The CpxRA signal transduction system of Escherichia coli: growth-related autoactivation and control of unanticipated target operons

Abstract

In Escherichia coli, the CpxRA two-component signal transduction system senses and responds to aggregated and misfolded proteins in the bacterial envelope. We show that CpxR-P (the phosphorylated form of the cognate response regulator) activates cpxRA expression in conjunction with RpoS, suggesting an involvement of the Cpx system in stationary-phase survival. Engagement of the CpxRA system in functions beyond protein management is indicated by several putative targets identified after a genomic screening for the CpxR-P recognition consensus sequence. Direct negative control of the newly identified targets motABcheAW (specifying motility and chemotaxis) and tsr (encoding the serine chemoreceptor) by CpxR-P was shown by electrophoretic mobility shift analysis and Northern hybridization. The results suggest that the CpxRA system plays a core role in an extensive stress response network in which the coordination of protein turnover and energy conservation may be the unifying element.

FIG. 1

Representation of the cpxP-cpxRA intergenic region. Putative recognition sequences for ς⁷⁰ (single lines above or below the sequence) and ribosome binding (RBS) are shown. The perfect CpxR-P recognition consensus sequence is boxed in a bold line, whereas the consensus sequence with a 2-bp mismatch is boxed in a thin line. The consensus mismatches are indicated by black circles.

FIG. 2

Representation of the Φ(cpx-lacZ) operon fusion constructs integrated at the λ attachment site (attB) of strain ECL3501. The black box depicts the deleted region within cpxRA. The hatched boxes depict the in-frame deletion (418 bp) within cpxR. The Φ(cpx-lacZ) constructs contain 309 bp upstream from the cpxR translational start point and include the cpxRA promoter region (P) (Fig. 1).

FIG. 3

Effect of CpxR-P on Φ(cpxRA⁺-lacZ) expression. Growth curves and β-galactosidase activity profiles of strain ECL3502 [Φ(cpxR⁺A⁺-lacZ)] (A) and strain ECL3503 [Φ(cpxR^ΔA⁺-lacZ)] (B) are shown. The cells were grown in glucose minimal medium (pH 7.0) at 37°C.

FIG. 4

Effect of RpoS on Φ(cpxRA⁺-lacZ) expression. Growth curves and β-galactosidase activity profiles of strain ECL3506 [Φ(cpxR⁺A⁺-lacZ) rpoS::Tn10] (A) and strain ECL3507 [Φ(cpxR^ΔA⁺-lacZ) rpoS::Tn10] (B) are shown. The cells were grown in glucose minimal medium (pH 7.0) at 37°C.

FIG. 5

Effect of CpxR-P on Φ(cpxR⁺A*-lacZ) expression. Growth curves and β-galactosidase activity profiles of strain ECL3504 [Φ(cpxR⁺A*-lacZ)] (A) and strain ECL3505 [Φ(cpxR^ΔA*-lacZ)] (B) are shown. The cells were grown in glucose minimal medium (pH 7.0) at 37°C.

FIG. 6

Negative regulation of motABcheAW by the Cpx system. (A) Promoter region of motABcheAW (8, 14, 15). The CpxR-P recognition site is boxed. The Eς^F promoter and transcriptional start site are assigned on the basis of the Eς^F recognition consensus sequence (2, 15). RBS, ribosome binding site. (B) Swarm patterns. (a) ECL3502 [Φ(cpxR⁺A⁺-lacZ)]. (b) ECL3503 [Φ(cpxR^ΔA⁺-lacZ)]. (c) ECL3504 [Φ(cpxR⁺A*-lacZ)]. (d) ECL3505 [Φ(cpxR^ΔA*-lacZ)]. (C) Electrophoretic mobility shift analysis of the motABcheAW promoter DNA region with CpxR or CpxR-P. Lane 1, motABcheAW promoter DNA; lane 2, promoter DNA plus CpxR; lane 3, promoter DNA plus CpxR-P. (D) Profiles of expression of motABcheAW in the four Φ(cpx-lacZ) strains, as determined by Northern analysis. Error bars indicate standard deviations.

FIG. 7

Negative regulation of tsr by the Cpx system. (A) Promoter region of tsr (14). The CpxR-P recognition site is boxed. The Eς⁷⁰ promoter site is putative, but involvement of Eς⁷⁰ in tsr expression has been reported (14). RBS, ribosome binding site. (B) Electrophoretic mobility shift analysis of the tsr promoter DNA region with CpxR or CpxR-P. Lane 1, tsr promoter DNA; lane 2, promoter DNA plus CpxR; lane 3, promoter DNA plus CpxR-P. (C) Profiles of expression of tsr in the four Φ(cpx-lacZ) strains, as determined by Northern analysis. Error bars indicate standard deviations.

FIG. 8

Representation of CpxRA signal transduction. CpxA and CpxR are shown as dimers. OM, outer membrane; PS, periplasmic space; IM, inner membrane; X, unknown activator. For explanations, see the text.