CpxP, a stress-combative member of the Cpx regulon

Abstract

The CpxA/R two-component signal transduction system of Escherichia coli can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random lacZ operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus, cpxP, whose transcription is stimulated by activation of the Cpx pathway. cpxP specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that cpxP transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that cpxP and cpx mutant strains display hypersensitivity to growth in alkaline conditions.

FIG. 1

cpxP-lacZ transcription is induced in a CpxA-dependent fashion by overproduction of the outer membrane lipoprotein NlpE. β-Galactosidase activities were determined for SP1 (MC4100 ara⁺ λplacMu53[cpxP-lacZ]) (lanes 1 and 2) and SP7 (SP1 cpxA::cam) (lanes 3 and 4). The strains whose β-galactosidase activities are depicted in lanes 1 and 3 were transformed with plasmid pBR322 (control for pLD404). The strains whose β-galactosidase activities are depicted in lanes 2 and 4 were transformed with pLD404 (overproduces NlpE). All strains were grown in Luria broth containing 50 μg of ampicillin per ml as described in Materials and Methods.

FIG. 2

Ac∼P can mediate the transcriptional induction of cpxP-lacZ in the absence of CpxA. β-Galactosidase activities were determined for strains SP34 (MC4100 ara⁺ λplacMu53[cpxP-lacZ] zej::Tn10) (lanes 1 and 2), SP35 (SP34 Δ[pta ackA hisQ hisP]) (lanes 3 and 4), SP36 (SP34 cpxA::cam) (lanes 5 and 6), and SP37 (SP34 cpxA::cam Δ[pta ackA hisQ hisP]) (lanes 7 and 8). Strains whose β-galactosidase activities are depicted in odd-numbered lanes were grown in Luria broth (LB); strains whose β-galactosidase activities are depicted in even-numbered lanes were grown in Luria broth supplemented with 0.4% glucose to stimulate Ac∼P production.

FIG. 3

The cpxP locus. (a) The cpxP open reading frame shown in relation to the cpx operon. The cpx operon and cpxP are divergently transcribed, as shown by the arrows. The size of this genomic region is also shown in nucleotides. (b) Nucleotide and deduced amino acid sequences of the cpxP open reading frame. The site of the cpxR::Ω insertion is marked with Ω. The start codon of cpxR (which is shown in reversed typeface) is depicted by a leftward-pointing arrow. The deduced primary amino acid sequence of cpxP is shown below the nucleotide sequence. A putative Shine-Dalgarno sequence (GGGAG) is enclosed within a box. The residues comprising the putative CpxP signal sequence are shown in boldface. The position of the λplacMu53[cpxP-lacZ] fusion joint is marked by a downward-pointing arrow within the 13th codon of the cpxP open reading frame. An asterisk marks the stop codon of the cpxP open reading frame. A putative rho-independent transcriptional terminator stem loop is underlined with inverted arrows. The adjacent sequence of eight consecutive thymine nucleotides in this putative rho-independent transcriptional terminator is underlined. The nucleotide sequence shown in panel b corresponds to positions 67200 to 68039 of the published DNA sequence for the E. coli chromosomal region from 87.2 to 89.2 min.

FIG. 4

The Cpx signal transduction system regulates transcription of a cpxP-lacZ fusion situated at the λatt site on the E. coli chromosome. Lanes 1, 3, and 5 show β-galactosidase activities of strains transformed with pBAD18 (control for pND18); lanes 2, 4, and 6 show β-galactosidase activities of strains transformed with pND18 (overexpresses nlpE). Lanes 1 and 2, SP702 (MC4100 ara⁺ zej::Tn10 Δ[pta ackA hisQ hisP] λRS88[cpxP-lacZ]); lanes 3 and 4, SP704 (SP702 cpxA::cam); lanes 5 and 6, SP706 (SP702 cpxR::Ω). All strains were grown in Luria broth containing 0.4% l-arabinose and 50 μg of ampicillin per ml (see Materials and Methods for details). These experiments were performed with strains deleted for pta and ackA. Since NlpE synthesis is driven from the araB promoter (11) in these experiments, full transcriptional induction requires growth in arabinose. Hence, Ac∼P synthesis must be eliminated to prevent hyperphosphorylation of CpxR in the cpxR⁺ cpxA background.

FIG. 5

CpxP-AP possesses relatively high alkaline phosphatase activity. Strain SP627a (MC4100, ara74::cam) was transformed with pBR322 (control for pND24) (lanes 1 and 5), pND24 (produces CpxP-AP) (lanes 2 and 6), pBAD18 (control for pCH215) (lanes 3 and 7), and pCH215 (produces SecY-AP) (lanes 4 and 8). The transformants were grown in Luria broth containing 50 μg of ampicillin per ml supplemented with 0.4% l-arabinose to induce the synthesis of SecY-AP from pCH215. (a) The CpxP-AP fusion protein displays relatively high alkaline phosphatase activity. The alkaline phosphatase activities of each of these four transformant strains were determined in the presence of 5 mM IAA (lanes 1 to 4). (b) The amounts of alkaline phosphatase-cross-reacting species generated by the CpxP-AP and SecY-AP fusion proteins are comparable. Immunoblot analysis was performed on whole-cell protein extracts generated from SP627a transformed with pBR322, pND24, pBAD18, and pCH215 (lanes 5 to 8). The whole-cell extracts were separated by SDS-polyacrylamide gel electrophoresis (equal OD₆₀₀ units were loaded in each lane) and subjected to immunoblot analysis with anti-alkaline phosphatase and anti-OmpR antisera. OmpR serves as an additional loading control.

FIG. 6

Subcellular fractionation of CpxP-AP. Whole-cell protein extracts were prepared from strain SP627a (MC4100 ara74::cam) transformed with plasmid pBR322 (control for pND24) (lane 1). Whole-cell, spheroplast, and periplasmic extracts were prepared from SP627a transformed with pND24 (lanes 2 to 4). The protein extracts were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis with anti-alkaline phosphatase, anti-MalE, and anti-OmpR antisera. Abbreviations: WCE, whole-cell extract; SPH, spheroplast extract; PER, periplasmic extract. MalE and OmpR serve as model periplasmic and cytoplasmic proteins, respectively. Both strains were grown to late-log phase in M63 minimal medium supplemented with 0.4% maltose and 50 μg of ampicillin per ml. Protein extracts were then generated as described in Materials and Methods.

FIG. 7

CpxP combats extracytoplasmic stress. Ten microliters of 40% maltose was added to filter discs that had been placed on lawns of strains WBS164 (MC4100 Φ(lamB-lacZX90) Hyb42-1[λp1(209)]) (lanes 1 and 2), SP9 (WBS164 degP::Tn10) (lanes 3 and 4), SP10 (WBS164 λplacMu53[cpxP-lacZ]) (lanes 5 and 6), and SP24 (WBS164 degP::Tn10 λplacMu53[cpxP-lacZ]) (lanes 7 and 8). Strains in odd-numbered lanes were transformed with pBR322 (control for pLD404); strains in even-numbered lanes were transformed with pLD404 (overproduces NlpE and activates the Cpx signal transduction pathway). The values displayed along the y axis (zone of clearing) represent the amount of growth inhibition caused by the addition of maltose. The zone of clearing value is defined as the diameter of growth inhibition around the maltose-saturated filter disc minus the diameter of the filter disc itself (7 mm). The maltose disc assays were performed on M63 minimal agar containing 50 μg of ampicillin per ml and 0.2% glycerol as a carbon source.

FIG. 8

Transcription of the cpxP-lacZ fusion is induced by alkaline pH. The β-galactosidase activities of SP1 (MC4100 ara⁺ λplacMu53[cpxP-lacZ]) (squares) and SP7 (SP1 cpxA::cam) (circles) were determined after strains had been grown at the indicated pH values. Strains were grown in buffered Luria broth ranging from pH 5.3 to 8.4. Luria broth was buffered with 100 mM sodium phosphate as described in Materials and Methods.

FIG. 9

cpxP and cpx null strains are hypersensitive to alkaline pH. Strain SP744 (MC4100 (squares), SP754 (SP744 cpxA::cam) (diamonds), SP762 (SP744 cpxR::Ω) (circles), and SP774 (SP744 λplacMu53[cpxP-lacZ]) (triangles) were grown to saturation in Luria broth. Serial dilutions of each culture were then plated on Luria broth buffered with 100 mM Tris-HCl (ranging from pH 7.0 to 9.5). Strains were incubated at 37°C for 48 h, and the number of CFU/milliliter of each culture at each pH value was determined as described in Materials and Methods. The results are plotted as the log₁₀ of the CFU/milliliter values.

FIG. 10

Homology between the CpxP and Spy proteins. Identical amino acids are shaded; similar amino acids are connected with plus signs.