Vol. 175, No. 8JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 5009-50210021-9193/93/085009-13$02.00/0Copyright C 1993, American Society for Microbiology

Identification and Transcriptional Analysis of the Escherichiacoli htrE Operon Which Is Homologous to pap

and Related Pilin OperonsSATISH RAINA,1,2,3* DOMINIQUE MISSIAKAS,12 LISA BAIRD,2 SUSHIL KUMAR,3

AND COSTA GEORGOPOULOS1'2Department of Cellular, Viral and Molecular Biology, University of Utah School ofMedicine, Salt Lake City,

Utah 841321; Departement de Biochimie Medicale, Centre Medical Universitaire, 1 rue Michel-Servet,1211 Geneva 4, Switzerland2*; and Division of Genetics, Indian Agricultural

Research Institute, New Dehli 110012, India3

Received 1 February 1993/Accepted 4 June 1993

We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE. The ecpD geneencodes a 27-kDa protein which is 40o identical at the amino acid level to the pilin chaperone PapD family ofproteins. Immediately downstream of the ecpD gene is the htrE gene. The htrE gene encodes a polypeptide of95 kDa which is processed to a 92-kDa mature species. The HtrE protein is 38% identical to the type II pilinporn protein PapC. The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the regionfrom kbp 153 to 157 of the E. coli physical map. The htrE gene was identified on the basis of a TnS insertionmutation which resulted in a temperature-sensitive growth phenotype above 43.5°C. The transcription of thisoperon is induced with a temperature shift from 22 to 37 or 42°C but not to higher temperatures, e.g., 50°C.Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoHgene product (&32). The transcription of this operon was further shown to require functional integration hostfactor protein, since himA or himD mutant bacteria possessed lower levels ofecpD htrE transcripts. Among thethree transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positiveregulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of manygrowth phase-regulated genes.

Escherichia coli is a normal inhabitant of the intestinaltract of humans and has been recognized as an importantpathogen involved in a variety of intestinal and extraintesti-nal diseases. These include urinary tract infections, bactere-mia, bacterium-related traveler's diarrhea, and newbornmeningitis (reviewed in references 4 and 15).The initiation of many of these infections is thought to be

mediated by molecular recognition between adhesins onbacterial cell surfaces and specific receptor molecules foundon mucosal cells of the host (4). The majority of adhesinsproduced by extraintestinal E. coli are associated withstructures known as fimbria or pili, whose genes are usuallyfound on plasmids (15). The fimbrial adhesins are distin-guished by their receptor specificity. Most uropathogenic E.coli strains express P pili that mediate Gal (a 1-4) Gal-sensitive hemagglutination (26). Type I pili are expressed bymany pathogenic as well as nonpathogenic E. coli strains andare characterized by their D-mannose-sensitive agglutination(10, 13).The genetic organization of both type I and P pilin loci is

similar. Many genes necessary for the biosynthesis andexpression of P and type I pili have been cloned. Of these,the papC and papD genes of the P pilin locus and theiranalogs,fimD andfimC, encode proteins which are involvedin translocation and assembly of pili (21, 27, 35). PapC is an88-kDa outer membrane protein thought to form the assem-bly center for pilus polymerization and act like a molecularusher in differentially recognizing various chaperone-subunitcomplexes (8). The 28.5-kDa PapD protein has been local-

* Corresponding author.

ized in the periplasmic space and is known to function as achaperone mediating the assembly of pilin subunits (19, 24).Another interesting feature of pilin operons is their intri-

cate regulation. For example, it has been shown that the typeI pili are subject to phase variation and that mutations inhimA or himD reduce the frequency of this switch (1). Phasevariation of the P pili locus is controlled at the transcriptionallevel by a methylation inhibition mechanism exerted near thePap pilin transcriptional start site (46). The gene lrp, encod-ing the leucine-responsive regulatory protein, has beenshown to be required for inhibition of this methylation,thereby positively regulating the pap P pilin operon (5).

Recently another class of pili, called curlins on the basis oftheir morphology as seen by electron microscopy, has beenidentified in E. coli (38). The structural genes encoding curlinadhesin pili have been shown to be expressed in bothpathogenic and nonpathogenic E. coli. However, the genesinvolved in the assembly and transport of these pili have notbeen identified so far.Over the last few years, we have been characterizing a

group of transposon insertional mutations in E. coli, isolatedfollowing saturation mutagenesis with various transposons,such as TnS and Tn1O. These presumably null mutationswere isolated on the basis of either inability to form coloniesat 43°C or lethal hypersensitivity to a normally sublethal heatshock at 50°C (28, 39, 40). Here we report on one such TnSinsertion mutation which defines a new gene, htrE. The htrEgene maps at 3.3 min on the E. coli genetic map. It wassequenced and shown to encode an approximately 95-kDaprotein which is highly homologous to the PapC proteininvolved in the assembly and transport of P type pili. Likethepap gene cluster, htrE was found to be part of an operon

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TABLE 1. Bacterial strains and plasmids

Strain orRelevant characteristics Reference or sourceplasmid

StrainsCA8000 HfrH thi Laboratory collectionW3110 Wild type Laboratory collectionMC4100 A(argF-lac)U169 araDl39 rpsL150 deoCI rel41 ptsF25 flbB5501 rbsR Laboratory collectionJC8679 recB21 recC22 sbcA23 X- F- supE A. J. ClarkSR10 CA8000 htrEl394::TnS This workSR126 CA8000ponB::TnlO This workSR532 CA8000 htrEl353::fI-Tetr This workSR335 CA8000 htrEl467::f-Kanr This workSR381 CA8000 htrEA(553-2224)::A-Tet' This workAMS150 katF::TnlO 32DM555 MC4100 katF::TnlO constructed by P1 transduction This workK1299 AhimA82 12K1926 himD7 12CV975 Irp-35::TnlO 5SR1021 MC4100 AhimA82 This workSR1022 MC4100 himD7 This work

PlasmidspREG Cosmid cloning vector 24pSK/KS Bluescript plasmid StratagenepSR7 pSK carrying a 7.0-kb KpnI fragment; htrE+ ecpD' This workpSR10 pSK carrying a 3.9-kb DraI-HindIII fragment; htrE+ ecpD+ This workpSR174 pSK carrying a 3.0-kb EcoRI-HindIII fragment; htrE+ This workpSR176 pSK carrying a 1.5-kb DraI-EcorV fragment; ecpD+ This workpDM655 pKS carrying 750-bp HincII-HindIII promoter region This work

and cotranscribed with another gene, highly homologous tothe papD gene, encoding a pilin chaperone protein. Wedesignated this gene ecpD (E. colipapD) on the basis of itshomology to the uropathogenic E. coli papD gene. Thetranscription of the htrE operon was found to be inducedfollowing a temperature shift from 22 to 37 or 42°C, but in anE&32-independent manner. We further show that the tran-scription of this operon requires the DNA-bending proteinintegration host factor (IHF)-along with KatF, encoded bythe rpoS gene and thought to be a sigma factor for growthphase-regulated genes.

MATERIALS AND METHODSBacterial strains and plasmids. The E. coli bacterial strains

and plasmids used in this study are listed in Table 1. Theinability to form colonies at high temperatures is referred toas the temperature-sensitive (TS) phenotype.Media. M9 minimal medium was prepared as described by

Miller (32) and was supplemented with glucose (0.2%),thiamine (2 ,g/ml), MgSO4 (1 mM), MgCl2 (3 mM), CaCl2(0.1 mM), and FeCl3 (0.3 ,uM). For [35S]methionine labelingexperiments, the M9 medium, supplemented with a mixtureof the 20 amino acids (each at 20 ,ug/ml, except methionineand cysteine), was used. When necessary, the media weresupplemented with ampicillin (100 ,ug/ml), tetracycline (15,ug/ml), spectinomycin (50 ,g/ml), or kanamycin (50 pg/ml).Cloning of the htrE gene. A Sau3A E. coli chromosomal

DNA library, prepared in cosmid vector pREG153 (23), wasused to isolate cosmids carrying the htrE+ gene. The abilityto complement the TS phenotype and recombine htrE mu-tations onto the plasmid was taken as preliminary proof thatthe cosmid clone carried the htrE+ gene. The htrE+ genewas also subcloned from the transducing bacteriophage X115of Kohara's collection (22). The gene was further subclonedby standard DNA manipulation techniques (43).Mapping of the htrE gene. Nick-translated cosmid DNA or

subclones carrying the htrE gene were used to probe the XE.coli genomic library as previously described (28).Mapping of TnS insertions in the htrE gene. The exact

position of the TnS insertions in h*rE1394::TnS was deter-mined by first transferring these TnS insertion mutations byhomologous recombination onto plasmid pSR174 containingthe htrE+ gene. The Kanr-containing sequences and theIS50L portion of TnS, along with the adjacent htrE DNA,were recloned and sequenced as previously described (39).

Construction of htrE::fl-Tetr insertion or substitution mu-tations in the htrE gene. Additional null alleles were con-structed as shown in Fig. 1, by in vitro DNA manipulationsof pSR7 or pSR10. The plasmids were then linearized byappropriate enzyme digestions and used to transform strainJC8679 recB recC sbcA. The transformants were tested forampicillin sensitivity to eliminate bacteria carrying undi-gested, intact plasmid. The htrE1353::f-Tetr allele carriesthe fl-Tetr cassette (11) inserted at the unique AsuII restric-tion site within the htrE gene. The htrE::fl-Kanr allele wasconstructed by inserting the fl-Kanr cassette at the BgtIsite. A substitution derivative of the htrE gene was achievedby first constructing plasmid pSR535 htrEA(553-2224)::fl-Tetr. In this construct, the fl-Tetr cassette replaces mostof the htrE gene sequence located between the EcoRVrestriction sites.Primer extension and mung bean nuclease protection assay

of RNA transcripts. RNA was isolated by the hot-phenolextraction procedure (43). Approximately 5 ng of 32P-end-labeled primer (5'TATATIACGCGAGTACCCGAAATG3')complementary to nucleotide positions 83 to 106 of the ecpDcoding region and 10 ,ug of RNA isolated from differentisogenic bacteria were coprecipitated and resuspended in 10pl of 50 mM Tris-HCl (pH 8.3)-100 mM KCL. The cDNAsynthesis and resolution of primer extension products werecarried out as previously described (39). The transcriptionalstart site(s) was also determined by hybridization of total

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n TetRFIG. 1. Restriction map of the htrE operon and construction of htrE insertion and deletion derivatives. The right column indicates the

complementation of the htrEl394::TnS mutant bacteria. (a and b) Restriction maps of pSR7 and pSR10, both carrying the htrE and ecpDgenes; (c) pSR174 carrying only the htrE gene; (d) pSR176 carrying the 1.5-kbp DraI-EcoRV fragment which contains the ecpD gene alongwith its promoters and 500 bp of the coding region of htrE gene; (e) the location and orientation of the Tn5 insertion in the htrE gene; (f, g,

and h) in vitro insertions of fl-Kanr at the BgIH site and fl-Tetr at theAsuIl site and substitution of a 1.7-kb EcoRV fragment of the htrE geneby the fl-Tetr cassette.

cellular RNA to a uniformly radiolabeled probe containingthe 5' end ecpD-specific sequences followed by digestion ofRNA-DNA hybrids with mung bean nuclease. First thepromoter-containing region along with the flanking se-

quences was subcloned from pSR176(ecpD+) as a 750-bpHincII-HindlI insert into the SmaI-HindIII sites of phage-mid vector pBluescript KS, resulting into pDM655. To labelthe antisense strand, 1 ,ug of single-stranded DNA preparedfrom pDM655 with the help of helper phage M13KO7 was

extended from the SK primer in the presence of [a-32PIdCTP. From the replicated, double-stranded DNA a 315-bpHinclI-Dral promoter-containing probe was isolated by di-gestion with BamHI and HindIII and then hybridized to

approximately 20 pg of RNA under the conditions describedpreviously (30). The RNA-DNA hybrids were digested with300 U of mung bean nuclease. The protected fragment wassized and compared with pBR322 MspI-digested DNA as

well as results of a dideoxy sequencing reaction carried outwith SK primer by using pDM655 as a template.

Genetic manipulations. Isogenic sets of various E. colimutant derivatives were constructed by standard P1 trans-duction experiments (32). For most of the comparativestudies dealing with the regulation of the htrE operon,MC4100 and its isogenic derivatives were used, except incertain cases as outlined in Table 1. The AhimA82 mutationwas transduced from K1299 (12) into MC4100 by using the

VOL. 175, 1993

145 kbp

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Tetr insertion in the himA gene. The himD7 (hip) mutationwas transduced from K1926 (12) by using a nearby Tn1OKanr marker. This was achieved by using the TnlO Kanrinsertion from CAG18528, with TnlO Kanr zbj-3110 locatedat 20 min (45). Similarly, the katF::TnlO insertion mutationfrom AMS150 (31) was transduced into MC4100 by standardgenetic techniques. The isogenic rpoH' and rpoH strainshave previously been described (39).

Sequencing. Nucleotide sequence analysis of the 3.9-kbDraI-HindIII chromosomal insert in pSR10 was performedby either subcloning various restriction fragments or usingnested sets of exonuclease III-generated subclones. Both ofthe DNA strands were sequenced by the dideoxyribonucle-otide chain termination reaction technique. The sequencingreactions were carried out with Sequenase kit (U.S. Bio-chemicals, Cleveland, Ohio).

Nucleotide sequence accession number. The GenBank ac-cession number for the sequences described here is L00680.This includes both the ecpD and the htrE genes along withthe DNA sequences from the flanking regions.

RESULTS

A pool of approximately 5 x 104 TnS insertion events wasobtained in CA8000 bacteria (39) and screened for isolateswith a TS phenotype, i.e., inability to form colonies at43.50C. One such TS isolate, subsequently designatedhtrE1394::TnS, was chosen for the present study. The htrEmutation was first shown by P1 transduction to confer a TSphenotype in various genetic backgrounds. In all such ge-netic backgrounds tested, the htrE mutation conferred thefollowing phenotypes: (i) inability to form colonies above43.5°C in either rich (Luria-Bertani) or minimal (M9) me-dium, (ii) reduced colony-forming ability on Luria-Bertanimedium in the presence of a high salt concentration, e.g., 0.7M NaCl, and (iii) lethal hypersensitivity to a normallysublethal heat shock at 50°C. All three phenotypes cotrans-duced 100% with the TnS-encoded kanamycin resistancemarker, demonstrating that all three phenotypes are theconsequence of a single mutational event.

Cloning of the htrE gene. To isolate the wild-type htrEgene, an E. coli genomic library cloned in the low-copy-number cosmid vector pREG153 (36) was used to select forthose clones which allow htrEl394::TnS bacteria to formwild-type-like colonies at 43.5°C. In subsequent experi-ments, we also used the X115 transducing bacteriophage,from Kohara's E. coli library (22), to clone the wild-typehtrE gene (see below). Further subclones were constructedin either the low-copy-number vector pGB2 (6) or thepUC-derived Bluescript vector, pSK+ (Stratagene). A com-mon 7.0-kbp KpnI fragment, obtained from either the htrE+cosmids or X115, was shown to rescue all the mutantphenotypes exhibited by the htrEl394::TnS mutation. Fur-ther subcloning experiments resulted in the identification ofa 3.9-kbp DraI-HindIII fragment (pSR10) which comple-mented the TS phenotype of htrE bacteria (Fig. 1).Mapping of the htrE gene. To map the htrE gene, we used

the 32P-labeled 7.0-kbp K;pnI fragment derived from plasmidpSR7 (htrE+) or the larger htrE+ cosmid clones to hybridizeto the X E. coli DNA library (22). Hybridization with thehtrE' cosmid clones was observed with X clones 113 to 116.The smaller 7.0-kbp KpnI probe hybridized only to bacte-riophage X115, which corresponds to kbp 150 to 160 of the E.coli physical map (22). To further confirm the map positionassignment, the htrE mutants were lysogenized with differ-ent X transducing bacteriophages spanning the region from

kbp 140 to 170. Only recombinant bacteriophage X115 wasfound capable of (i) complementing the TS phenotype and(ii) recombining with the kanamycin resistance marker of thehtrEl394::TnS mutation. By comparing the restriction pat-tern of pSR7(htrE+) or a smaller clone, pSR10(htrE+), withthe E. coli restriction map, the htrE gene was localized moreprecisely within the region from kbp 153 to 157. Theseresults were further verified by carrying out P1 transductionswith drug resistance markers known to be located in thisarea. Results of such transduction experiments placed thehtrE gene counterclockwise to thepcnB gene, the two genesbeing approximately 90% linked by P1 transduction. ThepcnB gene product has been previously shown to be in-volved in copy number control of various plasmids (29). Byusing another nearby gene, ponB, which encodes penicillin-binding protein 1B and is located clockwise to pcnB, acotransduction frequency of 60% was observed between aTnJO insertion in ponB from SR126 and the Kanr markerfrom htrE. All of the data obtained are consistent with themapping results presented above.

Construction of0 insertion or substitution mutations in thehtrE gene. Since in the original screen we isolated only asingle htrE allele, htrEl394::TnS, we also constructed threeadditional null alleles. All three alleles were constructed onplasmid pSR7 or pSR10 and transferred onto the chromo-some as described in Materials and Methods. The three newnull alleles were then transduced by P1 into various wild-type E. coli strains. In all genetic backgrounds tested, similarphenotypes were observed, including TS bacterial growthand hypersensitivity to a 50°C incubation. Thus, it is con-cluded that the htrE gene is dispensable for E. coli bacterialgrowth at temperatures up to 43°C but is required for E. colibacterial viability at temperatures above 43.5°C (Fig. 2). Inall cases tested, it was observed that bacterial cell growthstopped at the nonpermissive temperature, but neither a lossin colony-forming ability nor cell lysis occurred when aculture of htrE mutant bacteria was shifted to a high temper-ature (43.5°C). However, incubation at 50°C resulted in rapidbacterial cell lysis (Fig. 2), as compared with isogenicwild-type bacteria.

Sequencing of the htrE operon. The htrE gene, along withflanking sequences, was sequenced from the minimal sub-clone pSR10. The sequence analysis of a 3.8-kbp fragment ofDNA revealed the existence of two open reading frames(ORFs). The first ORF, designated ecpD (E. coli PapD),starts with an ATG as the initiator codon at position 186 andterminates with a TAA codon at position 924 (Fig. 3). Thesecond ORF defines the htrE gene. The ORF starts with aGTG as the initiator codon at position 961 and terminateswith a TAG at position 3550. The sequence upstream of theecpD gene shows certain interesting features, such as thepresence of a 13-bp inverted repeat located between nucle-otide positions 120 and 158 as presented in Fig. 3. Alsoobserved was a potential secondary structure which couldform a stem-loop-like structure, between nucleotide posi-tions 179 and 212.The exact position of the TnS insertion event was deter-

mined by the cloning and sequencing strategy describedearlier (39). TnS was shown to be inserted between nucle-otides 2354 and 2355, which should disrupt the htrE ORF atamino acid position 465 (Fig. 3).The predicted molecular masses of the proteins encoded

by the two ORFs are approximately 27 and 95 kDa, respec-tively. The 27-kDa protein is expected to be highly hydro-phobic, with a predicted pI of 10.2. However, the 95-kDaprotein is predicted to be mostly hydrophilic, with a pI of

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time (min) time (min)FIG. 2. Growth and viability curves of htrE' and htrE1394::TnS isogenic bacteria. Exponentially growing cultures at 30'C were diluted

1:10 into prewarmed Luria-Bertani medium and incubated at 30, 43.5, or 50"C. Bacterial growth was monitored by measuring CFU per

milliliter at various times following the temperature shift by plating serial dilutions and by measuring the optical density at 595 nm forexperiments with incubation at 50'C.

4.6. These predictions were verified by labeling the proteinsencoded by plasmids carrying ecpD and htrE under the T7promoter expression system and by then performing two-dimensional gel electrophoresis (see below).

Identification of the hlrE and ecpD gene products. Bacterialcultures of BL21(DE3) carrying pSR10, pSR174, or pSR176were labeled with [35S]methionine as described in the legendto Fig. 4. The presence of plasmid pSR10 resulted in thelarge overproduction of 95- and 27-kDa proteins (Fig. 4). Inthe presence of pSR174 (Fig. 5A), the synthesis of the95-kDa species corresponding to the HtrE protein was

induced. Plasmid pSR176 led only to overproduction of the27-kDa polypeptide corresponding to the EcpD protein.Both of these polypeptides appeared to be processed. Toverify that the 95-kDa protein was indeed processed, thebacterial cultures carrying pSR176 were pulse-labeled with[35S]methionine for 1 or 3 min and then chased with a

1,000-fold excess mixture of cold methionine and cysteinefor 5. The resolution of such labeled extracts followingseparation by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) (7.5% acrylamide) showed thatthe 95-kDa protein is processed to a 92-kDa form. Both ofthese protein species were separated on isoelectric focusinggels and shown to migrate at the expected isoelectric points.The processing of the putative signal sequence of the 95-kDaprotein resulted in a protein of a more acidic nature (Fig.SB). These data correspond well with those expected fromthe DNA sequence analysis, although more-direct evidencewould be from N-terminal sequencing of these products.Similarly, we showed by pulse-chase experiments that the27-kDa polypeptide corresponding to the EcpD protein wasalso processed to an approximately 26-kDa mature product(Fig. 6).

Homology to other known proteins. Sequence similaritysearches against the Swiss-Prot and GenPept data basesrevealed strong homology of the HtrE and EcpD proteins toa family of prokaryotic proteins involved in pilin transportand assembly. This family includes proteins from at least fivedistinct E. coli serotypes and homologous proteins fromHaemophilus influenzae and Klebsiellapneumoniae (18, 19).Of these, the HtrE protein is homologous to the PapC-likefamily of proteins, whereas the EcpD protein resembles thePapD-like family of proteins. In all cases analyzed so far, thePapD protein has been localized to the periplasm, whilePapC is anchored onto the membrane and has been recentlyshown to act like a molecular usher (8). The 27-kDa proteinencoded by the ecpD ORF shows an overall identity of 34 to40% to PapD-like proteins. The overall similarity, taking intoaccount conservative substitutions, rises to about 60% (Fig.7). Similar scores were observed for homology of the HtrEprotein at the amino acid level to the PapC-like family ofproteins. PapD is the specific periplasmic chaperone re-

quired for the assembly of P pili (24, 27). The crystalstructure of PapD has revealed that it has the overalltopology of an immunoglobulin-like fold (17). Alignment ofthe EcpD protein sequence to that of the seven previouslysequenced pilus chaperone sequences from E. coli, H.influenzae, andK pneumoniae shows identity in 22 of the 23conserved amino acid residues (Fig. 7). An additional 58amino acid residues have been shown to be identical in atleast four of the seven proteins. Our EcpD protein sequenceshows identity in 53 of these 58 amino acid residues, with theremaining 5 reflecting only conservative substitutions. Mostof these conserved amino acid residues found in PapD havebeen shown to participate in maintaining the overall struc-ture of the various domains.

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173 ACCATTACATGTGTATCATTAATGATTGAATAATATTTTCTATTI'

P1 p2 p3 IR

145 AArTr

217

289

361

433

IR M F F N T K H T T A

TATGCTCAACCTGTATGGCTTmTAGTTCATCGTCTATTGCGGACATTGTCATTTCGGGTACTCGCGTAAL C F V T C M A F S S S S I A D I V I S G T R V

TATATAAAAGCGATCAAAAAGGTrCAA CGTACTGGAAAATAAAGGGAATAACCCTGCCAGAI Y K S D Q K S V N V R L E N K G N N P L L V Q

GTTGGTTAGATACTGGCGATGACAACGCTGAGCCTGGCAGTATTACAGTCCCTTTTACTCGTACGCCGCCAGS W L D T G D D N A E P G S I T V P F T R T P P

TATCGCGTATTGATGC CAGCACCTCACTGCCTAAAGV S R I D A K R G Q T I K L M Y T A S T S L P K

505 ACAGAGAGAGAGCGT AACGTACTGGAAGTTCCACCAAAACCAGATGCAGAAAAGGTCGCGAATCD R E R A F W F N V L E V P P K P D A E K V A N

577 AAAGCCTGCTGCAACTGGCATTTCGCACACGTATAAAACTTTTCTATCGCCCGGATGGATTGAAGGGAAATCQ S L L Q L A F R T R I K L F Y R P D G L K G N

649 CCTCTGAAGCCCCGTTAGCCCTGAGTGGTCTGGTCAGGTCAGAAGGTAAGGCGTCATTACGAGTGACCAP S E A P L A L K W F W S G S E G K A S L R V T

721 AT TPYAAYVSTFTACTACGTCT SSAGCAGTGGTGAETTAGAAGCTAGCGGTAAACGCTATCCGATTGATGN P T P Y Y V S F S S G D L E A S G K R Y P I D

793 TGAAAATGATTGCACCATTTAGTGATGAGGTCATGAAAGTCAATGGCCTTAATGGCAAAGCGAATTCTGCAAV K M I A P F S D E V M K V N G L N G K A N S A

865 AAGTGCATTTTTACGCCATTAATGACTTTGGTGGCGCAATTGAAGGTAATGCCAGGCTGTAATCAGGCAAGGK V H F Y A I N D F G G A I E G N A R L --

937 ATATAATTC CCGTATCGCCM T I E Y T K N Y H H L T R I A

1009

1081

ACGVTTGCGCGCTGCTGTATTGCAATACTGCTTTCAGTGCTGAACTCGTTGAATATGACCATACCTTCCTGT F C A L L Y C N T A F S A E L V E Y D H T F L

ATFGGGCAGAATGCATCTAATATTGATCTCACGCGGTACAGTGAAGGTAACCCCGCTATACCGGGTGTTTACM G Q N A S N I D L T R Y S E G N P A I P G V Y

1153 GACGTCAGTGTTTATGTAAACGACCAACCAATCATTAACCAAAGTATTACATTTGTCGCAATTGAAGGAAAAD V S V Y V N D Q P I I N Q S I T F V A I E G K

1225 AAGAACGC CCGATATAAATAACK N A Q A C I T L K N L L Q F H I N S P D I N N

1297 GAAAAAGCCGTTGCTTGC GGGATGAAACGCTCGGCAATTGCCTCAATTTGACGGAAATTATCCCTCAGE K A V L L A R D E T L G N C L N L T E I I P Q

1369 GCTTCTGTTCGTTATGACGTTAACGATCAACGTCTGGATATAGACGTTCCTCAAGC-TGGrATGAAAAATA S V R Y D V N D Q R L D I D V P Q A W V M K N

FIG. 3. Nucleotide sequence of the htrE and ecpD genes and the flanking regions. The three transcriptional start sites P1, P2, and P3 areshown by arrows. The inverted repeats present near the promoter region (IR) are underlined. The insertional site of TnS is indicated by atriangle.

ecpD and htrE form an operon. Our in vivo complementa-tion experiments showed that the 3.0-kbp EcoRI-HindIIIfragment cloned in either a low- or high-copy-number vectorwas unable to complement any of the known htrEl394::TnSphenotypes (Fig. 1). Although a protein of 95 kDa wasexpressed in vivo from an inducible promoter (such as the T7expression system), such a synthesis was not detected in theabsence of such a system. These results suggested that a keyregulatory element was missing from this clone, whichincludes 260 bp of sequence upstream of the GTG initiationcodon. This idea is further supported by the fact that clonescarrying complete copies of both ecpD and htrE comple-mented htrEl394::TnS mutant bacteria, suggesting that allthe necessary regulatory elements required for transcriptionof ecpD and htrE are located at the 5' end of the ecpD gene.To identify the regulatory regions and the transcriptionalstart site(s), we carried out primer extension experiments,using RNA isolated from a variety of genetic backgroundsand under different growth conditions.Mapping of htrE transcripts. The ecpD and htrE transcrip-

tional start sites determined by the primer extension exper-iments and mung bean nuclease protection assay wereshown to be located at nucleotide positions 152 (P1), 163(P2), and 174 (P3). The sequences located upstream of theputative P1 and P3 transcriptional start sites have goodhomology to the -10 and -35 E&70 consensus sequences(Fig. 8 to 11). In the absence ofmRNA capping experiments,we cannot rule out the possibility that one or more of thesesites represent either transcriptional termination or mRNAprocessing sites. Among the three transcriptional start sites,only P2 varied in the relative amounts of transcripts made,depending upon the growth conditions as well as the strainbackground used.

Since hirE was essential for growth at high temperatures,we looked at the relative transcript levels under a wide rangeof temperatures. Interestingly, the level of transcripts ob-served at temperatures ranging from 37 to 42°C was signifi-cantly higher than that at the lower temperatures (Fig. 8).Judging from the amount of primer extension products madefrom RNA isolated from wild-type bacterial cultures, it is

TAAAGCGTCTTACGTTCGTGCCGTCGCAGACCAAACAGCAACTriCTGrTATGTAAAAACTAACACTCGTATC

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1441 TACCAAAACTATGTTGATCCATCGTTATGGGAAAACGGCATTAATGCGGCCATGTTGrCATACAACCTCAACY Q N Y V D P S L W E N G I N A A M L S Y N L N

1513 GGATATCATAGTGAAACCCCTGGTCGAAAAAATGAAAGCATTTATGCTGCATTTAACGGTGGGATGAATTTAG Y H S E T P G R K N E S I Y A A F N G G M N L

1585 GGTGCATGGCGACTGCGTGCCTCGGGCAACTACAACTGGATGACCGATTCTGGCAGTAATTATGATTTTAAGG A W R L R A S G N Y N W M T D S G S N Y D F K

1657 AATCGGTATGTTCAGCGTGATATCGCCTCGCTGCGrTCTCAACTCATTCT1GGTGAGTCT=ATACGACGGGCN R Y V Q R D I A S L R S Q L I L G E S Y T T G

1729 GAAACCTTTGATTCCGTCAGTATCCGAGGCATTCGTTmATACAGTGACAGCCGCATGTTGCCTCCGACTTTAE T F D S V S I R G I R L Y S D S R M L P P T L

1801 GCCAGCTTmGCGCCTATCATTCATGGCGTTGCCAATACCAACGCCAAAGTCACTATTACGCAAGGTGGCTATA S F A P I I H G V A N T N A K V T I T Q G G Y

1873 AAGATTTATGAAACGACGGTGCCGCCAGGCGCTTTCGTCATTGATGATCTGAGTCCGTCAGGGTACGGCAGCK I Y E T T V P P G A F V I D D L S P S G Y G S

1945 GATCTTATTGITlACCATCGAAGAATCCGATGGCTCAAAGCGGACATTCTCGCAACcTTCTccATccGTTGTTD L I V T I E E S D G S K R T F S Q P F S S V V

2017 CAAATGTTACGCCCTGGCGTTGGACGTrGGGATATTAGCGGCGGTCAGGTCTTAAAAGATGATATTCAGGATQ M L R P G V G R W D I S G G Q V L K D D I Q D

2089 GAGCCTAATATTTCAAGCAAGCTACTACTACGGCCTGAATAACTATCTGACGGGTTATACCGGTATTCAGE P N L F Q A S Y Y Y G L N N Y L T G Y T G I Q

2161 ATAACCGATAATAACTATACCGCTGGTTGTTAGGTCTTGGTCTGAATACTTCAGTTGGTGCATTTTCTTTCI T D N N Y T A G L L G L G L N T S V G A F S F

2233 GATGTGACTCATTCCAATGTTCGTATCCCGGATGATAAAACATACCAGGGGCAAAGTTATCGTGTTTCCTGGD V T H S N V R I P D D K T Y Q G Q S Y R V S W

V2305 AACAAGTTATTCGAAGAAACAAGTACTTCACTGAATATCGCGGCCTATCGCTATTCGACACAGAATTACCTT

N K L F E E T S T S L N I A A Y R Y S T Q N Y L

2377 GGTCTTAATCCGAACAAGATCTTGAACCGAAATCCATGG L N D A L T L I D E V K H P E Q D L E P K S M

2449 CGTAATTACTCACGCATGAAAAATCAGGTTACGGTCAGTATTAACCAACCGTTGAAATTTGAGAAAAAAGATR N Y S R M K N Q V T V S I N Q P L K F E K K D

2521 TACGG= CATTTTATCTTTCCGGAAGTrGGTCCGATTACTGGGCTTCCGGACAAAATCGTAGCAATTACTCTY G S F Y L S G S W S D Y W A S G Q N R S N Y S

2593 ATTGGCTACAGTAACAGTACATCCTGGGGCAGCTACAGTGTCAGTGCCCAGCGTTCATGGAATGAAGACGGCI G Y S N S T S W G S Y S V S A Q R S W N E D G

2665 GACACTGACGATAGCG1TTATCTTAGTTTCACCATTCCAATTGAAAAATTACTTGGCACTGAACAACGTACTD T D D S V Y L S F T I P I E K L L G T E Q R T

2737 TCAGGTTTCCAGAGTATTGATACTCAAATAAGCAGTGACTTTAAGGGTAATAACCAACTCAACGTTAGCAGCS G F Q S I D T Q I S S D F K G N N Q L N V S S

2809 AGTGGCTATAGCGATAACGCTCGCGTCAGTTATAGCGTGAATACTGGCTATACGATGAATAAAGCCAGCAAAS G Y S D N A R V S Y S V N T G Y T M N K A S K

2881 GATTTGAGTTATGTTGGGGGTTATGCCAGCTATGAGTCACCATGGGGAACGCTGGCAGGTTCAATTTCTGCAD L S Y V G G Y A S Y E S P W G T L A G S I S A

2953 AATAGCGATAACAGCCGTCAAGTmTCTCTCAGCACCGACTN S D N S R Q V S L S T D G G F V L H S G G L T

3025 TTCAGTAATGATAGTPTTAGCGACTCCGATACACTGGCGGTAGTTCAGGCTCCAGGTGCTAAGGAGCGCGAF S N D S F S D S D T L A V V Q A P G A Q G A R

3097 ATAAATTATGGCAACAGTACTATCGATCGATGGGGTTATGGTGTCACCAGCGC rCTCCTTATCATGAAI N Y G N S T I D R W G Y G V T S A L S P Y H E

3169 AACCGTATCGCGCTGGATATCAACGATCTTGAGAACGA rGAATTAAAAAGTACCAGTGCAGTAGCTGTAN R I A L D I N D L E N D V E L K S T S A V A V

3241 CCGCGTCAGGG CTCAGTCGTTmTGAGAAACGAAGGG CAATAGCCATTATGAACATCACAP R Q G S V V F A D F E T V Q G Q S A I M N I T

3313 CGAAGTGATGGTAAAAATATTC CAGATATm ATGATGAGCAAGGCAATGTCATTGGTAATGTTR S D G K N I P F A A D I Y D E Q G N V I G N V

3385 GGACACCGTGGACAAGCATTrGTCGTGGTATTGAGCAGCAGGGAAATATCAGCATTAAATGGCTCGAACAAG H R G Q A F V R G I E Q Q G N I S I K W L E Q

3457 AGTAAACCCGTAAGTTGTCTTGCGCATTATCAACAAAGCCCAGAAGCACCAAAAATAGCACAATCTATTATTS K P V S C L A H Y Q Q S P E A P K I A Q S I I

3529 CTGAATGGAATTCTGTATATAGATTATGAAACCGAAGATGACACCTCCGATGGGGTTTACCTAATCAAGGL N G I L F I ---

3601 TAACGGCACATCACAGCCTCTTCATTTCCAGGCAACATTAAAGCAAGACGGGAATATTGCTATCGAACCCGG3673 CGAATTTAAAGCCACCAGTACTTTCCAGGTAACCTATCCCTGATAACGTAGCAGCACTGTCACCTTCTGGCA

FIG. 3-Continued.

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oi

ct) cn4 c4_

--O HtrE 95 kDa

-<-EcpD 27 kDa

FIG. 4. Identification of the htrE and ecpD gene products. Cul-tures of the BL21(DE3) strain transformed with plasmid pBluescriptSK+ alone or pSR10 containing both the hirE and ecpD genescloned under the T7 RNA polymerase promoter. Expression of T7RNA polymerase was induced by addition of IPTG (isopropyl-.-D-thiogalactopyranoside) (0.5 mM). Following a 30-min incubation,cells were treated with rifampin (200 pg/ml) for another 15 min andthen labeled with [35S]methionine (50 ,uCi/ml) for 10 min. Theproteins were resolved by SDS-12.5% PAGE.

A

pSR174 htrE+1 2 3 4 pSK

clear that very little ecpD-specific mRNA accumulated atlow temperatures, e.g., 220C. The amount of transcriptaccumulation gradually increased following a temperatureupshift. This type of temperature-induced transcriptionalregulation is reminiscent of Ea2 regulation of classical heatshock genes (34). However, unlike most of the heat shockgenes, the transcript levels were markedly reduced at 500C(Fig. 8).

Since it is known that o2 (the rpoH gene product) isabsolutely required for the transcription of the classical heatshock genes, we compared the mRNA levels from isogenicrpoHA and rpoH' strains. The results of the transcriptionalanalysis clearly showed that transcription of the htrE operonis not dependent on &32, since the levels of transcripts as wellas the usage of the putative transcription initiation sites wereidentical in all genetic backgrounds studied, irrespective ofthe presence or absence of rpoH. In addition, it is knownthat &2-promoted transcription depends on the presence ofcanonical -10 and -35 consensus sequences unique to thisclass of genes (34). Such Ea&2-dependent promoter consen-sus sequences were not found in the ecpD promoters, asshown in Fig. 8 and 9.IHF-mediated positive regulation of the htrE operon. It is

known that pilin operons are in either an on or off mode. Thephase variation of the type I pilin-like fim operon of E. coliK-12 is known to be mediated by the DNA-binding proteinIHF, known to bend DNA (1, 9). In contrast, type II pilins,exemplified by the Pap pilins, are regulated by PapI andPapB. PapI has been shown to be regulated positively by theglobal regulatory protein Lrp through methylation (5).

Since the ecpD and htrE gene products show a high levelof sequence similarity to the pilin chaperone PapD and thepilin porin protein PapC, respectively, we analyzed thelevels of transcripts in lrp+ and lrp::TnlO strains, using theprimer extension method (Fig. 9). In both strains and underall conditions studied, both the actual level of transcripts and

B

pSR174 htrE+

ecursor HtE::Mature Htrlz

FIG. 5. In vivo processing of the HtrE protein. (A) Cultures of the BL21(DE3) strain transformed with vector pBluescript SK+ alone orpSR174 containing the htrE gene under the T7 RNA polymerase promoter. Expression of T7 RNA polymerase was induced as described inthe legend to Fig. 4, and cultures were labeled for 1 and 3 min (lanes 1 and 3) with [35S]methionine (50 mCi/ml) and then chased for 5 min witha 1,000-fold excess of cold methionine (lanes 2 and 4). Proteins were precipitated with trichloroacetic acid (10%), washed with cold acetone,and then resolved by SDS-7% PAGE. (B) Two-dimensional PAGE of the same [35S]methionine-labeled cell extracts as used in panel Aprepared from bacteria carrying htrE on plasmid pSR174. In the first dimension, 2% ampholines (pH 3.5 to 10.5; Pharmacia) were used. Thearrows indicate the positions of the precursor and mature HtrE species.

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E. COLI htrE 5017

pSR176 ecpD v

1 2 3 4 X.

EcoFAN MNKFISIIALCV-FSSYANAAFTLNSTrYIYNEGQQSVSVNIHiESEHKYGG

MFFNTKHTTALCFVTCMAFSSSSIADIVISGIVIYKSDQKSVNVRLE

KpnMRK MKRIALFFCFI--FSFAAHA-NNIIVNGtFIYPGNEKEITVGLS' TADRPALA

EcoFAN

EcpD

KpnMRK

EcpD precursor -

EcpD mature

FIG. 6. Processing of the EcpD protein. Pulse-chased sampleswere prepared from cultures carrying the ecpD gene cloned underT7 RNA polymerase promoter (pSR176) or vector pSK alone.Cultures were labeled for 30 s (lane 1) or 1 min (lane 2) and thenchased with cold methionine for either 1 min (lane 3) or 5 min (lane4). The proteins were resolved by SDS-15% PAGE.

the position of transcription initiation remained identical,demonstrating no direct participation of the Irp gene productin the regulation of the ecpD operon. This is consistent withthe lack of Dam methylase recognition sites upstream of theecpD promoter or near its leader sequence. This result isquite surprising since both ecpD and htrE structural genesare highly conserved with their correspondingpapD-like andpapC-like gene families.

Next, we isolated and studied mRNA from isogenichimA+ and himA::TnlO (SR69 and SR1021) strains or fromhimD mutant bacteria (SR1022). The primer extension re-sults showed comparatively low levels ofmRNA transcriptsinitiating from all three putative promoters. Thus, it is clearthat functional IHF is needed for efficient transcription of theecpD-htrE operon. However, since some ecpD-htrE operon-specific transcripts were seen in strains lacking IHF, therequirement for the IHF protein is not absolute. The densi-tometry scanning of the autoradiograms showed a five- tosixfold decline in the levels of transcripts initiated fromeither of the three transcriptional start sites in strains carry-ing mutations in either himA or himD, which encode the twodifferent subunits of the IHF protein.katF-mediated positive regulation of the htrE operon. Since

the htrE gene is essential for E. coli growth at high temper-atures, particularly under more severe stress conditions suchas growth at 50°C, we used the Northern (RNA) technique tosee whether transcriptional regulation is mediated by thealternative sigma factor cs (coded by the katF [=rpoS] geneproduct). Interestingly, the level of ecpD-htrE operon tran-scripts was substantially reduced in the katF::TnlO mutantbacteria. To determine which of the three transcriptionalstart sites in particular was used by EU, a primer extensionanalysis was done. Transcriptional initiation was reducedprimarily from the P2 transcriptional start site (Fig. 10). The-10 region of the putative P2 promoter does in fact have

Q DNIDKNGE ----VVFFSP MVFKLNPKQKQIVIV-NINDNLP.RISI FWL

QS LDTGDDNAEPGSITVPFTR PVSRIDAKRGQTI KLMYTASTSLPKgRRRAFWFTA LDNGNADATPDTITTPFIIIPISRVDAKSGQTL IKLGSNAGLAK KWrLWWL

EcoFANVQE PAPKGD----GGSLSL NNRV LIYIALKNGRDEAENNIKLINSTD

EcpD VLE PKPDAEKVANQSLLQL FRTRI LFYDGLKGNPSEAPLALKWFWSNSE

KpnMRK LLE PVEASQKNEGQNILQL RSRFKFIYRAGL-GNRDAAAEKLALSANGSS

EcoFAN S ----CLE YYFAISDVKINGKSIDLNSDAKNKMGVFSPFS ----KVC-LGNV

EcpD GKASLRVT YYVSFSSGDLEASGKRYPIDVK ----MITAFSDEVMKVNGLNGK

KpnMRK --- LSVSIP FYITVSRIS-RNGGKALNSKTV ----MFAQSSQTIALSSAVSK

EcoFAN NTSGNITVTAFDY@VATSYTVQRSK

EcpD ANSAKVHFYAII FiGAIEGNARLKpnMRK GETLTVN--NINPADVAVKVTVK

FIG. 7. Comparison of amino acid sequences of E. coli EcpDand two of the representative PapD-like proteins, MrkB and FanE.The identity retained for the consensus sequence for PapD familyconsisting of 23 invariant residues is shown by shaded bars. Identi-cal residues are indicated by double dots; conserved residues areindicated by single dots.

similarity to -10 regions found in katF-dependent promot-ers. However, it may be noted that P2 promoter is still usedin katF::TnlO mutant bacteria, although at significantlyreduced rates. A similar dependence on the rpoS geneproduct has also been observed in case of curli, encoded bythe curlin subunit gene, csgA (37).

0q0 0 G A T Ce'4 o r- cl Q

P1

P2 -

P3-

FIG. 8. Mapping of 5' termini of htrE ecpD transcripts. Primerextension reactions of total cellular RNA hybridized to 32P-end-labeled DNA oligonucleotide probe, complementary to nucleotides83 to 106 in the ecpD sense strand. RNA was extracted fromwild-type bacteria grown at 22°C or after a 10-min shift to 30, 37, 42,or 50°C. Lanes G, A, T, and C correspond to the dideoxy sequenc-ing reactions carried out with the same oligonucleotide as theprimer.

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G

~- P1 -4- P1

- P3<- P3

FIG. 9. IHF-mediated transcriptional regulation of the htrE ecpD operon. (A) RNA was extracted from isogenic rpoHA (KY1621) andrpoH+ (SR407) strains at 30'C or from isogenic lrp::TnlO and lop+ strains. (B) Primer extension reactions were carried out with RNAextracted from isogenic himA::TnlO and himD mutant bacteria. The positions of the three transcriptional start sites are indicated by arrows.

DISCUSSIONFrom a library of random TnS insertions in the E. coli

chromosome, we characterized an insertion that results in aTS phenotype for bacterial growth at temperatures above43.50C. This TnS insertion defined a locus, htrE, constitutingan operon of at least two new genes, designated ecpD andhtrE. The htrE locus was mapped to 3.3 min, correspondingto the region from kbp 153 to 157 of the physical map of theE. coli chromosome. The ecpD gene is the first gene of theoperon and is located approximately 2.5 kbp counterclock-wise of the pcnB gene, involved in copy number control ofvarious plasmids (29). The direction of transcription of theecpD-htrE operon was shown to be counterclockwise rela-tive to the E. coli genetic map.Sequence analysis of a 3.9-kbp minimal clone which

complemented all phenotypes of htrE::Tn5 insertion mutantbacteria revealed the existence of two ORFs. The first ORFidentified encodes a protein with a predicted molecular massof 27-kDa and a predicted pI of 10.2. The second ORFidentified was found to encode a protein with a predictedmolecular mass of 95 kDa and a predicted pI of 4.58. Thepredicted isoelectric point of the HtrE and EcpD proteins isin close agreement with their observed migration on two-dimensional SDS-polyacrylamide gels. In addition, bothHtrE and EcpD proteins are synthesized as precursors andare processed to 92- and 26-kDa species, respectively. Theseresults are also in agreement with a predicted transportsignal sequence for both proteins. By sequencing from theTnS insertion site in the htrE gene, we demonstrated that thehtrE gene is encoded by the second ORF.The EcpD and HtrE proteins were found to be highly

homologous to the class of proteins involved in postsecre-tional assembly of pili, PapD and PapC, respectively, in E.coli or the related class of proteins from K pneumoniae,MrkB and MrkC, as well as nonfimbrial adhesins, includingAfaC. It has been proposed that PapC (or its homologs)plays an active role in the dissociation of pilin from the PapDchaperone (8). The 95-kDa HtrE protein and the 27-kDaEcpD protein show an overall identity of 34 to 40% over

their entire polypeptide with their homolog PapC and PapDclass of proteins (2, 7, 20, 33, 35, 42, 44). In the PapC familyof proteins, one of the conserved features is the presence oftwo highly conserved cysteine residues, spaced approxi-mately 27 amino acids apart. In the HtrE protein, these twocysteine residues are located at amino acid positions 94 and

TTATTCCCT

0

+ E..

G A T C

GTTTAATTAAC

Pi TC

-4-P2 TAT

..u--P3 *CAGGG

FIG. 10. Positive regulation of the P2 promoter by KatF. Primerextension reactions were carried out as described in the legend toFig. 8, using template RNA from isogenic katF::TnlO and katF+bacteria harvested after the entry into stationary phase.

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P3 --

FIG. 11. Mapping of the 5' termini by mung bean nuclease protec-tion assay. Mung bean nuclease mapping used a uniformly labeled,single-stranded, 315-nucleotide HincII-DraI DNA fragment as a probe.The probe (106 cpm) was hybridized to RNA extracted from MC4100bacteria grown at 37°C, digested with 300 U of mung bean nuclease,and electrophoresed (lane 1). Lanes G, A, T, and C correspond tothe dideoxy sequencing reactions carried out using pDM655 (Hinll-HindIII) as the template, initiated by the SK primer (Stratagene).

127. In the HtrE protein homologs, the first cysteine isapproximately 100 amino acid residues from each aminoterminus in all cases studied so far. A previous comparisonof seven members of the PapD family has shown that 23amino acid residues are identical in all cases (18). As can beseen in Fig. 7, this conservation has been retained in EcpD.This result strongly suggests a role for the EcpD proteinsimilar to that of the other members of the PapD family.

It may be noteworthy to point out here that in certain E.coli strains a novel class of pilins, called curlins, has beendetected (38). Although the genes encoding the structuralsubunit of curlin have been shown to map at 8 min on the E.coli chromosome, nothing is known about their assembly or

transport. It is possible that the htrE and ecpD genes are

involved in this pathway, since both the curlin operon andhtrE operons seem to be incomplete by themselves. If thehtrE operon is indeed used to transport pilin-like substrates,then certainly the corresponding structural genes are locatedelsewhere. However, the transcription of the csgA gene,

which encodes the curlin subunit, is under the transcrip-tional regulation of a cytoplasmic protein, Crl. The transcrip-tional activation of the csgA gene by the Crl protein occurs

only at lower temperatures, such as 26°C, but not at 37°C (3).Such a temperature-dependent transcriptional regulation isexactly the opposite of that found for the ecpD-htrE operon.

However, one feature common to the ecpD-htrE operon andthe curli-encoding csgA gene is the positive regulation by therpoS gene product (37).The main observations of the transcriptional regulation of

the ecpD-htrE operon were that (i) transcription was inducedwith a temperature upshift from 22 to 37°C but was notcontrolled by the E- regulon, (ii) transcription declined at

temperatures above 420C and was much lower at 50'C thanat 370C, a situation reminiscent of most housekeeping genes,and (iii) transcription was severely reduced at low tempera-tures, such as 220C. Similar results have also been obtainedfor pap transcription (14). However, unlike pap transcrip-tion, ecpD-htrE operon transcription is not regulated by Lrp(the product of the lrp [= mbf] gene). Instead, the mainregulation seems to be through the DNA-binding proteinIHF and the product of katF gene, which is known tofunction as an alternative sigma factor, judging from the factthat the levels of the htrE transcripts were reduced in bothhimA and himD mutant bacteria, as well as in bacteriacarrying katF::TnlO mutations.One of the main phenotypes associated with htrE null

mutations is hypersensitivity to a brief exposure to 500C, acondition that is normally sublethal for htrE' bacteria. Themechanism of thermotolerance at 500C is not understood atpresent. We have previously shown that htrC mutant bacte-ria have a similar phenotype (39). Clearly the transcriptionalregulation of the htrC gene, which is exclusively transcribedby Ea32, is different from that of the ecpD-htrE operon.Interestingly, it has been shown that mutations in the RNApolymerase sigma subunit e, encoded by rpoS (katf), aredeficient in stationary-phase thermotolerance (25). Further-more, the trehalose synthesis gene otsA (osmA), an osmot-ically inducible gene which is also transcribed by Eo, hasbeen also implicated in such a thermotolerance at hightemperatures (16). The P2 promoter of the htrE operonappears to be under positive regulation by Eoe. Also, htrEnull mutant bacteria do exhibit sensitivity to high osmolarity.Thus, some of the rpoS-regulated genes might be importantto survival both at high temperatures and under other stressconditions. However, further studies are needed to seewhether the ecpD-htrE operon is also subject to growthphase regulation and osmotic induction. Finally, it is worthmentioning here that some of the defects observed in htrEnull mutant bacteria may be due to the possibility that thehtrE-encoded PapC-like protein functions as a porin, per-haps involved in the transport of certain essential metabo-lites. Such a possibility is not unlikely since the PapChomolog of Salmonella typhymurium pathogenic bacteriahas been shown to transport molecules such as vitamin B12(41). The fact that htrE null mutants exhibit an osmosensitivephenotype is consistent with such a hypothesis.

ACKNOWLEDGMENTS

D.M. is a recipient of an EMBO fellowship. This work wassupported by grants from the Fond National Scientifique SuisseFN31-31129-91 and by NIH grant AI21039.

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