Genome-wide Profiling of Promoter Recognition by the Two-component Response Regulator CpxR-P inEscherichia coli

Peter De Wulf, Abigail M. McGuire, Xueqiao Liu, Edmund C. C. Lin
2002 Journal of Biological Chemistry  
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
more » ... 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. 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 envelopeprotein 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 CuCl 2 (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 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 ϩ A ϩ -lacZ that contains a complete cpxRA operon), ECL3503 (cpxR ⌬ A ϩ -lacZ in which cpxR sustained an in-frame deletion), and ECL3504 (cpxR ϩ A * -lacZ in which cpxA sustained a base change resulting in a Leu38 3 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 rpm Ϫ1 ) until an optical density (A 600 ) of 0.9 was reached. At this point of growth, the ⌽(cpxR ϩ A * -lacZ) transcription level in strain ECL3504 exceeded the level of ⌽(cpxR ϩ A ϩ -lacZ) expression in strain ECL3502 by a factor of 3, whereas the expression level of ⌽(cpxR ⌬ A ϩ -lacZ) was ϳ20% ⌽(cpxR ϩ A ϩ -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 A 600 of 0.9 for RNA isolation. To analyze the expression of rpoErseABC, the cells were grown at 30°C until reaching A 600 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
doi:10.1074/jbc.m203487200 pmid:11953442 fatcat:qxrgfxyyjfdsxnqqkvcoai5erm