterodimers. The activity of RcsB mutants with exchanges of residues of the active quintet and conserved residues, surface-exposed residues, and a mutation mapping in the DNA-binding domain was analyzed using reporters for RcsB-RcsB (A, strain T1052), RcsA-RcsB (B, strain T963), BglJ-RcsB (C, strain T572), and MatA-RcsB (D, strain T1987). The strains were transformed with empty plasmid pKESK22, as control, and plasmids expressing wild-type and mutant RcsB under control of the IPTG-inducible tac promoter. The pKETS6-derived RcsB plasmids are listed in Table 2. β-Galactosidase expression levels were normalized to the values obtained for RcsB-D56E, which was defined as 100%. Values of bars marked with 1 are from Ref. . Cultures for β-galactosidase assays were grown to A₆₀₀ of 0.5 in LB medium supplemented with 1 mm IPTG and 25 mg/ml kanamycin. Error bars represent S.D.

FIGURE 7.

Model of the complex Rcs phosphorelay. The lipoprotein RcsF monitors the lipoprotein transport by LolA and the outer membrane protein assembly by BamA. Upon perturbations, RcsF activates the sensor kinase RcsC via IgaA (7). Upon induction of the Rcs phosphorelay and autophosphorylation of RcsC, the phosphate is transferred via the receiver domain of RcsC to the histidine transfer domain of RcsD and from there to the receiver domain of RcsB. Regulation of target genes by RcsB homodimers and by RcsA-RcsB heterodimers is dependent on RcsB phosphorylation. RcsB also forms heterodimers with DctR, BglJ, GadE, and MatA. Transcriptional activation by heterodimers of RcsB with BglJ, GadE, and MatA is RcsB phosphorylation-independent. For DctR-RcsB, it is not known whether the activity is phosphorylation-dependent. The genes rcsA, dctR, bglJ, gadE, and matA, which are encoding the auxiliary regulators of RcsB, are all repressed by the nucleoid-associated global repressor H-NS, and activation of their expression provides an additional level of control of the Rcs output.