characterization of the cpx regulon in escherichia coli...

JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1798–1815 Vol. 191, No. 60021-9193/09/$08.00�0 doi:10.1128/JB.00798-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of the Cpx Regulon in Escherichia coli Strain MC4100�

Nancy L. Price and Tracy L. Raivio*Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 6 June 2008/Accepted 12 December 2008

The Cpx two-component signal transduction pathway of Escherichia coli mediates adaptation to envelopeprotein misfolding. However, there is experimental evidence that at least 50 genes in 34 operons are part of theCpx regulon and many have functions that are undefined or unrelated to envelope protein maintenance. Nocomprehensive analysis of the Cpx regulon has been presented to date. In order to identify strongly Cpx-regulated genes that might play an important role(s) in envelope protein folding and/or to further define therole of the Cpx response and to gain insight into what makes a gene subject to strong Cpx regulation, we havecarried out a uniform characterization of a Cpx-regulated lux reporter library in a single-strain background.Strongly Cpx-regulated genes encode proteins that are directly linked to envelope protein folding, localized tothe envelope but uncharacterized, or involved in limiting the cellular concentration of noxious molecules.Moderately Cpx-regulated gene clusters encode products implicated in biofilm formation. An analysis of CpxRbinding sites in strongly regulated genes indicates that while neither a consensus match nor their orientationpredicts the strength of Cpx regulation, most genes contain a CpxR binding site within 100 bp of thetranscriptional start site. Strikingly, we found that while there appears to be little overlap between the Cpx andBae envelope stress responses, the �E and Cpx responses reciprocally regulate a large group of stronglyCpx-regulated genes, most of which are uncharacterized.

The Cpx envelope stress response of Escherichia coli is con-trolled by a two-component system consisting of a sensor his-tidine kinase (CpxA) and a cytoplasmic response regulator(CpxR). The Cpx pathway senses a variety of disturbances(e.g., pH changes, overexpression of envelope proteins such asNlpE or P pilus subunits, and alterations in membrane and/orenterobacterial common antigen synthesis) within the bacterialcell envelope and upregulates expression of a number of genes,including periplasmic protein folding and degradation factors(66). Since all known activating signals are predicted to causeprotein misfolding and the originally identified regulon mem-bers encode protein folding and degradation factors, it hasbeen proposed that the role of the Cpx pathway is to sensechanges in protein folding and respond accordingly. There arecurrently 50 genes in 34 operons for which experimental evi-dence of Cpx regulation exists (Table 1). Cpx regulation ofsome genes such as degP, dsbA, and ppiA has been confirmedby multiple laboratories (10, 16, 17, 59); however, for theremaining putative Cpx regulon members, a vast array of meth-ods have been used to determine Cpx regulation, and thesestudies have been carried out in diverse genetic backgroundsunder widely varying growth conditions. No broad-scale studiessuch as microarray analysis have been published to date, mak-ing it difficult to evaluate the strength of Cpx regulation of oneregulon member relative to another, as well as the centralcellular role of the Cpx response. Furthermore, conflictingreports have emerged concerning the effect of the Cpx pathwayon some reported regulon members (23, 56). Together theseobservations highlight the need for a comprehensive analysis of

the proposed Cpx regulon in a uniform genetic background inresponse to defined Cpx-inducing cues.

In light of the diverse functions of many of the proposed Cpxregulon members, the physiological role of the Cpx stress re-sponse is becoming more ambiguous. The first-described mem-bers of the Cpx regulon encode protein folding and degrada-tion factors directly related to the maintenance of cell envelopeproteins (e.g., degP, dsbA, and ppiA). Multiple studies, includ-ing genomic profiling using a weighted matrix for CpxR bind-ing, led to the identification of other putative regulon membersthat are involved in a wide array of cellular functions, includingmotility and chemotaxis (motAB-cheAW, tsr, and aer), proteintranslocation (secA), virulence (mviM), adhesion (papoperon, csgBAC, and csgDEFG), amino acid biosynthesis(aroG and aroK), phospholipid elaboration (psd), DNA me-tabolism (ung), iron storage and accumulation (efeU and ftnB),and the response to copper stress (ycfS, yebA, yccA, yqjA, ybaJ,and ydeH) (10, 22, 23, 37, 40, 82). Other studies have linked theCpx system to various complex cellular processes such as bio-film formation (62) and bacterial pathogenesis (49, 55). Thesestudies suggest that the cellular functions of the Cpx responsemay be broader than initially defined. Alternatively, some ofthese genes may be relatively weakly Cpx regulated and onlyperipherally involved in the cellular function of the Cpx re-sponse. Without a broad comparison of the Cpx regulation ofall known regulon members, it is impossible to distinguishthese possibilities.

To date, there have been six envelope stress responsesdescribed in E. coli (48). Previous work has demonstratedthat while the �E, Cpx, and Bae envelope stress responsesappear to have unique functions in outer membrane protein(OMP) biogenesis, periplasmic protein folding, and effluxpump regulation, respectively, they also overlap to someextent. Overexpression of the P pilus PapG subunit in theabsence of its cognate chaperone has been shown to activate

* Corresponding author. Mailing address: M344A Biological Sci-ences Building, Department of Biological Sciences, University of Al-berta, Edmonton, AB, Canada T6G 2E9. Phone: (780) 492-3491. Fax:(780) 492-9234. E-mail: [email protected].

� Published ahead of print on 19 December 2008.

1798

on June 17, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

all three stress response systems (45, 63). Similarly, the Cpx,�E, and Bae envelope stress responses have been shown toshare some gene targets. The degP gene, encoding aperiplasmic serine endoprotease, is controlled by both theCpx and the �E stress responses, although CpxR does notrequire �E to activate degP transcription (16). Similarly, thespy (spheroplast protein Y) gene, the function of which isunknown, is controlled by the Cpx and Bae pathways (63,64). It is unknown to what extent the gene targets overlapamong envelope stress responses, since, with the exceptionof degP and spy, little work has been performed to determineif other stress regulon genes might be controlled by morethan one envelope stress response.

In order to describe the relationship among the Cpx-regu-lated genes described to date, to better understand the physi-ological role this response plays in the cell, and to describe the

extent of envelope stress response overlap, we characterizedthe transcriptional regulation of the proposed Cpx regulonusing a lux reporter library. This reporter library consists of thepromoter regions of 31 of the 34 experimentally defined Cpx-regulated promoters that were described at the time we startedthis study (Fig. 1; Table 1), fused to a promoterless luxCDABEoperon on a low-copy vector (7, 49). We used this library toexamine transcriptional activation of the Cpx-regulated genesin genetic backgrounds that eliminated or constitutively acti-vated the Cpx response and in response to inducers of thewild-type Cpx signal transduction pathway. We confirmed ourexperiments with the lux reporter library by using quantitativePCR (qPCR). We also determined whether known Cpx-regu-lated genes were controlled by the �E and Bae pathways.

Our studies confirmed the Cpx regulation of most genes,while we found little evidence for Cpx regulation of a few

TABLE 1. Genes and operons proposed to be under Cpx regulation

Gene category Gene or operon Function Proposed Cpxregulationa Strain(s)b Reference(s)

Envelope protein maintenance degP Periplasmic serine endoprotease Positive MC4100 59rdoA-dsbA Disulfide oxidoreductase Positive MC4100 16, 59ppiA Periplasmic peptidyl isomerase A Positive MC4100 59ppiD Periplasmic peptidyl isomerase D Positive MC4100 19psd Phosphatidyl serine decarboxylase Positive ECL3502 23secA Secretion subunit A Positive ECL3502 23spy Spheroplast protein Y Positive MC4100 63, 64

Envelope components ompC Outer membrane protein C Positive ECL3502, MC4100,MG1655

6, 23

ompF Outer membrane protein F Negative MC4100, MG1655 23nanC NAN (N-acetylneuraminic acid) channel Positive MG1655 12acrD Component of efflux pump Positive MC4100 36mdtABDC Multidrug transporter subunit Positive MC4100 36efeU Elemental ferrous iron uptake permease Negative BW25113 10, 82yccA Modulator of FtsH proteolysis Positive BW25113 82ycfS Periplasmic protein with unknown

functionPositive BW25113 82

yqjA DedA-like predicted inner membraneprotein

Positive BW25113 82

yebE Inner membrane protein (predicted) Positive BW25113 82

Signal transduction cpxP Small periplasmic protein Positive MC4100 15cpxRA Modulator of CpxA Positive MC4100 22, 65rpoE-rseABC Sigma E and regulators Negative ECL3502 23

Bacterial appendages (flagella,fimbriae, pilins), and

motAB-cheAW Flagellar motor and chemotaxisregulators

Negative ECL3502 22, 23

chemotaxis tsr Serine chemotaxis Negative ECL3502 22, 23aer Aerotaxis receptor Negative ECL3502 22, 23csgBAC Curlin fimbriae components Negative MC4100 25csgDEFG Curlin regulatory components Negative MC4100 40pap Uropathogenic E. coli P pilus subunits Negative MC4100 35, 37

Unrelated to envelopecomponents or stress or

aroG DAHP (3-deoxy-D-arabino-heptulosonate7-phosphate) synthase

ND BW25113 82

unknown function aroK Shikimate kinase I Positive ECL3502 23ftnB Ferritin-like protein Positive BW25113 82htpX Heat shock protease Positive MC4100 72mviM Proposed virulence factor Positive ECL3502 23ung Uracil-DNA glycosylase Positive

or negativeBW25113,

ECL305223, 56

ybaJ Unknown Positive BW25113 82ydeH Unknown Positive BW25113 82

a ND, not determined.b ECL3502, K-12 F� rph-1 �(lac)X74 �(cpxRA) �(cpxR�A�-lacZ); BW25113, lacIq rrnBT14 �lacZW316 hsdR514 �araBADAH33 �rhaBADLD78.

VOL. 191, 2009 E. COLI Cpx REGULON 1799


.org/D

ownloaded from

http://jb.asm.org/

reported regulon members. We noted changes in the patternsof expression of some reported Cpx-regulated genes relative tothose reported in the literature that may depend on strainbackground or growth condition. Comparison of the changesin induction of each reporter construct under Cpx-activatingconditions revealed gene classes that were strongly, moder-ately, and weakly Cpx regulated or not Cpx regulated. Most ofthe strongly or moderately Cpx-regulated genes fell into twogroups, those previously shown to influence envelope proteinfolding and the copper stress genes, functions of which aremostly unknown. Interestingly, a number of preliminary stud-

ies implicate these copper stress genes in envelope functions,including biofilm formation (5, 41–43, 68, 80). This implication,together with our data, suggests that the copper stress genes, ofmostly unknown function, may be involved in envelope proteinfolding in some fashion. The strength of the gene regulation bythe Cpx system appears not to be defined by the position ordegree of consensus of the CpxR binding site but correlateswell with its position. Finally, we also show that the majority ofCpx-regulated genes are not controlled by the Bae pathway;however, curiously, there appears to be reverse regulation be-tween the Cpx and the �E responses of almost all of the

FIG. 1. Evidence for Cpx regulation of the proposed Cpx regulon of E. coli MC4100. Numbers at the top indicate relative distance from thetranscription start point for RNAP containing the sigma factor indicated next to the �1 site. Dashed lines indicate genes for which the transcriptionstart site is not known. In these cases, �1 indicates the translational start site. Arrowheads indicate the position and orientation of the consensusCpxR binding site 5� GTAAAN5GTAAA 3�. The numbers within the arrowheads indicate the number of nucleotides that differ from the consensusCpxR binding site. Shaded rectangles show the locations of demonstrated CpxR binding sites. Large arrows represent transcripts for the indicatedgenes. Black arrows represent genes shown to be significantly Cpx regulated under both strong (cpxA24 allele) and mild (NlpE overexpression)Cpx-activating conditions; dark gray arrows show genes that show Cpx regulation only by strong Cpx-inducing cues; light gray arrows indicate genesthat show Cpx regulation only by mild Cpx induction; and white arrows show genes that are weakly or not Cpx regulated (as determined in thisstudy). Small, black arrowheads indicate the location of the upstream primer used to construct the lux reporter for that gene. Numbers on the rightindicate the average differences in expression in the various indicated backgrounds relative to the wild-type or respective vector controls. Theaverage differences shown are based on the average differences observed for at least two different experiments assaying three replicates each time.Various backgrounds tested are cpxA* (cpxA24 allele), NlpE OXP (NlpE overexpression), RpoE OXP (�E overexpression), and baeS* (baeS1allele).

1800 PRICE AND RAIVIO J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/

Cpx-regulated genes originally identified as those induced bycopper.

MATERIALS AND METHODS

Growth media. Strains were cultivated in Luria-Bertani (LB) medium, brainheart infusion (BHI) medium, or minimal medium (M63) supplemented with0.2% glucose, which has been previously described (52), and grown at 30°C or37°C with shaking. To ensure proper strain and plasmid maintenance, mediawere supplemented with various antibiotics when applicable, as follows: amikacin(3 �g/ml), ampicillin (100 �g/ml), chloramphenicol (25 �g/ml), kanamycin (50�g/ml), and spectinomycin (20 �g/ml) (Sigma-Aldrich Co., Canada, Ltd.).

Strains and plasmids. The strains and plasmids utilized are described in Table2. Strains bearing the cpxA* mutations (TR10 and TR11) were cultivated at 30°Cin the presence of amikacin (3 �g/ml) to prevent accumulation of revertants aspreviously described (65). The lux reporter pNLP10 plasmid was derived frompCS26 (7) by expanding the multiple-cloning site (MCS) region by using thecomplementing oligonucleotides 5�-CTCGAGCGATCGGAATTCTGCAGCCCGGGTACCGGATCC-3� and 5�-GGATCCGGTACCCGGGCTGCAGAATTCCGATCGCTCGAG-3� (underlined sequences refer to the XhoI and BamHIsites, respectively). The oligonucleotides were annealed and inserted into theXhoI/BamHI sites of pCS26. The promoter regions of various genes were clonedvia PCR and cloned into the MCSs of the pNLP10 vector to create the luxreporter library. The primers utilized to amplify promoter regions of interest arelisted in Table 2 and are indicated in the context of the relevant promoter in Fig.1. Each amplified promoter region contains the proposed CpxR binding site(s),the �35 and �10 boxes, and the translational start site of each gene. To ensureproper cloning of each promoter region into the lux pNLP10 reporter plasmid,the cloned promoter of each construct was PCR amplified using the primerspNLP10-F (5�-GCTTCCCAACCTTACCAGAG-3�) and pNLP10-R (5�-CACCAAAATTAATGGATTGCAC-3�). These primers amplify the MCS region ofpNLP10. Each PCR product was purified according to the manufacturer’s pro-tocols using a QIAquick PCR purification kit (Qiagen, Inc.), and sequencing wasperformed with the PCR products, using a DYEnamic ET terminator cyclesequencing kit (Amersham, Molecular Biology Service Unit, Department ofBiological Sciences, University of Alberta).

With the use of standard techniques, all lux reporters were transformed intostrains bearing cpx* alleles that constitutively activate the Cpx response (TR10and TR11) (14, 65), the cpxR1::spc null allele (TR51) (65), the NlpE overexpres-sion vector (pLD404) (74), and the wild-type (MC4100) and vector controlstrains for the NlpE overexpression vector (NLP7) (this study). Additionally, thelux reporter library was transformed into a strain overexpressing �E (MB7) (16)and a strain bearing the baeS1 mutation that constitutively activates the Baepathway (TR884) (63) (see Table 2).

Luminescence assay. Single colonies of each bacterial strain to be assayedwere inoculated in 5 ml of LB medium with kanamycin plus additional antibi-otics, when applicable, and grown overnight at 30°C or 37°C with aeration. Onthe following day, each culture was diluted (1:100) into fresh LB broth with theappropriate antibiotics and incubated in a 96-well plate at 30°C or 37°C withshaking. Bioluminescence counts per second (CPS) and optical density at 600 nm(OD600) were measured after 8 h, using a Wallac Victor 2 multilabel plate reader(Perkin Elmer Life Sciences), at which time the culture had reached mid- to latelog phase (OD600 of 0.5 to 0.6). Each experiment was performed in triplicate andrepeated at least twice. The data presented represent the mean CPS/OD600

values and standard deviations for one experiment.�-Galactosidase assay. Single colonies of each strain to be tested were inoc-

ulated in 5 ml of LB broth with the appropriate antibiotics when applicable andgrown overnight at 30°C or 37°C with shaking. On the following day, eachovernight culture was diluted (1:50) into fresh LB or M63 broth and grown at30°C or 37°C with shaking to late log stage (OD600 of �0.6). Cells were har-vested, and -galactosidase activity was assayed as previously described by Slauchand Silhavy (73) in a 96-well microtiter plate, using a Wallac Victor 2 multilabelplate reader (Perkin Elmer Life Sciences). Each experiment was performed inquintuplicate and repeated at least twice. The data presented represent the meanMiller unit and standard deviation for one experiment.

RNA isolation and cDNA synthesis. RNA was isolated from 50-ml LB sub-cultures carrying the appropriate antibiotics that had been inoculated with a1:100 dilution of an overnight culture. Strains were grown to late log stage(OD600 of 0.8) at 30°C or 37°C with shaking, at which time two 10-ml sampleswere harvested and RNA was isolated using a MasterPure RNA purification kitas described by the manufacturer (Epicentre Biotechnologies). Isolated RNAwas resuspended in 100 �l of nuclease-free water (Epicentre Biotechnologies).For cDNA synthesis, 10 ng of isolated RNA was mixed with 10 �l of 300 ng/�l

random primers (Invitrogen Canada Inc.), 3 �l each of all deoxynucleosidetriphosphates (10 mM; Invitrogen), and nuclease-free water to a final volume of30 �l. The random primers and RNA were allowed to anneal (70°C for 10 minand 25°C for 10 min, respectively). After the annealing step, the RNA-primerhybridization mixture was added to a Superscript II master mixture (InvitrogenCanada Inc.), bringing the final volume to 60 �l, and incubated for cDNAsynthesis (25°C for 10 min, 37°C for 1 h, 42°C for 1 h, 70°C for 10 min). ThecDNA was purified using a QIAquick PCR purification kit per the manufactur-er’s instructions (Qiagen Inc.) and eluted in 50 �l of nuclease-free water.

qPCR. Each cDNA sample was standardized to 400 pg/ml, using nuclease-freewater. The qPCR primers were designed to amplify approximately 50 nucleotidesof the 5� half of each gene of interest and are listed in Table 3. qPCR wasperformed with a 96-well microtiter plate containing a 12.5-�l reaction mixtureof Platinum Taq (0.03 U/�l), ROX and SYBR green dyes, all deoxynucleosidetriphosphates (0.2 mM), DMSO (dimethyl sulfoxide; 2%), Tween 20 (0.01%),glycerol (0.8%), MgCl2 (50 mM), KCl (50 mM), Tris (10 mM [pH 8.3]), 2.5 �l ofa 3.2 �M stock solution of each primer, and 2.5 �l of a 400-pg/ml cDNAtemplate. The qPCR was carried out by incubation at 95°C for 15 s, followed by60°C for 1 min. This cycle was repeated for a total of 40 times, using a 7500 FastReal-Time PCR system (Applied Biosystems). Relative amounts of PCR productwere determined by monitoring the number of cycles required to reach a thresh-old level of fluorescence (cycle threshold [CT]) for each gene and subtracting thisnumber from the CT value for an endogenous control gene known not to be Cpxregulated. We used smpA for this purpose, since we showed that a smpA::luxreporter fusion was less than twofold regulated by the strongly activated cpxA24allele, thus indicating that it is not a Cpx-regulated gene (data not shown). Theresulting delta CTs (�CTs) for given genes were compared between the wild-typeand cpxR null strains and the wild-type and cpxA24 strains to obtain delta-deltaCT values (��CTs), which represent the change in gene expression betweendifferent strains. The ��CT values were converted to a log2 scale to take intoaccount the exponential nature of the PCR and to compare the quantity of PCRproduct of one strain with that of the other.

Copper sensitivity assay. Single colonies were inoculated into LB broth con-taining the appropriate antibiotics, and cultures were grown overnight at 37°Cwith shaking. On the following day, bacterial cells were collected by centrifuga-tion and resuspended in 1/5 of the original volume of LB. Two hundred micro-liters of each culture was transferred to a 96-well microtiter plate, and serialdilutions (1:2, 1:5, 1:10, and 1:20) were made in LB broth. Five microliters ofeach culture (straight and dilutions) was spotted onto BHI agar plates containing4 mM and 8 mM CuCl2.

pH sensitivity assay. Single colonies were inoculated into LB broth, andcultures were grown overnight with shaking at 37°C. On the following day, eachovernight culture was subcultured (1:40 dilution) into LB broth with 100 mMphosphate buffer (pH 7) and allowed to grow at 37°C with shaking for 2 h. Thebacterial cells were collected by centrifugation and resuspended in nonbufferedLB broth to the original volume and then diluted (1:20) into LB broth bufferedwith 100 mM phosphate at either pH 7 or pH 9.2. From each culture, 200 �l wastransferred to a 96-well microtiter plate, and the cultures were allowed to growat 37°C with shaking while the OD600 was monitored with a Wallac Victor 2multilabel plate reader (Perkin Elmer Life Sciences).

Biofilm assay. The quantification of biofilm production was performed byfollowing a previously established protocol described by Pratt and Kolter (61).Briefly, in a 96-well microtiter plate, single colonies were inoculated into 200 �lLB broth in triplicate and grown statically at room temperature for 48 h. Theculture was aspirated, and wells were washed with water. Each well was stainedwith 1% crystal violet for 15 min at room temperature and then washed fourtimes with water. The wells were allowed to dry for 2 h before 200 �l ofethanol-acetone (80:20) was added, and the plate was shaken at room temper-ature for 5 min to dissolve the crystal violet from the well walls. A volume of 80�l was then transferred to a new 96-well plate, and the OD600 was read using aWallac Victor 2 multilabel plate reader (Perkin Elmer Life Sciences).

RESULTS

Construction of a Cpx regulon lux reporter library. Wewished to study the Cpx regulon under a uniform set of con-ditions in order to examine the expression of the reportedCpx-regulated genes relative to one another and therefore gainmore insight into the strength of the effect of the Cpx responseon individual regulon members. The promoter regions of 31 ofthe 34 reported Cpx-regulated promoters, including the pre-



.org/D

ownloaded from

http://jb.asm.org/

dicted Cpx-regulated promoter, the transcriptional start site,and the demonstrated or putative CpxR binding site(s), werePCR amplified and cloned upstream of the promoterless lux-CDABE genes in pNLP10 (Table 1 and Fig. 1). We introduced

the lux reporters into E. coli K-12 MC4100 strains that lackedthe Cpx signal transduction pathway entirely (cpxR null strain),that contained cpxA* mutations which constitutively activatedthe Cpx response (cpxA24 and cpxA102) (14, 16), or that con-

TABLE 2. Strains and plasmids utilized in this study

Strain or plasmid Description Reference

Strains2K1056 F� l �IN(rrnD-rrnE)1 rph-1 �(argF-lac)U169 61BW25113 F� �(araD-araB)567 �lacZ4787(::rrnB-3) LAM� rph-1 �(rhaD-rhaB)568 hsdR514 1, 20MC4100 F� araD139 �(argF-lac)U169 rspL150(Strr) relA1 flbB5301 fruA25 deoC1 ptsF25

rbsR11

W3110 F� IN(rrnD-rrnE)1 2JW0450 BW25113 ybaJ::kan 1JW0953 BW25113 yccA::kan 1JW1527 BW25113 ydeH::kan 1JW1835 BW25113 yebE::kan 1JW3065 BW25113 yqjA::kan 1JW3883 BW25113 cpxR::kan 1JW5820 BW25113 ycfS::kan 1MB7 MC4100(pND12) 16NLP7 MC4100(pBR322) This studyNLP8 MC4100(pLD404) This studyNLP498 W3110 cpxA24 This studyNLP621 2K1056 cpxR::kan This studyNLP623 2K1056 yebE::kan This studyNLP624 2K1056 ycfS::kan This studyNLP625 2K1056 yqjA::kan This studyNLP626 2K1056 ybaJ::kan This studyNLP627 2K1056 yccA::kan This studyNLP628 2K1056 ydeH::kan This studyNLP629 2K1056 dsbA::kan This studyNLP630 2K1056 csgB::kan This studyNLP631 2K1056 fimA::kan This studyTR10 MC4100 cpxA24 65TR11 MC4100 cpxA102 This studyTR51 MC4100 cpxR1::spc 65TR384 MG1655 zii::Tn10 This studyTR386 MG1655 cpxR1::spc This studyTR884 MC4100 RS88 �spy-lacZ� baeS1::Tn10-cam 63

PlasmidspBR322 Cloning vector, Ampr Tetr 8pCS26 Low-copy-number cloning vector with a promoterless luxCDABE operon; Kanr 7pLD404 pBR322 with PtetR-nlpE Ampr; overexpresses nlpE 74pND12 pBR322 with PtetR-rpoE Ampr; overexpresses rpoE 16pNLP10 pCS26 with expanded MCS inserted between XhoI and BamHI sites; Kanr This studypNLP11 pNLP10 with PdegP::luxCDABE Kanr This studypNLP12 pNLP10 with PrdoA dsbA::luxCDABE Kanr This studypNLP13 pNLP10 with PppiA::luxCDABE Kanr This studypNLP15 pNLP10 with Pspy::luxCDABE Kanr This studypNLP16 pNLP10 with PmotAB cheAW::luxCDABE Kanr This studypNLP17 pNLP10 with Ptsr::luxCDABE Kanr This studypNLP19 pNLP10 with PrpoE rseABC::luxCDABE, Kanr This studypNLP20 pNLP10 with PompC::luxCDABE, Kanr This studypNLP21 pNLP10 with PsecA::luxCDABE, Kanr This studypNLP22 pNLP10 with Paer::luxCDABE, Kanr This studypNLP23 pNLP10 with Pung::luxCDABE, Kanr This studypNLP24 pNLP10 with ParoK::luxCDABE, Kanr This studypNLP25 pNLP10 with PmviM::luxCDABE, Kanr This studypNLP26 pNLP10 with Ppsd::luxCDABE, Kanr This studypNLP35 pNLP10 with PppiD::luxCDABE, Kanr This studypNLP43 pNLP10 with PompF::luxCDABE, Kanr This studypNLP49 pNLP10 with PacrD::luxCDABE, Kanr This studypNLP50 pNLP10 with PmdtABCD::luxCDABE, Kanr This studypNLP54 pNLP10 with PycfS::luxCDABE, Kanr This studypNLP55 pNLP10 with PcsgBAC::luxCDABE, Kanr This studypNLP56 pNLP10 with PcsgDEFG::luxCDABE, Kanr This studypNLP57 pNLP10 with PftnB::luxCDABE, Kanr This studypNLP58 pNLP10 with PyccA::luxCDABE, Kanr This studypNLP59 pNLP10 with PydeH::luxCDABE, Kanr This studypNLP60 pNLP10 with PyqjA::luxCDABE, Kanr This studypNLP61 pNLP10 with PycdN::luxCDABE, Kanr This studypNLP62 pNLP10 with ParoG::luxCDABE, Kanr This studypNLP63 pNLP10 with PybaJ::luxCDABE, Kanr This studypNLP64 pNLP10 with PyebE::luxCDABE, Kanr This studypJW1 pNLP10 with PcpxP::luxCDABE, Kanr This studypJW2 pNLP10 with PcpxRA::luxCDABE, Kanr This studypVS175 fliC::lacZ in pRS551, Ampr 76pVS182 flhD::lacZ in pRS551, Ampr 76pVS183 EHEC fliA::lacZ in pRS551, Ampr 75



.org/D

ownloaded from

http://jb.asm.org/

TABLE 3. Primer sequences utilized for reporter constructs and qPCR

Construct Gene(s)Primer set sequence for:

lux reporter (promoter)a qPCRb

pJW1 cpxP 5�-CGGAATTCCGGCAGCGGTAACTATGCGC-3� 5�-GGCATCCGGGTGAAGAACTT-3�

5�-CGGGATCCGGGAAGTCAGCTCTCGGTC-3� 5�-AACTTATGCCGTCGAACATATGG-3�

pJW2 cpxRA 5�-CGGAATTCCGGCAGCGGTAACTATGCGC-3� NA5�-CGGGATCCGGGAAGTCAGCTCTCGGTC-3�

pNLP11 degP 5�-GGAATTCCCGCCATCGGCTGGCCTATGT-3� 5�-TGCACCGATGCTCGAAAA-3�

5�-CGGATCCGAGAGCCAGTGCACTCAGTGCT-3� 5�-CGGTTGTGCTACCTTCTACGTTAAT-3�

pNLP12 rdoA-dsbA 5�-GGAATTCCACGTCTGAATGAAGTTATTGAACT-3� NA5�-CGGATCCGAGTAAAAGCGCTGTTATTCATCC-3�

pNLP13 ppiA 5�-GGAATTCCTATGCCAGGCGGCGATTTTAG-3� NA5�-CGGATCCGATCGCCGCCAGGGTCGATTT-3�

pNLP15 spy 5�-CGGGATCCCGCGGTAGTGGTGTCTGC-3� NA5�-GGAATTCCTTGCTTTCTTATAAATTAATACAG-3�

pNLP16 motAB-cheAW 5�-GGAATTCCTGGACATTGGTGCGGTTTGTT-3� NA5�-CGGATCCGAACTGTACCGAGAACAACCAG-3�

pNLP17 tsr 5�-GGAATTCCAGTTGGCCGAAGCCGTTCTA-3� NA5�-CGGATCCGCCAGCAGTAAGCTGGTCACA-3�

pNLP19 rpoE-rseABC 5�-GGAATTCCCTCTTCAGGCAGTTAAATGGG-3� NA5�-CGGATCCGAAGGCTTTCTGATCTCCCTTC-3�

pNLP20 ompC 5�-GGAATTCCGATTGCTGGAAATTATGCGGATG-3� 5�-CCTACATGCGTCTTGGCTTCA-3�

5�-CGGATCCGAAACTTCAGCAGCGTTTGCTGC-3� 5�-ATATTCCCACTGGCCGTAACC-3�

pNLP21 secA 5�-GGAATTCCGCACGGTAATCCGTCATCTTT-3� 5�-GGATGCGCAAAGTGGTCAA-3�

5�-CGGATCCGCGATCGTTACGACTACCGAAA-3� 5�-TCGTCGGAGAGTTTTTCCATCT-3�

pNLP22 aer 5�-GGAATTCCTGCACGGTTTTTGTCGGGTAT-3� NA5�-CGGATCCGGGGTATTTTGCTGGGTGACAT-3�

pNLP23 ung 5�-GGAATTCCTGCCTCCCCGGCAAAATTATT-3� 5�-TCGGCCAGGATCCTTATCAC-3�

5�-CGGATCCGTAGGGTTGCTGCTTCTCTTCA-3� 5�-CGGGACGAACGGAAAATG-3�

pNLP24 aroK 5�-GGAATTCCCTGGTTCGGGCAATTATTTCG-3� 5�-AGTTAGCTCAACAACTCAATATGGAA-3�

5�-CGGATCCGGGCCCAACCAGAAAGATATTG-3� 5�-CATCAGCTCCGGTTCGTTTC-3�

pNLP25 mviM 5�-GGAATTCCAAGATGGTCCTTTTGTGGTGC-3� 5�-CCTACGCGCGCGAAAG-3�

5�-CGGATCCGAATGCCACCTAATCCCACTAC-3� 5�-ACGAATCGGCATAAGGAATGC-3�

pNLP26 psd 5�-CCTCGAGGTTGCAAACACGATACCGATCC-3� NA5�-GGAATTCCTTCGGCAGAATGTACTGTAGC-3�

pNLP35 ppiD 5�-GGAATTCCGGATGATGTAGCACTGGTAGG-3� 5�-GCAATTCGAGAACGCCTTCA-3�

5�-CGGATCCGACTGTTTGCAGCCGTGCGTAA-3� 5�-GAGTATTGATCGCCCAGCTGTT-3�

pNLP43 ompF 5�-GGAATTCCTGATTCCGTTCCCACGTACT-3� 5�-GCGCAATATTCTGGCAGTGA-3�

5�-CGGATCCGCACTGCCAGAATATTGCGCT-3� 5�-TATAGATTTCTGCAGCGTTTGC3A-3�

pNLP49 acrD 5�-GGAATTCCAATGCGATGAAGCACGCCAA-3� NA5�-CGCGGATCCAATGGGGCGATCAATAAAGA-3�

pNLP50 mdtA 5�-GGAATTCCGCTTCATCATGACCCTTTCC-3� NA5�-CGGATCCGCGATAACCACCACGATTACG-3�

pNLP54 ycfS 5�-GGAATTCTGGTGATCGAATTTGCCGAGA-3� NA5�-CGGATCCACGTTAGCCAGCGAGAAAAAC-3�

pNLP55 csgBAC 5�-GGAATTCCATTAAACATGATGAAACCCCGC-3� 5�-CTTCATTTAATCAGGCAGCCATAA-3�

5�-CGGATCCGAGGCGCACCCAGTATTGTTAA-3� 5�-CCCTGCCGTAACTGAGCACTAT-3�

pNLP56 csgDEFG 5�-GGAATTCCAGGCGCACCCAGTATTGTTA-3� NA5�-TGGATCCCATTAAACATGATGAAACCCCGC-3�

pNLP57 ftnB 5�-GGAATTCCAACAAGATACAAAAAGCACTATC-3� NA5�-CGGATCCTTGAGAAGCATTCCAGCGGTT-3�

pNLP58 yccA 5�-GGAATTCCCACGCTATCAATGCACTTTTG-3� NA5�-GGGATCCTGATGTACGGTCATGTGAAGAAC-3�

pNLP59 ydeH 5�-GGAATTCTAGAAGATGGCTATCGCGGAA-3� NA5�-AGGATCCCAGATGGCATCAATTTCCGTT-3�

pNLP60 yqjA 5�-GGAATTCCTACCGTCGATAACTGGTGTG-3� NA5�-CGGATCCGGCTTGCAGCAATTGGGTCAA-3�

pNLP61 efeU 5�-GGAATTCTGATAATCGCGGATGGACGAA-3� NA5�-CGGATCCGAAACATGCCACCCACCCTTA-3�

pNLP62 aroG 5�-GGAATTCCGATGCTCCTGTTATGGTCGT-3� NA5�-CGGATCCGATGCGACAGGAGGAAGTAAC-3�

pNLP63 ybaJ 5�-GGAATTCCGAACCACCAACTCAGGATCT-3� NA5�-CGGATCCGTAAGCTGTGCGATATCATGT-3�

pNLP64 yebE 5�-GGAATTCCATTCGATTGACTGTTGGCGG-3� NA5�-TGGATCCAGTTGATTTAACCAGTTAGCC-3�

a Underlined sequences refer to BamHI, EcoRI, or XhoI sites.b NA, nonapplicable; these genes were omitted from the qPCR testing.



.org/D

ownloaded from

http://jb.asm.org/

tained an NlpE overexpression plasmid (74) or its vector con-trol. We selected the E. coli MC4100 background because theCpx envelope stress response and its first identified regulonmembers were originally characterized in this strain (14, 16).

We first assessed expression of the well-characterized Cpx-regulated genes cpxP, degP, and rdoA-dsbA to ensure that ourreporters accurately reflected the known transcriptional regu-lation of these genes. The cpxP::lux reporter was induced 110-,97-, and 28-fold by the cpxA24 allele and the cpxA102 alleleand by NlpE overexpression, respectively, as previously ob-served (Fig. 2A) (15, 59, 65). The levels of cpxP::lux lumines-cence in the absence of CpxR reflected background levels ofluminescence, indicating an absence of transcription (Fig. 2A).This is consistent with previous reports which showed that cpxPtranscription is completely dependent on the presence of CpxR(15). Similarly, levels of degP::lux and rdoA-dsbA::lux expres-sion dropped in the cpxR null background, although to a lesserextent, indicating that CpxR is not required for basal-level

expression of these promoters (Fig. 2B and C), which is con-sistent with findings from earlier studies (16, 17). In contrast, instrains carrying either of two different cpxA* mutations, thedegP::lux and rdoA-dsbA::lux reporters showed large increasesin luminescence compared to that of the wild-type strain(Fig. 2B and C). Similarly, the overexpression of NlpE resulted inelevated expression of the cpxP::lux, rdoA-dsbA::lux, and degP::lux reporters, as previously shown (17). The cpxP, degP, andrdoA-dsbA genes demonstrated the same degree of sensitivityto alterations in Cpx signal transduction as previously demon-strated, with the cpxP::lux reporter demonstrating the highestlevel of sensitivity to changes in activity of the Cpx pathway(Fig. 2) (24). Furthermore, in agreement with previous obser-vations, we consistently observed that the cpxA24 allele stim-ulated the transcription of Cpx regulon members to a greaterdegree than the cpxA102 allele (Fig. 2 and data not shown)(65). As expected, given the greater range of measurement forluminescence than for -galactosidase activity, we observed

FIG. 2. The cpxP (A), degP (B), and rdoA-dsbA (C) promoters are strongly Cpx regulated. The relevant lux reporters were transformed intoMC4100 (wild type), TR51 (cpxR null), TR10 (cpxA24), TR11 (cpxA102), NLP7 [MC4100(pBR322), the vector control (VC) strain for NlpEoverexpression], and NLP8 [MC4100(pLD404), the NlpE overexpression (OXP) strain] (white bars). Reporter gene expression was measured atmid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS corrected for cell density (OD600). Actual CPS/OD600 values are indicated aboveeach bar. Each assay was performed in triplicate and repeated at least twice. The data from one experiment are reported as average CPS/OD600values. Error bars show standard deviations from the means.



.org/D

ownloaded from

http://jb.asm.org/

sensitivity with our strongly Cpx-regulated lux reporters thatwas increased compared to that of previously studied lac re-porters. For example, degP::lux expression was induced an av-erage 106-fold by the cpxA24 allele (Fig. 2), compared to ap-proximately 5- to 10-fold for the degP::lacZ reporter (17).Similarly, cpxP::lux expression was enhanced an average 26-fold by NlpE overexpression from the plasmid pLD404 (Fig. 2)compared to an approximate 6-fold increase conferred by thesame plasmid on cpxP::lacZ expression (67). Taken together,the data demonstrate that the lux reporters function as sensi-tive, accurate reporters of Cpx-regulated gene expression.

Analysis of Cpx regulon expression. Once our gene expres-sion assay was validated, we assessed Cpx regulation of theentire lux reporter library under the same uniform conditions.With the exception of cpxP, we noted that the cpxR null mu-tation did not elicit a strong (i.e., greater-than-twofold) changein gene expression for the majority of proposed Cpx regulonmembers (data not shown). Thus, most Cpx-regulated genesare not dependent on CpxR for expression. Therefore, wefocused our attention on a comparison of levels of Cpx-regu-lated gene expression in the presence of two Cpx-inducing cuesin the E. coli K-12 MC4100 background. One inducing condi-tion consisted of the cpxA24 mutation and the other was theoverexpression of NlpE from the plasmid pLD404 (14, 74). Ithas been established that the cpxA24 allele activates the Cpxresponse to a much greater extent than overexpression of NlpEfrom pLD404 (17, 65). Based on this analysis, we divided mostpromoters into one of three major groups: those that wereinduced or repressed (i.e., greater than twofold) by both strongand mild Cpx response induction conditions; those that werefound to be induced or repressed only by the cpxA24 mutation;and those in a group of gene clusters for which we did notobserve changes in gene expression in response to either strongor mild Cpx-inducing cues.

Genes affected by both cpxA24 and NlpE overexpression.Sixteen gene clusters were affected by both the cpxA24 alleleand NlpE overexpression. We classified genes in this groupthat showed an average induction or repression of greater thantwofold in the presence of the cpxA24 mutation, as well aswhen NlpE was overexpressed (Fig. 1). These Cpx-regulatedgenes included the above-mentioned cpxP, rdoA-dsbA, anddegP genes (Fig. 2), along with other genes that also showedpositive Cpx regulation (ycfS, yebE, ftnB, yccA, yqjA, psd, spy,ppiA, and cpxRA) (Fig. 3) and genes that exhibited negativeCpx regulation (ompF, rpoE-rseABC, aroK, and efeU) (Fig. 4).

Among the 16 gene clusters within this category, 8 have beenpreviously implicated in protein folding stress. These geneclusters were induced or repressed 6.7- to 113-fold by thecpxA24 allele and 2.0- to 45.4-fold by NlpE overexpression(Fig. 1, 2, and 3). In addition to cpxP, degP, and rdoA-dsbA,these included the psd, spy, ppiA, cpxRA, and rpoE-rseABCgenes (Fig. 1, 2, and 3). CpxP is a novel protein involved incontrolling both the Cpx signal transduction and the targetingof misfolded periplasmic proteins to the DegP protease (in-duced 113-fold by the cpxA24 allele and 25.5-fold by NlpEoverexpression) (Fig. 2A) (9, 15, 38). The rdoA-dsbA operonencodes the major periplasmic disulfide oxidase DsbA and aputative regulator (induced 97.9-fold by the cpxA24 allele and5.0-fold by NlpE overexpression) (Fig. 2C) (4, 79). degP en-codes a periplasmic protease/chaperone (induced 105.9-fold by

the cpxA24 allele and 2.1-fold by NlpE overexpression) (Fig.2B) (46, 77, 78). The psd gene product, phosphatidyl-serine-decarboxylase, is involved in phospholipid metabolism and al-terations in the ratio of inner membrane phospholipids havepreviously been shown to affect both protein folding and theCpx envelope stress response (induced 32.5-fold by the cpxA24allele and 2.3-fold by NlpE overexpression) (Fig. 3F) (33, 51).Spy is a novel periplasmic protein of unknown function that isinduced by multiple envelope stress responses and implicatedin OMP folding (induced 25.4-fold by the cpxA24 allele and2.0-fold by NlpE overexpression) (Fig. 3G) (32, 63). The ppiAgene encodes a peptidyl-prolyl isomerase that facilitates enve-lope protein folding (induced 2.3-fold by the cpxA24 allele and2.1-fold by NlpE overexpression) (Fig. 3H) (34, 47). The cpxRAlocus encodes the CpxR response regulator and CpxA histidinekinase that together regulate the Cpx envelope stress re-sponse (induced 2.0-fold by the cpxA24 allele and 2.6-foldby NlpE overexpression) (Fig. 3I) (67). Finally, the rpoE-rseABC operon encodes �E and its regulators, which controlthe OMP biogenesis-sensitive �E envelope stress response(repressed 57.2-fold by the cpxA24 allele and 3.1-fold byNlpE overexpression) (Fig. 4B) (21, 23, 53).

A second subgroup of strongly Cpx-regulated promotersconstituted those genes known for their collective Cpx-depen-dent induction in response to lethal copper stress (82, 83). TheycfS, yebE, ftnB, yccA, yqjA, and efeU genes encode proteins ofmostly unknown functions. Interestingly, ycfS and yebE wereupregulated by the cpxA24 allele and by NlpE overexpressionto a level comparable to that of cpxP (ycfS is upregulated93.3-fold by the cpxA24 allele and 45.4-fold by NlpE overex-pression [Fig. 3A]; yebE is upregulated 84.2-fold in response tothe cpxA24 mutation and 17.7-fold when NlpE is overexpressed[Fig. 3B]). The yccA and yqjA genes were also strongly acti-vated by the cpxA24 mutation (average 30- to 24-fold, respec-tively) and overproduction of NlpE (average 4- to 7-fold, re-spectively) (Fig. 3D and E). Interestingly, both yccA and yqjAhave been implicated in envelope functions. The yccA geneproduct influences the function of the FtsH inner membraneprotease, while strains bearing the yqjA yghB double mutation,another uncharacterized gene, possess altered phospholipidlevels and display cell division phenotypes (42, 43, 80). TheftnB gene, in addition to being copper regulated, has beenimplicated in iron sequestration by virtue of its homology toother ferritin proteins (81). We showed that ftnB expressionis induced an average of 100-fold and 8-fold by the cpxA24mutation and NlpE overexpression, respectively (Fig. 3C).Another gene cluster that is copper regulated and impli-cated in iron metabolism was also strongly Cpx regulated:efeU::lux expression was diminished 16.1-fold by the cpxA24allele and 4.6-fold by NlpE overexpression (Fig. 4C). TheefeUOB operon has recently been shown to encode an irontransporter (10, 30).

Two other genes affected by both the cpxA24 allele andNlpE overexpression were aroK, which encodes a shikimatekinase involved in aromatic amino acid synthesis (29), andompF, which encodes a large outer membrane porin (71).Expression of the aroK::lux reporter was diminished an av-erage of 7-fold in the presence of the cpxA24 allele and2.3-fold when NlpE was overexpressed (Fig. 4D), while



.org/D

ownloaded from

http://jb.asm.org/

ompF transcription was diminished 57.2-fold by the cpxA24allele and 4.4-fold by NlpE overexpression (Fig. 4A).

Genes that showed Cpx regulation via the cpxA24 mutationbut not through NlpE overexpression. We found five gene

clusters that showed altered regulation only in the presence ofthe cpxA24 allele. These genes included the uncharacterized,copper-responsive genes ybaJ and ydeH, the curlin regulatorygenes csgDEFG, another gene involved with aromatic amino

FIG. 3. The ycfS, yebE, ftnB, yccA, yqjA, psd, spy, ppiA, and cpxRA genes are strongly Cpx activated. The promoters of ycfS (A), yebE (B), ftnB (C), yccA (D),yqjA (E), psd (F), spy (G), ppiA (H), and cpxRA (I) were fused to a luxCDABE reporter cassette and transformed into MC4100 (wild type), TR10 (cpxA24) (graybars), NLP7 [MC4100(pBR322), the vector control (VC) strain for NlpE overexpression (OXP), and NLP8 [MC4100(pLD404)] the NlpE overexpression strain](white bars). Reporter gene expression was measured at mid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS, corrected for cell density (OD600).Actual CPS/OD600 values are indicated above each bar. Each assay was performed in triplicate and repeated at least twice. The data from one experiment arereported as average CPS/OD600 values. Error bars account for the standard deviations from the means.

FIG. 4. The ompF, rpoE-rseABC, efeUOB, and aroK genes are strongly repressed by the Cpx response. The promoters of ompF (A), rpoE-rseABC (B), efeU(C), and aroK (D) were fused to a luxCDABE reporter cassette and transformed into MC4100 (wild type), TR10 (cpxA24) (gray bars), NLP7 [MC4100(pBR322),the vector control (VC) strain for NlpE overexpression], and NLP8 [MC4100(pLD404), the NlpE overexpression (OXP) strain] (white bars). Reporter geneexpression was measured at mid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS, corrected for cell density (OD600). Actual CPS/OD600 values areindicated above each bar. Each assay was performed in triplicate and repeated at least twice. The data from one experiment are reported as average CPS/OD600values. Error bars show standard deviations from the means.



.org/D

ownloaded from

http://jb.asm.org/

acid synthesis, aroG, and a component of an efflux pump, acrD.Because ybaJ, ydeH, csgDEFG, aroG, and acrD were shown tobe Cpx regulated only by the constitutively activated Cpx sys-tem and not by the overexpression of NlpE, this suggests thatthese genes are not strongly Cpx regulated. Two genes origi-nally identified as Cpx-regulated genes based on copper ho-meostasis microarray assays, ybaJ and ydeH, showed an averageinduction of lux activity greater than 15-fold in the cpxA24mutant relative to wild type (Fig. 5A and B). However, nosignificant differences (more than twofold) in ybaJ or ydeHgene expression were observed when NlpE was overexpressed(Fig. 5A and B). Originally the components of curli encoded bythe csgBAC operon were suggested to be under Cpx regulatorycontrol (25). However, our csgBAC::lux reporter consistentlyshowed background levels of luminescence activity, despiteseveral attempts with various media and experimental condi-tions (data not shown). Later, it was established that the Cpxresponse was acting directly on the csgDEFG operon, in whichcsgD encodes regulatory components for the csgBAC operon

(40, 62). We observed that only the strongly Cpx-inducingcpxA24 allele altered luminescence of the csgDEFG::lux re-porter, an average of eightfold (Fig. 5C). aroG, which encodesa protein involved in the first step of aromatic amino acidsynthesis, also showed repressed expression only in the pres-ence of the cpxA24 mutation (average sixfold); the overexpres-sion of NlpE did not elicit a significant change in gene expres-sion (Fig. 5D). The transcription of acrD was upregulatedapproximately fivefold by the cpxA24 mutation, and we ob-served that NlpE overexpression had no significant effect onacrD::lux expression (Fig. 5E).

The mdtABCD operon shows Cpx regulation only throughNlpE overexpression. Among all the Cpx regulon memberstested, the mdtABCD operon appears to be in a uniquecategory of its own. As previously observed, we found thatdeletion of cpxR had very little effect on expression of themdtABCD::lux reporter (data not shown). In the presence ofthe cpxA24 allele, mdtABCD expression was unaffected (Fig.1 and 6). Strikingly, we witnessed a 25-fold decrease in

FIG. 5. The ybaJ, ydeH, csgDEFG, aroG, and acrD genes are regulated only by the cpxA24 allele. The promoters of ybaJ (A), ydeH (B), csgDEFG(C), aroG (D), and acrD (E) were fused to a luxCDABE reporter cassette and transformed into MC4100 (wild-type), TR10 (cpxA24) (gray bars),NLP7 [MC4100(pBR322), the vector control (VC) strain for NlpE overexpression], and NLP8 [MC4100(pLD404), the NlpE overexpression(OXP) strain] (white bars). Reporter gene expression was measured at mid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS, correctedfor cell density (OD600). Actual CPS/OD600 values are indicated above each bar. Each assay was performed in triplicate and repeated at least twice.The data from one experiment are reported as average CPS/OD600 values. Error bars account for the standard deviations from the means.

FIG. 6. The mdtABCD operon is strongly downregulated by NlpE overexpression and not by the cpxA24 allele. The promoter of mdtABCD wasfused to a luxCDABE reporter cassette and transformed into MC4100 (wild type), TR10 (cpxA24) (gray bars), NLP7 [MC4100(pBR322), the vectorcontrol (VC) strain for NlpE overexpression], NLP8 [MC4100(pLD404), the NlpE overexpression (OXP) strain] (white bars), and cpxR null strainscarrying either pBR322 or pLD404. Reporter gene expression was measured at mid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS,corrected for cell density (OD600). Actual CPS/OD600 values are indicated above each bar. Each assay was performed in triplicate and repeatedat least twice. The data from one experiment are reported as average CPS/OD600 values. Error bars account for the standard deviations from themeans.



.org/D

ownloaded from

http://jb.asm.org/

mdtABCD::lux expression when NlpE was overexpressed (Fig. 6).Surprisingly, this inhibition was dependent on CpxR, suggestingthat NlpE induction of the Cpx response, but not mutationalactivation, leads to repression of mdtABCD expression (Fig. 6).

Genes for which expression is unaltered by the cpxA24 alleleor NlpE overexpression. Several proposed members of the Cpxregulon exhibited weak, ambiguous, or no regulation by theCpx system in this study. These included the ompC, ppiD, andung genes. The ompC reporter showed only a slight increase(1.4-fold) in the constitutively active cpxA24 strain, and weobserved no differences in luminescence levels when NlpE wasoverexpressed (Fig. 7B). Although ppiD has been reported tobe part of the Cpx regulon (19), our data showed that neitherthe strong cpxA24 allele nor NlpE overexpression had anyeffect on ppiD::lux expression, even though this reporter wasdefinitely expressed (Fig. 7C). The ung gene, encoding uracil-N-glycosylase, has been reported to be both positively andnegatively regulated by the Cpx response (23, 56). Expressionof our ung::lux reporter was increased less than twofold by thecpxA24 mutation (Fig. 7A). Overexpression of NlpE had littleeffect on ung::lux expression, which suggests that the Cpx en-velope stress response has a very weak or no effect on ungexpression in our strain background.

lux reporters that were not expressed. We constructed twolux reporters that were not expressed under the conditionsused in our studies. The mviM::lux and secA::lux fusions yieldedlevels of luminescence at or below that of the background (datanot shown). In the case of mviM, a putative virulence factor,and secA, which is required for activity of the general secretoryapparatus, the promoters required for transcription are ill de-fined and may actually be upstream of the 5� genes (70). There-fore, the putative phosphorylated CpxR binding sites identifiedthrough bioinformatics directly upstream of these genes maynot actually be valid since they are apparently not located neara promoter.

The effect of the Cpx response on transcription of the mo-tility regulon. It has been suggested that the Cpx system actsdirectly to repress the class III motility genes encoding theflagellar motor and chemotaxis proteins (motAB-cheAW, tsrand aer) (22, 23). We constructed lux reporters to monitorexpression of the motAB-cheAW, tsr, and aer promoters. Sinceour MC4100 laboratory strain is nonmotile, we evaluated Cpxregulation of these constructs with a motile E. coli strain,W3110. Motility assays showed that the introduction of thecpxA24 mutation severely impaired motility compared to that

of the wild-type W3110 strain (data not shown), which is inagreement with previously published data (22). We then sep-arately transformed the lux reporters for the motAB-cheAW,tsr, and aer genes into the W3110 wild-type and cpxA24 mutantbackgrounds, as well as the cpxP::lux reporter to act as a pos-itive control. The lux assays demonstrated mild negative Cpxregulation of the motAB-cheAW, tsr, and aer lux reporters inthe cpxA24 strain background relative to that of the wild type,as previously reported (Fig. 8) (22, 23).

lux reporter expression reflects in vivo Cpx regulon tran-scription. In order to confirm our observations with the luxreporter library, we chose two positively Cpx-regulated genes(cpxP and degP), two negatively Cpx-regulated genes (ompFand aroK), and two genes for which we observed no Cpx reg-ulation (ompC and ppiD) and measured their transcript levelsin wild-type, cpxR null, and cpxA24 genetic backgrounds byusing quantitative PCR. Transcript levels were measured,quantified, and reported as log differences between the wild-type and the cpxA24 mutant (Fig. 9), relative to smpA, a genethat we showed was not Cpx-regulated, using an smpA::lux re-porter (data not shown; see Materials and Methods). Tran-script levels measured by qPCR analysis of the cpxA24 mutantrelative to the wild-type strain largely mirrored what we ob-served with our lux reporters. The greatest differences in ex-pression in the cpxA24 background were observed for the cpxP,degP, and ompF genes, which is consistent with our lux data(Fig. 9). The aroK gene transcripts were moderately dimin-ished in the cpxA24 background, relative to the level in the wild

FIG. 7. The ung, ompC, and ppiD genes are weakly or not Cpx regulated. The promoters of ung (A), ompC (B), and ppiD (C) were fused toa luxCDABE reporter cassette and transformed into MC4100 (wild-type), TR10 (cpxA24) (gray bars), NLP7 [MC4100(pBR322), the vector control(VC) strain for NlpE overexpression], and NLP8 [MC4100(pLD404), NlpE overexpression (OXP) strain] (white bars). Reporter gene expressionwas measured at mid- to late log phase (OD600 of 0.6 to 0.8) and reported as CPS, corrected for cell density (OD600). Actual CPS/OD600 valuesare indicated above each bar. Each assay was performed in triplicate and repeated at least twice. The data from one experiment are reported asaverage CPS/OD600 values. Error bars account for the standard deviations from the means.

FIG. 8. The Cpx response influences motility gene expression. In amotile E. coli background (W3110), the motAB-cheAW, tsr, and aerpromoters fused to lux reporters show mild repression in the presenceof the strong Cpx-inducing cue (W3110 carrying the cpxA24 allele).The cpxP::lux promoter reporter was utilized as a positive control.Reporter gene expression was measured at mid- to late log phase(OD600 of 0.6 to 0.8) and reported as CPS, corrected for cell density(OD600).



.org/D

ownloaded from

http://jb.asm.org/

type, as seen with aroK::lux expression (Fig. 9). Similarly, ompCexpression appeared to be weakly upregulated in the cpxA24background, again, as observed with the ompC::lux fusion (Fig.9). In contrast to what we observed with the ompC::lux andaroK::lux reporters, the qPCR results indicated that the pres-ence of the cpxA24 allele led to similar changes in ompC andaroK mRNA levels (Fig. 9). These data suggest that the post-transcriptional effects that our lux reporters could not detectmay influence expression of either ompC or aroK or expressionof both. Finally, the ppiD gene showed no significant changesin expression in either the cpxR null or cpxA24 genetic back-grounds, relative to that of the wild type (Fig. 9).

As several of the genes tested were shown to be induced byelevated levels of external copper, we selected three of thesegenes together with the cpxP::lux reporter and tested the effectof copper on their expression (82, 83). We found that our luxreporters (cpxP::lux, ycfS::lux, ftnB::lux, and yebE::lux) assayedin ion-deprived media were induced in the presence of nonle-thal amounts of copper (i.e., 50 �M copper in minimal mediapretreated with Chelex-100; data not shown), thus confirmingthat the Cpx system is induced by elevated external levels ofcopper. Taken together, the results from the qPCR and copperinduction lux assays demonstrate that our lux reporters accu-rately reflected what occurred at the level of transcription.

Analysis of envelope stress response overlap. To date, sixgene clusters of the Cpx regulon have been demonstrated to be

under the control of other envelope stress response systems.The degP, rpoE-rseABC, ompC, ompF, and yqjA genes areregulated by the �E envelope stress response, and the Baepathway controls spy gene expression (16, 18, 23, 59, 63). Ad-ditionally, the psd and ftnB (recently renamed from yecI) geneshave been reported to contain �E consensus promoters, butlittle evidence exists for their regulation by �E (69). Further-more, it has been reported that Cpx pathway activation cannegate some effects of eliminating �E and that the Cpx re-sponse has been shown to modulate BaeR-mediated transcrip-tion activation (13, 36), suggesting that there might be substan-tial overlap among envelope stress responses. To address thisquestion, we introduced our lux reporter library into strainscarrying the pND12 plasmid, which overexpresses �E (16), orthe baeS1 mutation, which constitutively activates the Bae re-sponse (63), and the appropriate control strains. We did notanalyze the motility genes in these experiments.

As expected, the degP::lux reporter was upregulated by �E

overexpression, showing twofold differences (Fig. 10). Simi-larly, yqjA showed strong �E regulation (Fig. 10) (18). We didnot detect altered expression of the ompF-lux or ompC-luxreporters in our experiments, although these genes have beenreported to be negatively regulated by �E (69). This may reflectthe fact that our experiments were performed in rich media,while past studies were carried out in minimal media, or dif-ferences in the strength of the �E overexpression vectors used.Alternatively, since the effects of �E on ompF and ompC aremediated through sRNAs, it may be that our lux fusions are notas sensitive to this type of regulation (31). An additional eightgene clusters were �E regulated in this assay. The majority ofthis regulation was negative (ycfS, yebE, ftnB, yccA, yqjA, ydeH,ybaJ, and efeU), with only the aroG promoter showing positiveregulation by �E (Fig. 10). The negative regulation variedbetween 1.5-fold (yccA) and 19.4-fold (yqjA), while aroG was2.2-fold upregulated when �E was overexpressed (Fig. 10).

Among the 28 lux reporters assayed, none that had notpreviously been shown to be Bae regulated demonstratedchanges in expression in the constitutively activated baeS1background compared to that in the wild-type strain MC4100.The acrD::lux, mdtA::lux and spy::lux reporters showed 7-, 70-,and 78-fold increases in expression in the presence of thebaeS1 allele, as expected (3, 54, 63) (data not shown).

FIG. 9. qPCR analysis of cpxP, degP, ompF, aroK, ompC, and ppiDtranscript levels. RNA was isolated from late log (OD600 of 0.8) cul-tures of MC4100 and TR10 (cpxA24) and converted to cDNA. ThecDNA was subjected to qPCR analysis, and PCR product was quan-tified as described in Materials and Methods. The data are presentedas the log10 of the relative quantity of PCR product present in thecpxA24 strain compared to that in the wild-type strain (MC4100).

FIG. 10. The Cpx and �E envelope stress responses reciprocally regulate the ycfS, yebE, ftnB, yccA, yqjA, ydeH, ybaJ, and efeU genes.Luminescence produced by the indicated lux reporters was measured in the wild-type, cpxA24, vector control, and �E overexpression strains. Theaverage CPS corrected for the OD600 value was determined based on three replicates, Cpx activation (white bars) was determined by dividing theaverage CPS/OD600 value in the cpxA24 by the CPS/OD600 value obtained with MC4100, and the �E (gray bars) was determined by dividingthe average CPS/OD600 value in the �E overexpression background by the CPS/OD600 value obtained in the vector control strain.



.org/D

ownloaded from

http://jb.asm.org/

The copper-regulated Cpx regulon genes play roles in otherknown phenotypes associated with the Cpx response. A subsetof genes that we identified here as bona fide Cpx regulonmembers are “y” genes known mostly only for their Cpx-de-pendent induction in response to elevated copper ion concen-trations (82, 83). Our results predict that they might play rolesin previously identified Cpx-related phenotypes, such as bio-film formation (40) and adaptation to alkaline pH (15). Todetermine if this was true, we assayed the copper-regulatedCpx regulon “y” gene mutants for their abilities to form bio-films in 96-well microtiter plates, as well as for their sensitivi-ties to copper and alkaline pH.

To ascertain sensitivity to copper, we spotted 5 �l of anovernight culture and various serial dilutions of wild-typeBW25113 and strains bearing mutations in the cpxR, ydeH,yccA, ycfS, yebE, yqjA, and ybaJ genes onto BHI plates con-taining 4 mM or 8 mM copper chloride. All strains grew wellon 4 mM CuCl2. As previously shown (82), the cpxR null strainwas copper sensitive and failed to grow on plates containing 8mM CuCl2. Of the strains carrying individual mutations in thecopper-regulated ydeH, yccA, ycfS, yebE, yqjA, and ybaJ genes,none exhibited a detectable copper-sensitive phenotype. Giventhat all of these genes have previously been shown to be in-duced by copper (82, 83), this was surprising. Our results sug-gest that no single gene is responsible for resistance to copperbut, rather, that multiple gene products may be involved inadaptation to this toxic element.

We next examined sensitivity to alkaline pH, since the Cpxresponse has been implicated in resistance to this stress (15). Wegrew the wild-type BW25113 strain and its isogenic derivativesbearing mutations in cpxR or the ydeH, yccA, ycfS, yebE, yqjA, andybaJ copper-regulated Cpx regulon genes in LB medium bufferedat pH 7.0 or 9.2 and monitored growth. As previously demon-strated, the cpxR null strain was sensitive to alkaline pH and failedto grow at pH 9.2 (Fig. 11A). Of the remaining mutants, only theyqjA mutant demonstrated a phenotype under these conditions.Deletion of yqjA resulted in a strain that was as sensitive toalkaline pH as the cpxR mutant (Fig. 11A), indicating that it playsa critical role in adaptation to this stressor.

Finally, we analyzed the ability of the wild-type 2K1056 strainand those bearing mutations in the same genes as described aboveto form biofilms by using a 96-well microtiter plate assay (61). Inaddition, we included strains bearing mutations in cpxR, dsbA,fimA, and csgB as positive controls, as these genes have beenshown to influence biofilm formation. As previously observed, wefound that the cpxR, dsbA, fimA, csgB, and ybaJ mutants exhibiteddefects in early biofilm formation (Fig. 11B) (5, 25, 27, 57, 61). Inaddition, yccA, yebE, and ycfS exhibited modest decreases in bio-film formation in this assay (Fig. 11B), suggesting that they mayalso contribute to early biofilm formation.

DISCUSSION

Cpx-regulated genes influence envelope protein folding, bio-film formation, limitation of noxious molecules, and undefinedenvelope functions. In this study, we identified a set of Cpx-regulated genes that were induced or repressed at least twofoldby either the strong, gain-of-function cpxA24 allele or theweaker inducer, NlpE overexpression from the plasmidpLD404, or both (Fig. 1). Interestingly, most of these genes can

be subdivided into two broad categories, those involved withenvelope protein maintenance and those, mostly of unknownfunction, shown to be induced in a Cpx-dependent fashion bycopper stress (Table 1; Fig. 1) (82, 83). We have already dis-cussed those genes previously implicated in envelope proteinfolding functions (see Results). Of those genes, cpxP is themost sensitive to Cpx-activating conditions, followed by rdoA-dsbA, degP, psd, rpoE-rseABC, spy, ppiA, and cpxRA. Our com-parison of the expression profiles of ppiA and cpxRA, relativeto those of the remainder of the Cpx regulon, suggests thatthese two gene clusters are only moderately responsive to Cpxregulation. The reason for the dampening of the �E envelopestress response by the Cpx response is not currently clear. Thismay represent an energy conservation measure; i.e., the cellcontrols the number of envelope stress responses that areinduced at any one time. Alternatively, it may be that the �E

response affects expression of some genes in a way that isdetrimental during times when the Cpx response is induced. Inthis regard, it is striking that we find that almost all of thecopper-responsive genes are upregulated by the Cpx responsebut are downregulated by the �E response (Fig. 10) (seebelow).

Interestingly, a second group of strongly Cpx-regulatedgenes consisted of a set of genes known mostly only for theirCpx-dependent induction in response to toxic levels of copper(ycfS, yebE, ftnB, yccA, yqjA, and efeU). Among these, the ycfSand yebE genes were induced by the cpxA24 mutation andNlpE overexpression to levels comparable to those of cpxP(Fig. 1, 2, and 3). This is striking because cpxP has been shownto be the most strongly Cpx-regulated regulon member (Fig. 1,2) (24). YcfS is predicted to be localized to the periplasm andcontains a potential inner-membrane-anchoring sequence to-gether with a LysM motif predicted to confer interaction withpeptidoglycan (41). YebE is predicted to be a single-pass innermembrane protein. Two other genes, yccA and yqjA, were alsoinduced strongly in the presence of the cpxA24 allele or theoverexpressed NlpE (Fig. 1 and 3). YccA is an inner mem-brane protein with seven transmembrane domains, and it hasbeen implicated in FtsH-mediated proteolysis of inner mem-brane proteins (42, 43). YqjA is also predicted to be a polyto-pic inner membrane protein, and mutations of yqjA and yghB(uncharacterized function) lead to altered levels of phospho-lipids in the inner membrane and cell division defects (80).Interestingly, cpxA* mutants are known to possess cell divisiondefects, so it seems possible that aberrantly high levels of yqjAtranscription may be involved in this phenotype (58). Thestrong Cpx regulation of ycfS, yebE, yccA, and yqjA, togetherwith preliminary studies and cellular locations linking theirproducts to the envelope, argues that they too may play a rolein the process of adapting to periplasmic protein misfolding.Interestingly, of all of the copper-regulated Cpx regulon genesanalyzed in this study, only these four exhibited phenotypespreviously shown to be associated with the Cpx envelope stressresponse. A yqjA mutant was as sensitive to alkaline pH as acpxR mutant (Fig. 11A), and the yccA, yebE, and ycfS mutantsdisplayed slightly diminished biofilm formation in a 96-wellplate assay (Fig. 11B). Although we are still unable to com-ment on the specific functions of these genes, these observa-tions implicate them in processes known to be influenced bythe Cpx response and not just copper adaptation, suggesting



.org/D

ownloaded from

http://jb.asm.org/

that they may indeed share similar functions with other Cpxregulon members.

Two other copper-regulated genes showed more moderateCpx regulation. We witnessed the activation of transcription ofybaJ and ydeH only in the presence of the cpxA24 allele (Fig. 1and 5). YdeH shows homology to diguanylate cyclases andcontains the highly conserved GG[D/E]EF motif characteristicof these enzymes (26). Diguanylate cyclases are involved insynthesis of the ubiquitous signaling molecule cyclic di-GMP,which is implicated in a wide variety of processes, includingvirulence, biofilm formation, and motility (39). Our observa-tions suggest that ydeH is not involved in the early stages ofbiofilm formation (Fig. 11B); however, it will be interesting todetermine if this moderately Cpx-regulated gene is involved invirulence and/or motility, to which the Cpx response has alsobeen linked. Another moderately Cpx-regulated gene cluster,csgDEFG, encodes the regulator of curlin biosynthesis, CsgD.The curli adhesin, like di-guanylate cyclases, is also implicatedin biofilm formation (62). Furthermore, ybaJ expression hasbeen shown to be upregulated in glass wool biofilms, and its

mutation leads to diminished biofilm formation, conjugation,and aggregation, concurrent with increased motility (Fig.11B) (5, 68). These observations, coupled with those de-scribed above, provide support for a role for the Cpx re-sponse in biofilm formation.

The ftnB gene, encoding a putative iron-storing ferritin, isalso induced by copper in a Cpx-dependent fashion (82). Weshow here that ftnB is strongly Cpx regulated (Fig. 1 and 3).Limiting iron levels is a common response to multiple stressesand likely reflects the ability of this metal to inflict damage onmacromolecules through production of harmful oxygen radi-cals. Thus, in the context of the Cpx envelope stress response,upregulation of this putative iron storage protein may repre-sent an effort to limit further damage to proteins by limitingthe possible production of oxygen radicals. In support of thistheory, we also showed that the efeUOB operon is stronglyrepressed by the Cpx response (Fig. 1 and 4). The efeUOBoperon is also copper regulated and is proposed to encode aniron import system (10, 30). Iron may not be the only poten-tially toxic chemical that the Cpx response attempts to limit

FIG. 11. (A) The Cpx-regulated yqjA gene is involved in adaptation to alkaline pH. Overnight cultures of wild-type BW25113 and the isogenicyqjA mutant were subcultured in LB broth with 100 mM phosphate buffer (pH 7) and allowed to grow at 37°C with shaking for 2 h. The bacterialcells were pelleted and resuspended in nonbuffered LB to the original culture volume and then diluted (1:20) in LB buffered with 100 mMphosphate buffer at either pH 7 or pH 9.2. Cultures were allowed to grow at 37°C with shaking, while the OD600 was monitored over the courseof 4 h. (B) The Cpx-regulated genes dsbA, ybaJ, yccA, yebE, and ycfS contribute to biofilm formation in 96-well polystyrene microtiter plates. Singlecolonies were inoculated in 200 �l of LB broth in a 96-well microtiter plate in triplicate and grown statically at room temperature for 48 h. Biofilmstaining and quantification was performed as previously described (61).



.org/D

ownloaded from

http://jb.asm.org/

during times of envelope stress, since the large OmpF porin isrepressed 52.2-fold when the cpxA24 allele is present and 4.4-fold when NlpE is overexpressed (Fig. 1 and 4). OmpF isdownregulated by multiple stresses to the cell, and it is gener-ally thought that this reflects an effort to limit the entry ofnoxious substances that may impose further stress (60).

The Cpx response regulates genes involved in aromaticamino acid biosynthesis in a strain-dependent fashion. Twogenes involved in aromatic amino acid biosynthesis, aroK andaroG, were shown here to be repressed approximately 6-fold inthe presence of the strongly activating cpxA24 allele (Fig. 1, 4,and 5). Thus, it appears that, in MC4100, aromatic amino acidbiosynthesis is downregulated in the presence of a very strongCpx-inducing cue. Both aroG and aroK appear to be relativelyweakly regulated, since aroK was repressed only 2.3-fold byNlpE overexpression (Fig. 4D) and aroG was not affected byNlpE overexpression at all (Fig. 5D). Although it is weak, theCpx regulation of aroK and aroG is likely direct, since CpxRhas been shown to bind upstream of at least aroG (Fig. 1) (82).Downregulation of amino acid biosynthesis might be a generalstrategy to diminish protein traffic in the envelope during timesof stress.

Previously, in contrast to our results, transcription of aroKwas shown in an MG1655 strain background to be weaklypositively regulated by the Cpx envelope stress response (23).When we introduced our aroK::lux reporter into MG1655strains, we noted an average 15-fold decrease in expression inthe cpxR null strain compared to that in the wild type (data notshown), thus confirming the positive regulation of aroK by theCpx response in the MG1655 background, as reported by DeWulf et al. (23). Similarly, we found that, while the expressionof aroG is negatively regulated by the Cpx envelope stressresponse in MC4100 (Fig. 5D), it showed a sevenfold decreasein luminescence in the MG1655 cpxR null strain relative to thatof the wild-type control (data not shown). Presently, we cannotsay what the strain difference is between MC4100 and MG1655that leads to the observed reciprocal effects on aroK and aroGexpression, but both sets of data implicate the Cpx response inthe control of aromatic amino acid biosynthesis and highlightpreviously observed differences between K-12 strains.

NlpE overexpression negatively regulates efflux pump ex-pression. We observed a moderate upregulation of acrD ex-pression in the presence of the cpxA24 allele (Fig. 5E), whilethe mdtABCD operon was unaffected (Fig. 6). Hirakawa et al.recently showed that deletion of cpxA had no effect on mdtAexpression but led to a fourfold increase in that of acrD (36).Since the Cpx pathway is induced in cpxA null mutants due tophosphorylation of CpxR by small-molecular-weight phospho-donors (17), these data indicate that activation of the Cpxresponse moderately stimulates acrD, but not mdtABCD, ex-pression and is in close agreement with our observations. CpxRcan bind upstream of BaeR at both the acrD and mdtABCDgene clusters, and genetic analysis indicates that CpxR func-tions to modulate the BaeR-mediated transcription activationof these genes but plays little role in the induction process inthe absence of Bae pathway activation (36). Our data supportthese conclusions and suggest that, in the absence of activationof the Bae response, the Cpx pathway has a weak positive effecton acrD expression and no effect on mdtABCD transcription.Curiously, we observed that NlpE overexpression had a signif-

icant negative influence only on expression of the mdtABCDoperon (Fig. 6). Furthermore, this effect was CpxR dependent(Fig. 6). Why then does the cpxA24 allele not inhibit mdtABCDexpression? At present, the only logical explanation is thatanother signal transduction pathway is involved which workstogether with the Cpx response to downregulate mdtABCDexpression when NlpE is overexpressed. This would be akin tothe situation observed when both the Cpx and the Bae re-sponses are induced, leading to the activation of mdtABCDexpression, except that in this case, induction of the Cpx re-sponse and the other unknown pathway would lead to repres-sion.

ung, ompC, and ppiD are weakly regulated, or not regulated,by the Cpx response in MC4100. The ung, ompC, and ppiD luxreporters were minimally affected by Cpx pathway activation(less than twofold) (Fig. 7), suggesting that these genes areweakly, or not, regulated by the Cpx envelope stress response.Batchelor et al. witnessed threefold increases in expression ofa fluorescent ompC::cfp reporter when NlpE was overex-pressed or a cpxA* allele was present (6). Thus, both sets ofexperiments predict that ompC is positively regulated by theCpx response in either a moderate or a weak manner. ppiD wasidentified as a Cpx regulon member based primarily on itsinduction in the presence of overexpression of a putative phos-phatase, PrpA, that was suggested to regulate Cpx signal trans-duction (19). There is also a predicted CpxR consensus bindingsite located upstream of the �70 promoter, although CpxR hasnot been demonstrated to bind there (19, 23) (Fig. 1). Previousstudies demonstrated that elimination of CpxR had littleeffect on the expression of a ppiD::lacZ reporter, while acpxAT252R* mutation that constitutively activated the Cpxresponse caused an approximate threefold increase in its ex-pression (19). Since both sets of experiments were performedin an MC4100 background, the only explanation we can offerhere is that the cpxA* allele used by Dartigalongue and Rainawas much stronger than ours, enabling an effect on ppiD ex-pression to be seen. If ppiD is regulated by the Cpx response,it must be very weakly regulated, since cpxA24 is one of themost strongly characterized cpxA* alleles and it seemingly hasno effect (Fig. 7C). De Wulf and colleagues showed weakpositive regulation of the ung gene (23), while Ogasawara et al.showed that strains overexpressing CpxR exhibited an elevatedmutation rate that could be explained by direct binding ofCpxR to, and repression of transcription from, the ung pro-moter region (56). We suspect that since the evidence pre-sented by Ogasawara et al. (56) for repression of ung transcrip-tion is very convincing, the differences we and De Wulf andcolleagues observed are due to fundamental strain differences,similar to what we observed with aroK and aroG expression(see above).

Cpx and �E, but not Cpx and Bae, regulons exhibit a sig-nificant degree of overlap. One of the most striking findings ofthis study was the inverse regulation of copper stress genes bythe Cpx and �E envelope stress responses. While the Cpxresponse upregulates all of the copper stress genes, the �E

response downregulates the great majority of them (Fig. 10).Only one of the genes, yccA, found to be copper regulated bythe Cpx response in this study, was not significantly downregu-lated by �E. It is possible that this negative effect is due to theoverexpression of �E outcompeting �70 for core RNAP; how-



.org/D

ownloaded from

http://jb.asm.org/

ever, we do not believe this to be the case, since most reporterswere unaffected by �E overexpression. Another explanation weconsidered was that since Cpx envelope stress response induc-tion simultaneously downregulated rpoE-rseABC transcriptionand upregulated expression of the copper stress genes, the Cpxresponse might facilitate positive regulation of the copperstress genes simply by downregulating rpoE-rseABC expression(Fig. 4). However, this is unlikely since phosphorylated CpxRhas been shown to bind the DNA upstream of the copper stressgenes (82). Instead, it is tempting to conclude that the inverseregulation of the copper genes by the �E and Cpx envelopestress responses reflects some function of these genes that isdetrimental to OMP assembly and/or beneficial for dealingwith misfolded periplasmic proteins. At present, we cannot saywhat this function might be.

Last, we uncovered no new genes that were coregulated bythe Cpx and Bae envelope stress responses. These experimentsindicate that the extent of overlap between Cpx and Bae regu-lon members is quite limited and suggest that these individualenvelope stress response systems serve discrete, unique cellularroles.

CpxR binding site consensus or orientation does not definethe strength of Cpx gene regulation, but its location is impor-tant. An overall comparison of our results showed that theorientation and degree of consensus of the CpxR binding sitedoes not determine the strength of Cpx regulation (Fig. 1). Thelocation of the CpxR binding site, however, does seem to beimportant. With the exception of psd, genes shown to be Cpxregulated by both the cpxA24 allele and NlpE overexpressionpossess a CpxR binding site within 100 bp upstream from thetranscriptional start site (Fig. 1). We found only one exceptionto this rule: the ung gene contains a CpxR binding site within100 bp of the transcriptional start site, but we show here thatthis gene is weakly or not Cpx regulated (Fig. 7). Thus, thepresence of a CpxR binding site within 100 bp of the transcrip-tional start site is positively correlated with strong Cpx regu-lation.

The extent of matching to the CpxR consensus binding site5�GTAAAN5GTAAA 3� does not appear to be predictive. Forexample, two of the three genes that showed the strongest Cpxregulation (ycfS and yebE) have one (ycfS) to three (yebE)nucleotide changes in the proposed (and demonstrated) CpxRbinding site consensus sequence. Conversely, the aroK consen-sus binding site is a perfect match, but aroK appears to be onlyweakly Cpx regulated (Fig. 1). Orientation of the CpxR bind-ing site is also nondefinitive for strength of Cpx regulation. Inexamining all of the genes that showed strong Cpx regulation,half of them contain a CpxR consensus binding site that isoriented in a forward direction (ftnB, rdoA-dsbA, yccA, yqjA,psd, ppiA, cpxRA, and aroK), while the other half have CpxRbinding sites oriented in the reverse orientation (cpxP, ycfS,yebE, degP, spy, ompF, rpoE-rseABC, and efeU), relative totranscription. Furthermore, several of these genes have CpxRbinding sites in both orientations (cpxP, degP, and cpxRA) (Fig.1). Together, these findings suggest that the orientation of theCpxR binding site in the promoter region does not determinethe strength of regulation by the Cpx envelope stress response.

How does CpxR activate transcription? Comparison ofCpxR binding sites in the context of our data, however, doesprovide some interesting theories regarding the mechanism of

transcription activation by CpxR. Since, among response reg-ulators, CpxR is most similar to OmpR, it seems likely that itactivates transcription through interactions with the alpha sub-unit of RNAP (50). The variable orientations of the CpxRbinding site suggest that perhaps CpxR can contact more thanone face of the alpha subunit, similar to CAP (44). An alter-native explanation is that CpxR can form both symmetrical andhead-to-tail dimers. Finally, with the exception of the ompFgene, it appears that for genes that are strongly regulated byother regulators (e.g., BaeR regulation of the acrD andmdtABCD genes and OmpR regulation of the ompC gene), CpxRmay play an accessory role, since Cpx’ effects on these geneclusters are weak, at best. Perhaps CpxR facilitates or stabilizesDNA binding or RNAP interactions of the other regulator atthese promoters.

ACKNOWLEDGMENTS

We thank the Alberta Heritage Foundation for Medical Research,the Natural Sciences and Engineering Research Council, and the Ca-nadian Institutes of Health Research for funding. We also thank theMolecular Biology Service Unit in the Department of Biological Sci-ences at the University of Alberta and, in particular, Troy Locke forassistance with qPCR.

REFERENCES

1. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. Dat-senko, M. Tomita, B. Wanner, and H. Mori. 2006. Construction of Esche-richia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.Mol. Syst. Biol. 2:1–11.

2. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coliK-12. Bacteriol. Rev. 36:525–557.

3. Baranova, N., and H. Nikaido. 2002. The baeSR two-component regulatorysystem activates transcription of the yegMNOB (mdtABCD) transporter genecluster in Escherichia coli and increases its resistance to novobiocin anddeoxycholate. J. Bacteriol. 184:4168–4176.

4. Bardwell, J. C., K. McGovern, and J. Beckwith. 1991. Identification of aprotein required for disulfide bond formation in vivo. Cell 67:581–589.

5. Barrios, A. F., R. Zuo, D. Ren, and T. K. Wood. 2006. Hha, YbaJ, and OmpAregulate Escherichia coli K12 biofilm formation and conjugation plasmidsabolish motility. Biotechnol. Bioeng. 93:188–200.

6. Batchelor, E., D. Walthers, L. J. Kenney, and M. Goulian. 2005. The Esch-erichia coli CpxA-CpxR envelope stress response system regulates expressionof the porins ompF and ompC. J. Bacteriol. 187:5723–5731.

7. Beeston, A. L., and M. G. Surette. 2002. pfs-dependent regulation of auto-inducer 2 production in Salmonella enterica serovar Typhimurium. J. Bacte-riol. 184:3450–3456.

8. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker,and H. W. Boyer. 1977. Construction and characterization of new cloningvehicles. II. A multipurpose cloning system. Gene 2:95–113.

9. Buelow, D. R., and T. L. Raivio. 2005. Cpx signal transduction is influencedby a conserved N-terminal domain in the novel inhibitor CpxP and theperiplasmic protease DegP. J. Bacteriol. 187:6622–6630.

10. Cao, J., M. R. Woodhall, J. Alvarez, M. L. Carton, and S. C. Andrews. 2007.EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated,low-pH Fe2� transporter that is cryptic in Escherichia coli K-12 but func-tional in E. coli O157:H7. Mol. Microbiol. 65:857–875.

11. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selectedpromoters in Escherichia coli using bacteriophages lambda and Mu. J. Mol.Biol. 104:541–555.

12. Condemine, G., C. Berrier, J. Plumbridge, and A. Ghazi. 2005. Function andexpression of an N-acetylneuraminic acid-inducible outer membrane channelin Escherichia coli. J. Bacteriol. 187:1959–1965.

13. Connolly, L., A. De Las Penas, B. M. Alba, and C. A. Gross. 1997. Theresponse to extracytoplasmic stress in Escherichia coli is controlled by par-tially overlapping pathways. Genes. Dev. 11:2012–2021.

14. Cosma, C. L., P. N. Danese, J. H. Carlson, T. J. Silhavy, and W. B. Snyder.1995. Mutational activation of the Cpx signal transduction pathway of Esch-erichia coli suppresses the toxicity conferred by certain envelope-associatedstresses. Mol. Microbiol. 18:491–505.

15. Danese, P. N., and T. J. Silhavy. 1998. CpxP, a stress-combative member ofthe Cpx regulon. J. Bacteriol. 180:831–839.

16. Danese, P. N., and T. J. Silhavy. 1997. The �E and the Cpx signal transduc-tion systems control the synthesis of periplasmic protein-folding enzymes inEscherichia coli. Genes. Dev. 11:1183–1193.



.org/D

ownloaded from

http://jb.asm.org/

17. Danese, P. N., W. B. Snyder, C. L. Cosma, L. J. Davis, and T. J. Silhavy.1995. The Cpx two-component signal transduction pathway of Escherichiacoli regulates transcription of gene specifying the stress-inducible periplas-mic protease, DegP. Genes. Dev. 9:387–398.

18. Dartigalongue, C., D. Missiakas, and S. Raina. 2001. Characterization of theE. coli �E regulon. J. Biol. Chem. 276:20866–20875.

19. Dartigalongue, C., and S. Raina. 1998. A new heat-shock gene, ppiD, en-codes a peptidyl-prolyl isomerase required for folding of outer membraneproteins in Escherichia coli. EMBO J. 17:3968–3980.

20. Datsenko, K., and B. Wanner. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci.USA 97:6640–6645.

21. De Las Penas, A., L. Connolly, and C. A. Gross. 1997. The �E-mediatedresponse to extracytoplasmic stress in Escherichia coli is transduced by RseAand RseB, two negative regulators of �E. Mol. Microbiol. 24:373–385.

22. De Wulf, P., O. Kwon, and E. C. C. Lin. 1999. The CpxRA signal transduc-tion system of Escherichia coli: growth-related autoactivation and control ofunanticipated target operons. J. Bacteriol. 181:6772–6778.

23. De Wulf, P., A. M. McGuire, A. Liu, and E. C. C. Lin. 2002. Genome-wideprofiling of promoter recognition by the two-component response regulatorCpxR-P in Escherichia coli. J. Biol. Chem. 277:26652–26661.

24. DiGiuseppe, P. A., and T. J. Silhavy. 2003. Signal detection and target geneinduction by the CpxRA two-component system. J. Bacteriol. 185:2432–2440.

25. Dorel, C., O. Vidal, C. Prigent-Combaret, I. Vallet, and P. Lejeune. 1999.Involvement of the Cpx signal transduction pathway of E. coli in biofilmformation. FEMS Microbiol. Lett. 178:169–175.

26. Galperin, M. Y., A. N. Nikolskaya, and E. V. Koonin. 2001. Novel domainsof the prokaryotic two-component signal transduction systems. FEMS Mi-crobiol. Lett. 203:11–21.

27. Genevaux, P., P. Bauda, M. S. BuBow, and B. Oudega. 1999. Identificationof Tn10 insertions in the dsbA gene affecting Escherichia coli biofilm forma-tion. FEMS Microbiol. Lett. 173:403–409.

28. Reference deleted.29. Griffin, H. G., and M. J. Gasson. 1995. The gene (aroK) encoding shikimate

kinase I from Escherichia coli. DNA Seq. 5:195–197.30. Grosse, C., J. Scherer, D. Koch, M. Otto, N. Taudte, and G. Grass. 2006. A

new ferrous iron-uptake transporter, EfeU (YcdN), from Escherichia coli.Mol. Microbiol. 62:120–131.

31. Guisbert, E., V. A. Rhodius, N. Ahuja, E. Witkin, and C. A. Gross. 2007. Hfqmodulates the �E-mediated envelope stress response and the �32-mediatedcytoplasmic stress response in Escherichia coli. J. Bacteriol. 189:1963–1973.

32. Hagenmaier, S., Y. D. Stierhof, and U. Henning. 1997. A new periplasmicprotein of Escherichia coli which is synthesized in spheroplasts but not inintact cells. J. Bacteriol. 179:2073–2076.

33. Hawrot, E., and E. P. Kennedy. 1975. Biogenesis of membrane lipids: mu-tants of Escherichia coli with temperature-sensitive phosphatidylserine de-carboxylase. Proc. Natl. Acad. Sci. USA 72:1112–1116.

34. Hayano, T., N. Takahashi, S. Kato, N. Maki, and M. Suzuki. 1991. Twodistinct forms of peptidylprolyl-cis-trans-isomerase are expressed separatelyin periplasmic and cytoplasmic compartments of Escherichia coli cells. Bio-chemistry 30:3041–3048.

35. Hernday, A. D., B. A. Braaten, G. Broitman-Maduro, P. Engelberts, andD. A. Low. 2004. Regulation of the pap epigenetic switch by CpxAR: phos-phorylated CpxR inhibits transition to the phase ON state by competitionwith Lrp. Mol. Cell 16:537–547.

36. Hirakawa, H., Y. Inazumi, T. Masaki, T. Hirata, and A. Yamaguchi. 2005.Indole induces the expression of multidrug exporter genes in Escherichia coli.Mol. Microbiol. 55:1113–1126.

37. Hung, D. L., T. L. Raivio, C. H. Jones, T. J. Silhavy, and S. J. Hultgren. 2000.Cpx signaling pathway monitors biogenesis and affects assembly and expres-sion of P pili. EMBO J. 20:1508–1518.

38. Isaac, D. D., J. S. Pinkner, S. J. Hultgren, and T. J. Silhavy. 2005. Theextracytoplasmic adaptor protein CpxP is degraded with substrate by DegP.Proc. Natl. Acad. Sci. USA 102:17775–17779.

39. Jenal, U., and J. Malone. 2006. Mechanisms of cyclic-di-GMP signaling inbacteria. Annu. Rev. Genet. 40:385–407.

40. Jubelin, G., A. Vianney, C. Beloin, J. M. Ghigo, J. C. Lazzaroni, P. Lejeune,and C. Dorel. 2005. CpxR/OmpR interplay regulates curli gene expression inresponse to osmolarity in Escherichia coli. J. Bacteriol. 187:2038–2049.

41. Kaserer, W. A., X. Jiang, Q. Xiao, D. C. Scott, M. Bauler, D. Copeland, S. M.Newton, and P. E. Klebba. 2008. Insight from TonB hybrid proteins into themechanism of iron transport through the outer membrane. J. Bacteriol.190:4001–4016.

42. Kihara, A., Y. Akiyama, and K. Ito. 1998. Different pathways for proteindegradation by the FtsH/HflKC membrane-embedded protease complex: animplication from the interference by a mutant form of a new substrateprotein, YccA. J. Mol. Biol. 279:175–188.

43. Kihara, A., Y. Akiyama, and K. Ito. 1999. Dislocation of membrane proteinsin FtsH-mediated proteolysis. EMBO J. 18:2970–2981.

44. Lawson, C. L., D. Swigon, K. S. Murakami, S. A. Darst, H. M. Berman, and

R. H. Ebright. 2004. Catabolite activator protein: DNA binding and tran-scription activation. Curr. Opin. Struct. Biol. 14:10–20.

45. Lee, Y. M., P. A. DiGiuseppe, T. J. Silhavy, and S. J. Hultgren. 2004. P pilusassembly motif necessary for activation of the CpxRA pathway by PapE inEscherichia coli. J. Bacteriol. 186:4326–4337.

46. Lipinska, B., S. Sharma, and C. Georgopoulos. 1988. Sequence analysis andregulation of the htrA gene of Escherichia coli: a sigma 32-independentmechanism of heat-inducible transcription. Nucleic Acids Res. 16:10053–10067.

47. Lui, J., and C. T. Walsh. 1990. Peptidyl-prolyl cis-trans-isomerase fromEscherichia coli: a periplasmic homolog of cyclophilin that is not inhibited bycyclosporin A. Proc. Natl. Acad. Sci. USA 87:4028–4032.

48. MacRitchie, D. M., D. R. Buelow, N. L. Price, and T. L. Raivio. 2008.Two-component signaling and gram negative envelope stress response sys-tems. In R. Utsumi (ed.), Advances in experimental medicine and biology,vol. 631. Springer, Austin, TX.

49. MacRitchie, D. M., J. D. Ward, A. Z. Nevesinjac, and T. L. Raivio. 2008.Activation of the Cpx envelope stress response down-regulates expression ofseveral locus of enterocyte effacement-encoded genes in enteropathogenicEscherichia coli. Infect. Immun. 76:1465–1475.

50. Martinez-Hackert, E., and A. M. Stock. 1997. Structural relationships in theOmpR family of winged-helix transcription factors. J. Mol. Biol. 269:301–312.

51. Mileykovskaya, E., and W. Dowhan. 1997. The Cpx two-component signaltransduction pathway is activated in Escherichia coli mutant strains lackingphosphatidylethanolamine. J. Bacteriol. 179:1029–1034.

52. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY.

53. Missiakas, D., M. P. Mayer, M. Lemaire, C. Georgopoulos, and S. Raina.1997. Modulation of the Escherichia coli �E (RpoE) heat-shock transcrip-tion-factor activity by the RseA, RseB, and RseC proteins. Mol. Microbiol.24:355–371.

54. Nagakubo, S., K. Nishino, T. Hirata, and A. Yamaguchi. 2002. The putativeresponse regulator BaeR stimulates multidrug resistance of Escherichia colivia a novel multidrug exporter system, MdtABC. J. Bacteriol. 184:4161–4167.

55. Nevesinjac, A. Z., and T. L. Raivio. 2005. The Cpx envelope stress responseaffects expression of the type IV bundle-forming pili of enteropathogenicEscherichia coli. J. Bacteriol. 187:672–686.

56. Ogasawara, H., J. Teramoto, K. Hirao, K. Yamamoto, A. Ishihama, and R.Utsumi. 2004. Negative regulation of DNA repair gene (ung) expression bythe CpxR/CpxA two-component system in Escherichia coli K-12 and induc-tion of mutations by increased expression of CpxR. J. Bacteriol. 186:8317–8325.

57. Otto, K., and T. J. Silhavy. 2002. Surface sensing and adhesion of Escherichiacoli controlled by the Cpx-signaling pathway. Proc. Natl. Acad. Sci. USA99:2287–2292.

58. Pogliano, J. A., J. M. Dong, P. De Wulf, D. Furlong, D. Boyd, R. Losick, K.Pogliano, and E. C. C. Lin. 1998. Aberrant cell division and random FtsZring positioning in Escherichia coli cpxA* mutants. J. Bacteriol. 180:3486–3490.

59. Pogliano, J. A., S. Lynch, D. Belin, E. C. C. Lin, and J. Beckwith. 1997.Regulation of Escherichia coli cell envelope proteins involved in proteinfolding and degradation by the Cpx two-component system. Genes. Dev.11:1169–1182.

60. Pratt, L. A., W. Hsing, K. E. Gibson, and T. J. Silhavy. 1996. From acids toosmZ: multiple factors influence synthesis of the OmpF and OmpC porins inEscherichia coli. Mol. Microbiol. 20:911–917.

61. Pratt, L. A., and R. Kolter. 1998. Genetic analysis of Escherichia coli biofilmformation: roles of flagella, motility, chemotaxis and type I pili. Mol. Micro-biol. 30:285–293.

62. Prigent-Combaret, C., E. Brombacher, O. Vidal, A. Ambert, P. Lejeune, P.Landini, and C. Dorel. 2001. Complex regulatory network controls initialadhesion and biofilm formation in Escherichia coli via regulation of the csgDgene. J. Bacteriol. 183:7213–7223.

63. Raffa, R. G., and T. L. Raivio. 2002. A third envelope stress signal transduc-tion pathway in Escherichia coli. Mol. Microbiol. 45:1599–1611.

64. Raivio, T. L., M. W. Laird, J. C. Joly, and T. J. Silhavy. 2000. Tethering ofCpxP to the inner membrane prevents spheroplast induction of the Cpxenvelope stress response. Mol. Microbiol. 37:1186–1197.

65. Raivio, T. L., D. L. Popkin, and T. J. Silhavy. 1999. The Cpx envelope stressresponse is controlled by amplification and feedback inhibition. J. Bacteriol.181:5263–5272.

66. Raivio, T. L., and T. J. Silhavy. 2001. Periplasmic stress and ECF sigmafactors. Annu. Rev. Microbiol. 55:591–624.

67. Raivio, T. L., and T. J. Silhavy. 1997. Transduction of envelope stress inEscherichia coli by the Cpx two-component system. J. Bacteriol. 179:7724–7733.

68. Ren, D., L. A. Bedzyk, S. M. Thomas, R. W. Ye, and T. K. Wood. 2004. Geneexpression in Escherichia coli biofilms. Appl. Microbiol. Biotechnol. 64:515–524.

69. Rhodius, V. A., W. C. Suh, G. Nonaka, J. West, and C. A. Gross. 2006.



.org/D

ownloaded from

http://jb.asm.org/

Conserved and variable functions of the �E stress response in related ge-nomes. PLoS Biol. 4:e2.

70. Riggs, P. D., A. I. Derman, and J. Beckwith. 1988. A mutation affecting theregulation of a secA-lacZ fusion defines a new sec gene. Genetics 118:571–579.

71. Sato, T., and T. Yura. 1979. Chromosomal location and expression of thestructural gene for major outer membrane protein Ia of Escherichia coli K-12and of the homologous gene of Salmonella typhimurium. J. Bacteriol. 139:468–477.

72. Shimohata, N., S. Chiba, N. Saikawa, K. Ito, and Y. Akiyama. 2002. Cpxstress response system of Escherichia coli senses plasma membrane proteinsand controls HtpX, a membrane protease with a cytosolic active site. GenesCells 7:653–662.

73. Slauch, J. M., and T. J. Silhavy. 1991. cis-acting ompF mutations that resultin OmpR-dependent constitutive expression. J. Bacteriol. 173:4039–4048.

74. Snyder, W. B., L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy.1995. Overproduction of NlpE, a new outer membrane lipoprotein, sup-presses the toxicity of periplasmic LacZ by activation of the Cpx signaltransduction pathway. J. Bacteriol. 177:4216–4223.

75. Sperandio, V., C. C. Li, and J. B. Kaper. 2002. Quorum-sensing Escherichiacoli regulator A: a regulator of the LysR family involved in the regulation ofthe locus of enterocyte effacement pathogenicity island in enterohemor-rhagic E. coli. Infect. Immun. 70:3085–3093.

76. Sperandio, V., A. G. Torres, J. A. Giron, and J. B. Kaper. 2001. Quorum

sensing is a global regulatory mechanism in enterohemorrhagic Escherichiacoli O157:H7. J. Bacteriol. 183:5187–5197.

77. Spiess, C., A. Beil, and M. Ehrmann. 1999. A temperature-dependent switchfrom chaperone to protease in a widely conserved heat shock protein. Cell97:339–347.

78. Strauch, K. L., and J. Beckwith. 1988. An Escherichia coli mutation prevent-ing degradation of abnormal periplasmic proteins. Proc. Natl. Acad. Sci.USA 85:1576–1580.

79. Suntharalingam, P., H. Spencer, C. V. Gallant, and N. L. Martin. 2003.Salmonella enterica serovar Typhimurium rdoA is growth phase regulatedand involved in relaying Cpx-induced signals. J. Bacteriol. 185:432–443.

80. Thompkins, K., B. Chattopadhyay, Y. Xiao, M. C. Henk, and W. T. Doerrler.2008. Temperature sensitivity and cell division defects in an Escherichia colistrain with mutations in yghB and yqjA, encoding related and conserved innermembrane protein. J. Bacteriol. 190:4489–4500.

81. Velayudhan, J., M. Castor, A. Richardson, K. L. Main-Hester, and F. C.Fang. 2007. The role of ferritins in the physiology of Salmonella enterica sv.Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair andvirulence. Mol. Microbiol. 63:1495–1507.

82. Yamamoto, K., and A. Ishihama. 2006. Characterization of copper-induciblepromoters regulated by CpxA/CpxR in Escherichia coli. Biosci. Biotechnol.Biochem. 70:1688–1695.

83. Yamamoto, K., and A. Ishihama. 2005. Transcriptional response of Esche-richia coli to external copper. Mol. Microbiol. 56:215–227.



.org/D

ownloaded from

http://jb.asm.org/

characterization of the cpx regulon in escherichia coli...

Documents