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J. Biol. Chem., Vol. 277, Issue 29, 26652-26661, July 19, 2002

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Genome-wide Profiling of Promoter Recognition by the Two-component Response Regulator CpxR-P in Escherichia coli^*

Peter De Wulf^§¶, Abigail M. McGuire^¶**, Xueqiao Liu, and Edmund C. C. Lin^§§

From the  Department of Microbiology and Molecular Genetics and ^** Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, April 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In Escherichia coli, the two-component Cpx system comprising the CpxA sensor kinase and the CpxR response regulator modulates gene expression in response to a variety of stresses including membrane-protein damage, starvation, and high osmolarity. To date, the few known CpxR-P target operons were mostly identified by genetic screens. To facilitate the discovery of all target operons, we derived a 15-bp weighted matrix for CpxR-P recognition that takes into account the relative base frequency at each nucleotide position. This matrix essentially consists of two tandem 5'-GTAAA-3' motifs separated by a 5-bp linker. All of the 15-bp stretches on both strands of the E. coli MG1655 genome were then scored for their degree of matching with the matrix and classified in statistical deviation groups. The effectiveness of this screening is indicated by the identification of eight new target operons (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) among eleven candidates tested. Moreover, the matrix score correlates with the likelihood that a site is a true target and with the relative site affinity for CpxR-P in vitro. Our data indicate that some 100 operons are under direct CpxR-P control and that the signal transduction pathway interacts with several other control circuits in manners hitherto unanticipated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cpxRA operon of Escherichia coli encodes the CpxA sensor kinase/phosphatase and the CpxR cognate response regulator. Together with RpoE (^E) and RpoH (³²), this system governs the expression of genes involved in relieving envelope-protein distress (1-3). The Cpx system also regulates biofilm formation (4), motility and chemotaxis (5), host cell invasion, and virulence (6-8). The expression of cpxRA increases sharply at the onset of the stationary growth phase. This induction results from the activation of the operon by RpoS (^S) in synergy with autogenous activation by CpxR-P (5, 9). Thus, an intricate stress response network that integrates the Cpx, RpoE, RpoH, and RpoS regulatory pathways seems to have evolved in E. coli. A clue for an extensive physiological role of the Cpx system is provided by numerous seemingly unrelated phenotypes of cpxA* mutants (10) that synthesize a sensor protein defective in its CpxR-P phosphatase activity (11). An in-frame cpxR mutation abolished all of the anomalous phenotypes tested (12). These phenotypes include randomized positioning of the FtsZ ring during cell division (13), tolerance to the antibiotic amikacin (14), failure to grow on succinate (10), ability to grow at high pH (15), resistance to CuCl₂ (12), sensitivity to high temperature (16), and reduced swarming ability (12).

Because of the complexity and extensiveness of the Cpx response and its overlap with responses elicited by several other signal transduction pathways, it is especially difficult to identify all of the direct target operons of the Cpx system. Without knowing most of these operons, it is hard to understand the overall selective advantage of the Cpx response. The aim of this study is to develop a CpxR-P recognition weight matrix that helps to identify reliably target promoters by the presence of a specific nucleotide sequence closely conforming to the matrix.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Matrix Screening-- The method of Berg and von Hippel (17) was used to score genomic sites with the CpxR-P weight matrix developed with the AlignACE program (18). The matrix screening method predicts the affinity of CpxR-P for a given DNA sequence based on the sequence statistics of 10 input CpxR-P-controlled promoter sequences. Both strands of the E. coli K-12 MG1655 genome sequence obtained from GenBank^TM accession number U00096 were searched. Near-symmetric sites with high scores in both the forward and reverse directions were counted only once, and the higher of the two scores was then used (19).

Strains and Growth Conditions-- The E. coli strains used in this study (Table I) are deleted in the cpxRA operon at its natural locus. Each strain bears at its attB site a -phage containing a particular cpx operon followed in-frame by the lacZ-coding sequence (5). To confirm the cpx genotype of strains ECL3502 (cpxR⁺A⁺-lacZ that contains a complete cpxRA operon), ECL3503 (cpxRA⁺-lacZ in which cpxR sustained an in-frame deletion), and ECL3504 (cpxR⁺A^*-lacZ in which cpxA sustained a base change resulting in a Leu38  Phe substitution rendering CpxA* phosphatase-defective), we assayed their reporter -galactosidase activities (20). Therefore, the strains were cultured at 30 or 37 °C in LB medium (20 ml in baffled shake flask of 250 ml and rotated at 300 rpm¹) until an optical density (A₆₀₀) of 0.9 was reached. At this point of growth, the (cpxR⁺A^*-lacZ) transcription level in strain ECL3504 exceeded the level of (cpxR⁺A⁺-lacZ) expression in strain ECL3502 by a factor of 3, whereas the expression level of (cpxRA⁺-lacZ) was ~20% (cpxR⁺A⁺-lacZ) expression level in strain ECL3502 (data not shown).

Northern Analysis of Gene Expression-- To analyze the expression of ung, ompC, psd, mviM, aroK, hlpA, secA, ubiC, flgM, aer, and envZ, the cells were grown at 37 °C until reaching A₆₀₀ of 0.9 for RNA isolation. To analyze the expression of rpoErseABC, the cells were grown at 30 °C until reaching A₆₀₀ of 0.9 and then shifted to 50 °C for 10 min for RNA extraction. Total RNA was isolated (RNeasy Total RNA System, Qiagen) and electrophorized in Tris acetate/EDTA-agarose (1%) containing guanidine thiocyanate (20 mM). The electrophorized and denatured RNA was blotted overnight onto Hybond-N nylon membranes (Amersham Pharmacia Biosciences). All of the RNA was then cross-linked (UV Stratalinker 2400, Stratagene), and the target mRNAs were hybridized with radiolabeled DNA probes. Radiolabeled DNA probes were PCR-generated (TaqPlus Precision polymerase mixture, Stratagene) from the chromosome of strain ECL3502 (cpxR⁺A⁺) in the presence of 500 µM [-³²P]dATP.

The transcription of ung was assayed with a 458-bp DNA fragment (from bp 35 to bp 493 in the coding sequence) that was PCR-generated with primers UNG1 (5'-CTGAAGAGAAGCAGCAACC-3') and UNG2 (5'-CCCACAACAAAAACACCAC-3').

The transcription of ompC was assayed with a 602-bp DNA fragment (from bp 144 to bp 746 in the coding sequence) that was PCR-generated with primers OMPC1 (5'-AGATGTAGATGGCGACCAG-3') and OMPC2 (5'-TCGTATTTCAGACCACCAG-3'). The transcription of psd was assayed with a 716-bp DNA fragment (from bp 89 to bp 805 in the coding sequence) that was PCR-generated with primers PSD1 (5'-CAGGATGGCTGACAAAAC-3') and PSD2 (5'-CAACCAGATTCACTTTACCC-3').

The transcription of mviM was assayed with a 674-bp DNA probe (from bp 156 to bp 830 in the coding sequence) that was PCR-generated with primers MVIM1 (5'-TTATGCCGATTCGTTATCC-3') and MVIM2 (5'-ACACATTCAATGAAGTGCC-3').

The transcription of aroK was assayed with a 486-bp DNA fragment (from bp 25 to bp 511 in the coding sequence) that was PCR-generated with primers AROK1 (5'-AGCGACCAGATATGCAGAG-3') and AROK2 (5'-CAAGATAAACGACAACGCC-3').

The transcription of hlpA was assayed with a 351-bp DNA fragment (from bp 31 to bp 382 in the coding sequence) that was PCR-generated with primers HLPA1 (5'-GGTTTAGCACTGGCAACTTC-3') and HLPA2 (5'-CAACGGATTTCACAGCAG-3'). The transcription of secA was assayed with a 743-bp DNA fragment (from bp 346 to bp 1089 in the coding sequence) that was PCR-generated with primers SECA1 (5'-GCTTACCTGAACGCACTAAC-3') and SECA2 (5'-CACACCTTCTTTCGCTTC-3').

The transcription of ubiC was assayed with a 299-bp DNA fragment (from bp 70 to bp 369 in the coding sequence) that was PCR-generated with primers UBIC1 (5'-CTGCGATACAA TGCCTTTAC-3') and UBIC2 (5'-TTCACCATCGGCACATAAC-3').

The transcription of flgM was assayed with a 211-bp DNA fragment (from bp 24 to bp 235 in the coding sequence) that was PCR-generated with primers FLGM1 (5'-TCTGAAGCCTGTAAGCACC-3') and FLGM2 (5'-TGTCCATTTTTAGTTCACCG-3'). The transcription of rpoErseABC was assayed with a 610-bp DNA fragment (from bp 41 to bp 651 in the coding sequence) that was PCR-generated with primers RPOE1 (5'-CCTGATAAGCGGTTGAACTTTGTTATC-3') and RPOE2 (5'-GATAGCGCGTGGAAATTTGGTTTGG-3'). The transcription of aer was assayed with a 987-bp DNA fragment (from bp 322 and bp 1309 in the coding sequence) that was PCR-generated with primers AER1 (5'-ATCAGTGGCTATATGTCGATTCGTACCC-3') and AER2 (5'-CCACAATATCTTCCATCGTCCGTCCG-3'). The transcription of envZ was assayed with a 920-bp DNA fragment (from bp 1958 to bp 2878 in the coding sequence) that was PCR-generated with primers ENVZ1 (5'-TTGGTTGTGCCTCCCGCTTTCC-3') and ENVZ2 (5'-CGGACCGTCATCTTCCACCTGG-3').

Following hybridization, radioactive signals were visualized after overnight incubation of the hybridized membranes on phosphorimaging screens (Amersham Biosciences). The screens were scanned by the PhosphorImager 445SI (Amersham Biosciences), and the obtained signals were quantified with ImageQuant 5.0 (Amersham Biosciences). The level of radiation background was determined by quantifying a part of the membrane lying outside of the target mRNA signals. The transcription of each genetic locus was performed by at least three independent experiments. The levels of transcription were averaged, and their standard deviations were calculated. For graphical representation, the transcription levels were normalized to the averaged values measured in wild-type strain ECL3502 (cpxR⁺A⁺).

Electrophoretic Mobility Shift Analysis of CpxR(-P) Binding to Promoter DNA-- The ability of CpxR and CpxR-P to recognize and bind to the promoters of rpoErseABC, aer, and ompR-envZ was examined according to a previously described procedure (5). A CpxR(-P)/DNA ratio ranging from 10 to 25 (100-250 pM/10 pM) was tested in the presence of a 500-fold molar excess of competitor DNA (sheared salmon sperm DNA, Promega) and a 100-fold molar excess of competitor protein (bovine serum albumin, Sigma).

CpxR was overproduced as His₆-CpxR, purified, and phosphorylated (when required) with 50 mM acetyl phosphate (3). For analysis of CpxR(-P) binding to the rpoErseABC promoter, a 214-bp promoter fragment (bp 17-231 upstream of the start codon, Fig. 5A) was PCR-amplified with primers RPOE/EMS1 (5'-CCAAACCAAATTTCCACGCGCTATC-3') and RPOE/EMS2 (5'-TTACTCTTCAGGCAGTTAAATGGGC-3'). For analysis of CpxR(-P) binding to the aer promoter, a 238-bp promoter fragment (bp 36-274 upstream of the start codon, Fig. 6A) was PCR-amplified with primers AER/EMS1 (5'-TCTTGTCAGAGTTTATGTCGGCCCCGC-3') and AER/EMS2 (5'-GTTGTTATTTTGCACGGTTTTTGTCGGG-3'). For analysis of CpxR(-P) binding to the ompR-envZ promoter, a 206-bp promoter fragment (bp 6-212 upstream of the start codon) was PCR-amplified with primers OmpR/EMS1 (5'-TCTGTTTGATAATGCGCACATTGGG-3') and OmpR/EMS2 (5'-GTACTCCCAAAGGTTCGCAACAATTTG-3'). All of the promoter fragments were radiolabeled during PCR by supplying 500 µM [-³²P]dATP (PerkinElmer Life Sciences) to the reaction.

CpxR(-P), radiolabeled promoter DNA, competitor protein, and competitor DNA were incubated for 30 min at 37 °C in a reaction buffer containing 10 mM Tris, pH 7.4, 5% glycerol, 50 mM KCl, 10 mM MgSO₄, 20 mM potassium glutamate, 1 mM EDTA, and 1 mM dithiothreitol. The samples were then electrophoresed on 5% non-denaturing acrylamide Tris borate/EDTA mini-gels in Tris borate/EDTA buffer. The gels were vacuum-dried and exposed to x-ray film (Kodak BioMax).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Derivation of a CpxR-P Weight Matrix for Target Operon Recognition-- On the basis of DNase I protection analyses of the degP, ppiA, and yihE-dsbA promoter regions, a 14-bp CpxR-P recognition box (5'-GTAAA(N)₅GTAA-3') was first proposed (3). When the genome of E. coli K-12 MG1655 was later scanned for this box, 50 sites positioned within 450 bp upstream of start codons were found (5). Three operons, motABcheAW, tsr, and cpxRA, containing such a site in their promoter region were then chosen for genetic and biochemical verification as actual CpxR-P targets. The motABcheAW and tsr operons proved to be under direct negative control, whereas the cpxRA operon turned out to be under autogenous positive control (5).

A genetic screening for Cpx-controlled operons using transcriptional lacZ fusions yielded ppiD (encoding periplasmic isomerase D) as a target (2). Using the previously reported 14-bp recognition sequence as a reference, three 7-bp boxes (5'-GGTAAA(G/C)-3') separated by 41 and 13-bp links were designated as CpxR-P binding sites. Deleting the region containing these boxes rendered the expression of ppiD independent of CpxR-P (2). However, the contribution of each individual box was not determined.

The discrepancy in length and base composition between the originally proposed 14-bp recognition sequence (5'-GTAAA(N)₅GTAA-3') and the subsequently proposed 7-bp box (5'-GGTAAA(G/C)-3') for CpxR-P recognition prompted us to characterize the site by a weight matrix approach that takes into account the base frequency at each nucleotide position within the recognition sequence. For input purpose, we assembled 10 known target operons (Table II) identified on the basis of four different criteria. The first group consisted of three operons that were shown to be regulated by CpxR-P in vivo, and their promoter sequences were shown to be protected from DNase I activity by CpxR-P in vitro (degP, ppiA, and yihE-dsbA) (1, 3, 11). The second group consisted of three operons that were shown to be regulated by CpxR-P in vivo, and their promoter sequence (motABcheAW, tsr, and ygjT) was shown to be electrophoretically retarded by CpxR-P in vitro (5).¹ A third group was represented by ppiD whose expression was liberated from CpxR-P control after deletion of the three 7-bp CpxR-P boxes in the promoter (2). The fourth group consisted of three operons that were shown to be regulated by CpxR-P in vivo, and their promoter sequence (cpxRA, cpxP, and csgBAC) was shown to contain the proposed 14-bp CpxR-P box (4, 5, 9, 15).² Using the promoter regions of these 10 operons, we aligned 500-bp fragments from 400 bp upstream to 100 bp downstream of the start codon with the motif-finding program AlignACE (18), which is based on the Gibbs sampling algorithm (21). A highly conserved 15-bp stretch was identified in each of the input promoters (Table II), and the base frequency at each nucleotide position was then used to calculate a CpxR-P recognition weight matrix (17). This matrix was found to extend the originally proposed 14-bp CpxR-P recognition box (3) by 1 bp (Fig. 1). The modified 15-bp stretch (hereafter referred to as "potential CpxR-P recognition site") essentially consists of two highly conserved pentamers in tandem (the second one being a bit less conserved) separated by five random bases, 5'-GTAAA(N)₅GTAAA-3' (Fig. 1).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Sequence logo for the CpxR-P recognition weight matrix in E. coli. The base conservation measured in bits is shown as the relative height of each base (39). The total sequence conservation is 13.9 ± 0.7 bits.

Screening the E. coli Genome with the CpxR-P Recognition Weight Matrix-- Each of the 4,639,206 successive 15-bp stretches of the E. coli K-12 MG1655 genome (22) was scored on both strands for its degree of matching with the CpxR-P recognition weight matrix using the log transformation method of Berg and von Hippel (17). The distribution of scores from all of the potential CpxR-P recognition sites is displayed in Fig. 2A. The average of all of these scores, i.e. the genomic mean, was assigned a Z score of 0. A histogram window comprising the highest scoring sites (Z  +3.5) is shown in Fig. 2B. As one would expect, these sites include those that are present in the 10 CpxR-P input target promoters, because the probability of being a true CpxR-P regulatory site should statistically increase with an increasing Z score. The mean score µ_i of the potential CpxR-P recognition sites in the 10 input promoters has a Z value of +4.85. We next determined the standard deviation, _i, of these input sites and ranked them in four deviation groups relative to µ_i (Fig. 2B).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Histogram of the genomic Z scores of all 15-bp stretches on both strands of the genome and scores of operons relevant to the study. A, the distribution of the scores of all potential CpxR-P recognition sites (15-bp stretches). The average score is assigned a genomic Z value of 0. The units in the abscissa represent the number of standard deviations above or below the genomic mean of Z = 0. B, potential CpxR-P recognition sites with Z scores above 3.5. µi denotes the mean Z score of the CpxR-P binding sites present in the input promoter regions. i denotes the mean ± S.D. above or below µi. Arrows with open heads indicate the Z score positions of the recognition sites used to develop the CpxR-P weight matrix. Arrows with solid heads indicate the Z score positions of potential CpxR-P recognition sites identified as true targets in this study. Arrows with gray heads indicate the Z score positions of the recently reported CpxR-P targets spy (23) and minCDE.3

The first standard deviation group (Fig. 2B, Table III) scoring above µ_i + 1_i contained only two potential CpxR-P recognition sites, both of which belong to the input operons. One site (designated as cpxRA1/cpxP1) is intergenically positioned between the divergently transcribed cpxRA and cpxP operons (5). The other site (designated as ygjT1) is positioned in the promoter region of ygjT. The second standard deviation group (Fig. 2B, Table III) scoring between µ_i and µ_i + 1_i contained 21 potential CpxR-P recognitions sites. Among these, 13 sites (or 62%) are located in intergenic regions as categorized according to Robison et al. (19). Representatives include the motABcheAW, tsr, and ppiA input operons. The third standard deviation group (Fig. 2B, Table IV) scoring between µ_i and µ_i  1_i comprises 138 potential CpxR-P recognition sites. Among these, 44 sites (or 32%) are located in intergenic regions. Representatives include the yhiE-dsbA, degP, csgBAC, and ppiD input operons. The fourth standard deviation group (Fig. 2B, not tabulated) scoring between µ_i - 1_i and µ_i - 2_i comprises 743 potential CpxR-P recognition sites. Among these, 186 sites (or 25%) are located in intergenic regions. Representatives include the second potential CpxR-P recognition site that is intergenically positioned between the cpxRA and cpxP operons (designated as cpxRA2/cpxP2) (5) and the second potential CpxR-P recognition site in front of ygjT (designated as ygjT2). In total, 245 intergenic sites score higher than µ_i  2_i. A list of all potential CpxR-P recognition sites in the genome and their scores will be made available at arep.med.harvard.edu/ecoli_matrices.

View this table: [in this window] [in a new window]   Table IV Selection of putative CpxR-P target operons with known or putative function that contain in their promoter region a potential CpxR-P binding site that scores between µi and µi  1 ia

Although some potential CpxR-P recognition sites are located in coding sequences, it is apparent that there is a strong tendency for high scoring sites to be located in promoter regions. This trend may in part reflect the A/T-rich bias of both the potential CpxR-P recognition sites and promoter regions. An in-depth discussion of the significance of weight matrix discrimination between non-coding and coding sequences in E. coli is found in Robison et al. (19).

Putative target operons were also categorized according to the number of potential CpxR-P recognition sites that is located in their promoter region. Thirty-two promoters, 25 of which precede coding sequences with known or assigned function (Table V), were found to contain at least two potential CpxR-P recognition sites, each scoring greater than µ_i - 2_i. Among the 25 promoters, those of ompF and yhdG contain three sites with two sites overlapping by 5 bp.

Evaluation of the CpxR-P Matrix-screening Method-- To evaluate the usefulness of the computational screening method in predicting CpxR-P-regulated operons, we selected 11 candidates for testing by Northern analysis. For the purpose of broad representation, test operons with diverse known functions bearing the potential CpxR-P recognition site at diverse locations were selected from three different standard deviation groups (Fig. 2B). The first group (µ_i  µ_i + 1_i) is represented by ung (µ_i + 0.63_i, encodes uracil-DNA glycosylase), ompC (µ_i + 0.56_i, encodes outer membrane protein 1b), and psd (µ_i + 0.43_i, encodes phosphatidyl serine decarboxylase). The second group (µ_i  µ_i - 1_i) is represented by mviM (µ_i - 0.19_i, encodes a virulence factor), aroK (µ_i - 0.22_i, encodes shikimate kinase I), hlpA (µ_i - 0.22_i, encodes a periplasmic chaperone), and rpoErseABC (µ_i - 0.53_i, encodes the ^E transcriptional factor and its regulators). The third group (µ_i- 1_i  µ_i - 2_i) is represented by secA (µ_i - 1.32_i, encodes a translocation ATPase for protein export), ubiC (µ_i - 1.34_i, encodes chorismate lyase), flgM (µ_i - 1.49_i, encodes the anti-²⁸ factor), and aer (µ_i - 1.75_i, encodes the aerotaxis sensor protein). As a negative control, envZ (µ_i - 1.93_i, encodes the EnvZ osmosensor kinase) was used, because the operon is known not to be regulated by CpxR-P (3).

An expression of the 12 operons was analyzed in strains ECL3502 (cpxR⁺A⁺), ECL3503 (cpxRA⁺), and ECL3504 (cpxR⁺A^*). It should be noted that ECL3504 (cpxR⁺A^*) was used as a strain with an elevated level of CpxR-P (a consequence of the phosphatase defect of CpxA*) instead of a transformant of ECL3503 (cpxRA⁺) that bears a plasmid expressing cpxR⁺, because excessive copies of CpxR are toxic (data not shown). The expression data (Fig. 3) showed that in the highest scoring group (µ_i  µ_i + 1_i), all of the three target operons (ung, ompC, and psd) are activated by CpxR-P. In the middle scoring group (µ_i  µ_i - 1_i), three targets (mivA, aroK, and rpoErseABC) of the four tested are CpxR-P-regulated. In the lowest scoring group (µ_i - 1_i  µ_i - 2_i), two targets (secA and aer) of four (not counting fifth target envZ, which is the negative control) are CpxR-P-regulated. It should be reiterated that both of the two operons scoring above µ_i + 1_i (cpxRA and ygjT) have already been shown to be CpxR-P-regulated (5). Although the total number of potential CpxR-P recognition sites tested is modest, the results indicate that the weight matrix approach is not only helpful in the search for target operons but also useful in estimating the likelihood of a candidate operon being a true target. It might be of interest to mention that the spy gene (encoding a periplasmic protein of unknown function) recently shown to be under the positive control of CpxR-P on the basis of expression levels in cpxR⁺A⁺, cpxR⁺A^*, and cpxR backgrounds (23) has a potential CpxR-P recognition site that scores µ_i + 0.13_i (Fig. 2B, Table II). Thus, the CpxR-P control seems to be direct. Likewise, the CpxR-P repression of minCDE (µ_i - 1.14_i) recently found is also likely to be direct.³

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Transcriptional analysis of matrix-identified CpxR-P target operons. Expression profiles of 11 putative CpxR-P target operons determined in strains ECL3502 (cpxR+A+), ECL3503 (cpxRA+), and ECL3504 (cpxR+A*). The ompR-envZ operon served as the negative control (3). The genomic Z score of each operon is shown in parentheses. Transcription levels were determined in strains ECL3502 (cpxR+A+, white bars), ECL3503 (cpxRA+, gray bars), and ECL3504 (cpxR+A*, black bars). Error bars indicate the means ± S.D. calculated from at least three independent experiments.

In Vitro Binding of CpxR-P to rpoErseABC and aer Promoter Fragments-- To confirm that the regulation of mRNA levels of operons possessing a potential CpxR-P recognition site in the promoter region is the result of direct CpxR-P control, we tested appropriate DNA fragments of rpoErseABC and aer for in vitro binding of the regulator protein. We chose these two operons, because of their roles in adaptation to environmental changes, for example, RpoE mediates a response to envelope-protein distress (Fig. 4), and Aer mediates aerotaxis (5).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   The RpoS (S), Cpx, RpoE (E), and RpoH (32) response network. The top row shows the various stimuli of each pathway. Envelope distress includes the presence of aggregated, misfolded, or damaged periplasmic proteins (1, 3), the overproduction of NlpE (new lipoprotein E) (35), a deficiency in phosphatidylethanolamine (34), and the accumulation of enterobacterial common antigen intermediate (lipid II) (36). The dashed arrows represent the activation of CpxA and RpoE or the stimulation of RpoS and RpoH synthesis. CpxP is thought to bind the periplasmic domain of CpxA, thereby inhibiting CpxA kinase activity (9). The dissociation of CpxP from CpxA allows CpxA to receive the signal(s) and catalyze the phosphorylation of CpxR at the expense of ATP as indicated by the dotted arrows. RpoE anchored to the inner membrane by RseA and RseB is prevented from functioning (27, 28). The dotted arrow shows the release of RpoE into the cytoplasm for action. The solid arrows represent transcriptional activation, and the solid lines with a bar at the end represent transcriptional repression. The RpoE- and RpoH-controlled operons designated with an asterisk also contain a high scoring potential CpxR-P recognition site in their promoter region. The operons highlighted in boldface (ung, ompC, psd, mviM, aroK, secA, aer, and rpoErseABC) were shown in this study to be under the control of CpxR-P.

To test the direct control of rpoErseABC expression by CpxR-P, we generated a 214-bp fragment of the rpoErseABC promoter containing a single potential CpxR-P recognition site at its center for use in EMS⁴ analysis. The putative CpxR-P binding site begins at 59 bp downstream of the transcriptional start of the first promoter (P1⁷⁰) and covers the 35^E recognition sequence of the second promoter (P2^E) (Fig. 5A). The results indicated that an effective binding to the rpoErseABC promoter occurred only when CpxR (200-250 pM) was phosphorylated (Fig. 5B). No binding was detected when a 206-bp fragment of the ompR-envZ promoter was used as a negative control (data not shown). The overlap of the putative CpxR-P binding site with the 35^E recognition sequence of P2^E is consistent with the repression of the operon by CpxR-P (Fig. 3). To what extent CpxR-P can attenuate transcription from the putative P1⁷⁰ site remains to be determined, because the condition that triggers rpoErseABC expression from this putative promoter is unknown.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Negative regulation of rpoErseABC expression by CpxR-P. A, promoter and coding sequences of rpoErseABC (27, 28). Bold lines show sequence fragments used in EMS and Northern hybridization analyses. Boxes show that potential CpxR-P recognition site is located on the complementary strand. Hooked arrows mark the transcriptional start sites (the P170 start site is putative). Arrowheaded lines mark the coding sequences. The binding sites for 70 and E are underlined. B, EMS analysis of rpoErseABC promoter DNA with CpxR and CpxR-P.

To test the direct involvement of CpxR-P in controlling aer expression by EMS analysis (Fig. 6A), we generated a 238-bp promoter fragment containing the single potential CpxR-P recognition site at its center. This putative binding site begins at 27 bp upstream of the transcriptional start of the first promoter (P1⁷⁰) and covers the central part of the ⁷⁰ binding site. The data showed that effective binding occurred only when CpxR (250 pM) was phosphorylated (Fig. 6B). Again, no binding was detected when a 206-bp fragment of the ompR-envZ promoter was used as a negative control (data not shown). The location of the potential CpxR-P recognition site within the ⁷⁰ binding region of the aer promoter (Fig. 6A) is consistent with the repression of the operon by CpxR-P (Fig. 3).

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Negative regulation of aer expression by CpxR-P. A, promoter and coding sequences of aer. Bold lines show sequence fragments used in EMS and Northern hybridization analyses. Boxes mark regulator binding sites. Hooked arrows show the transcriptional start sites (22). Arrowheaded lines show coding sequences. The binding sites for 70 are underlined. B, EMS analysis of aer promoter DNA with CpxR and CpxR-P.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effectiveness of the CpxR-P Recognition Matrix Sequence-- In light of the CpxR-P recognition weight matrix developed in this study, the apparent contradiction between the originally proposed 14-bp CpxR-P recognition box based on three DNase I protection experiments (3) and the subsequently proposed 7-bp box based on a single deletion experiment (2) can be readily resolved by the following explanation. In the former case, the involvement of the fifteenth base pair was missed, because that position is the least conserved within the two tandem pentamers. In the latter case, the extensive 147-bp deletion of the ppiD promoter made it difficult to identify the actual CpxR-P binding sequence. The ppiD promoter most probably has a single CpxR-P binding site that uses a part of the second 7-bp box as the first 5'-pentamer of the 15-bp recognition sequence (Tables II and IV).

According to the matrix analysis used, the ideal CpxR-P site should have the highest Z score, barring the influence of contextual reading. An indication of the soundness of the 15-bp weight matrix is the degree of correlation that exists between the relative affinity of CpxR-P for a recognition site and the Z score of that site. The concentrations of CpxR-P required for the mobility retardation of the promoters of motABcheAW (µ_i + 0.82_i), tsr (µ_i + 0.69_i), rpoE (µ_i - 0.53_i), and aer (µ_i  1.75_i) are ~150, 150, 200, and 250 pM, respectively (Figs. 2, 5B, and 6B) (5). When both CpxR-P binding parameters were plotted against each other, a first order correlation was observed (Fig. 7).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Correlation between the affinity of a CpxR-P site for CpxR-P with the matrix-recognition score of that site. The affinity is determined by the concentration of CpxR-P required for retardation of the electrophoretic mobility of the promoter sequence containing a CpxR-P binding site. The data were obtained from published work (motABcheAW, tsr) (for review see Ref. 5) and present (aer, rpoErseABC) studies.

A further indication of the soundness of the 15-bp weight matrix is the increase in the percentage of intergenically located potential CpxR-P recognition sites with an increasing Z score above µ_i - 2_i and the probability of these sites being true targets. However, not all of the targets scoring above a certain level can be expected to be true binding sites. This is because the set of 10 input promoters used might not accurately reflect the statistical distribution of all of the real sites. The site location may not be appropriate, and/or the surrounding sequence context is not permissive. All of the factors considered, a rough prediction of ~100 true CpxR-P target operons in E. coli seems to be reasonable. This estimate is based on the presence of 245 potential CpxR-P recognition sites in the intergenic regions scoring greater than µ_i - 2_i and the assumption that half of these sites are functional. The combined use of CpxR-P matrix screening with a microarray expression analysis would provide a more definitive identification of target operon numbers. Questionable candidates can then be singled out for further testing by in vitro DNA analysis.

Recognition of 5-bp Tandem Repeats by CpxR-P-- In contrast to most transcriptional regulators, CpxR-P does not recognize an inverted repeat sequence. Instead, it recognizes two tandemly organized pentamers that are separated by a 5-bp linker. Although the linker sequence composition is random, its length is of critical importance. For instance, a cydDC promoter fragment containing the two conserved pentamers separated by a 4-bp linker (5'-GTAACAAAAGTAAA-3') and a proP promoter fragment containing the two conserved pentamers separated by a 6-bp linker (5'-GTAAATTTGGCGTAAA-3') were both not recognized by CpxR-P in EMS analyses. No interaction was evident even when the regulator was provided in concentrations up to 500 pM (data not shown). The presence of a C instead of an A as the fifth nucleotide of the first pentamer in the cydDC fragment should not prevent CpxR-P binding, because the 5'-GTAAC-3' sequence is also the first pentamer in the bona fide CpxR-P recognition site of the aer promoter (Fig. 6A).

Another two-component response regulator that also recognizes a tandem repeat sequence is OmpR-P, which is the closest homologue of CpxR-P in E. coli. OmpR-P binds to two 10-bp half-sites that are not separated by a linker. OmpR-P acts as a dimer with each monomer interacting with the DNA helix in the major groove (24). CpxR-P most probably binds its target site in a similar non-symmetric manner. The higher degree of conservation of the first pentamer (5'-GTAAA-3') suggests that the CpxR-P monomer binds the first major groove more tightly than the second major groove (25).

Coordination of the RpoE and RpoH Stress Response Pathways by the Cpx System-- Even though the Cpx and RpoE (^E) systems (Fig. 4) both respond to protein-envelope distress and are stimulated by unassembled P-pilus subunits to activate the degP operon encoding a periplasmic protease (1, 3, 26), the two systems are distinct in terms of the true nature of specific signals, mechanism of signal transduction, and collective target operons (27, 28, 29). The negative control of rpoErseABC by CpxR-P would imply that when confronted with the two stress signals simultaneously, the Cpx response takes precedence over the RpoE response. Interestingly, curtailing the RpoE-mediated response would in turn dampen the RpoH (³²) response, because rpoH transcription at rpoHP3 is RpoE-dependent (30). CpxR-P may also directly repress rpoH transcription from rpoHP1, because the regulator binds an rpoHP1 promoter fragment containing a potential CpxR-P recognition site (µ_i + 0.38_i) that overlaps the ⁷⁰ site of rpoHP1.¹ Thus, the Cpx, RpoE, and RpoH systems respond in a concerted manner hitherto unanticipated (Fig. 4).

Attenuation of Taxis and Motility by the Cpx System-- We previously reported that the Cpx system negatively regulates the expression of tsr and motABcheAW, two operons that are involved in chemotaxis and motility (5). The tsr operon encodes the leucine/serine chemoreceptor (31, 32), whereas motABcheAW encodes the flagellar motor proton channels A and B, the kinase CheA, and the docking protein CheW (31, 32). This study provides evidence also for negative CpxR-P control of aer, which encodes the aerotaxis sensor protein (31, 32). In addition, a high scoring potential CpxR-P recognition site (µ_i - 0.92_i) was also identified in the promoter region (tap-cheR-cheB-cheY-cheZ) of another operon directly involved in chemotaxis encoding aspartate and dipeptide chemoreceptor Tap, methyltransferase CheR, methylesterase CheB, response regulator CheY, and phosphatase CheZ, respectively. The localization of the potential CpxR-P recognition site relative to the putative -binding sites in the promoter region of the tap operon makes it likely that the operon is also negatively regulated by CpxR-P. Thus, the Cpx system may play a broad role in modulating bacterial movement. This generalization is further supported by the presence of high scoring potential CpxR-P recognition sites in the promoters of fliM (µ_i - 1.90_i, encoding the anti-²⁸ flagellum-specific transcription factor), flgBCDEFGHIJKL (µ_i - 1.69_i) and fliLMN (µ_i - 1.90_i,) that are two operons encoding flagellum subunits, fliOPQR (µ_i - 1.99_i, encoding proteins that export the flagellum subunits), and flhA (µ_i - 1.12_i), flgA,( µ_i - 1.69_i), and flgD (µ_i - 1.69_i) that encode proteins that assemble the flagellum subunits. The location of these sites in the promoter sequences and the presence of multiple putative CpxR-P binding sites within the coding sequences of these polycistronic operons strongly suggest a negative control by CpxR-P of these potential targets.

Functional Diversity of CpxR-P Target Operons-- The first signal transduction role suggested for the Cpx system was to alleviate envelope-protein distress (1, 3, 26, 33). However, a broader role for the system became progressively apparent. For instance, the transcriptional activation of the cpxRA by RpoS recruits the Cpx system in the starvation response (5). In this connection, it is worthy of note that the expression of the cpxRA operon is also autogenously activated at the onset of stationary growth, thereby assuring an adequate Cpx response under conditions of limited resources (5). The results from this study showed that CpxR-P transcriptionally dampens the expression of the RpoE system. The other roles of the Cpx system being uncovered are the down-regulation of taxis and motility as well as cell proliferation as indicated by CpxR-P repression of minCDE.³ These down-regulations may serve to conserve resources during times of stress.

In addition to envelope distress (1, 3, 33-36), the Cpx system is triggered by a wide variety of other stimuli (Fig. 4). These include high pH stress (15, 37), contact with host cells (8), and envelope distress (1, 3, 33-36). Therefore, it is not surprising that matrix-identified target operons of CpxR-P encode proteins that are involved in highly diverse physiological activities (Tables III-V). Some of these can be readily associated with the roles of the Cpx system already discovered. They include 1) the management of protein distress (e.g. dnaK, ftsJ-hflB, hlpA, hslTS, rpoE, secA, rpoH, spy, and tig), 2) motility and taxis (e.g. aer, tap-cheR-cheB-cheY-cheZ, and flgM), 3) adaptation or the recovery from stationary phase (e.g. chaA, chaBC, and yjiY), 4) biofilm formation (e.g. csgDEFG and rfaY), and 5) pathogenesis (e.g. mviM, slt, and sspA). However, the remaining putative target operons cannot be associated with currently known roles of the Cpx system. From what we now know, it appears that the Cpx systems in E. coli and their cousins (38) have been shaped by evolution to confer broad resistance to hostile conditions both in the free environment and inside the mammalian host. The recruitment of the Cpx system in pathogenesis may be a relatively recent evolutionary event. For instance, the papA-K operon encoding the P-pilus is activated by CpxR-P in uropathogenic E. coli (8, Fig. 4).

	ACKNOWLEDGEMENTS

We thank George M. Church for encouragement and helpful discussions and Piet de Boer for allowing us to cite unpublished results.

	FOOTNOTES

^* This work was supported by Public Health Service Grant GM40993 from the National Institute of General Medical Sciences, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139.

^¶ Both authors contributed equally to this work.

Former Postdoctoral D. Collen Fellow of the Belgian American Educational Foundation.

Former predoctoral fellow of the Howard Hughes Medical Institute.

^§§ To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1925; Fax: 617-738-7664; E-mail: elin@hms.harvard.edu.

Published, JBC Papers in Press, April 12, 2002, DOI 10.1074/jbc.M203487200

¹ P. De Wulf, unpublished data.

² A. M. McGuire, unpublished data.

³ J. Johnson and P. de Boer, personal communication.

	ABBREVIATIONS

The abbreviation used is: EMS, electrophoretic mobility shift.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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