Proc. Natl. Acad. Sci. USAVol. 90, pp. 2970-2974, April 1993Microbiology

Molecular characterization of an operon required for pertussistoxin secretionALISON A. WEISS*, FREDERICK D. JOHNSONt, AND DRUSILLA L. BURNStt*Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298; and tDivision of Bacterial Products, Center forBiologics Evaluation and Research, 8800 Rockville Pike, Bethesda, MD 20892

Communicated by Stanley Falkow, December 15, 1992

ABSTRACT Mutants of BordeteUa pertussis which aredefective in secretion of pertussis toxin were isolated andcharacterized. The region of the B. pertussis chromosomeidentified by mutagenesis as playing a role in transport ofpertussis toxin was sequenced. Analysis of this region revealedeight open reading frames, seven of which predict a proteinexhibiting homology with one of the VirB proteins ofAgrobac-terium tumefaciens, which are involved in the transport of theT-DNA molecule across bacterial and plant membranes. Thusa set of accessory proteins are most likely involved in thesecretion of pertussis toxin, and these proteins appear to bemembers of a family of proteins involved in the secretion ofmacromolecules from bacteria.

Gram-negative bacteria have a unique feature that distin-guishes them from all other living cells, including Gram-positive bacteria. They possess two membranes separated bya cellular compartment called the periplasmic space. Theouter membrane presents a formidable barrier to compoundsentering and exiting the bacteria. Gram-negative bacteria thatare pathogens often produce protein toxins which are se-creted from the bacterial cell and subsequently interact withand induce damage to eukaryotic cells. After synthesis, theprotein toxin must cross the inner membrane, the periplasmicspace, and the outer membrane. We have examined theexport of one particular toxin, pertussis toxin (PT), from theGram-negative organism Bordetella pertussis. Since inacti-vated PT is a major component of new-generation pertussisvaccines, knowledge of the mechanism of secretion of PTmay lead to improved and less expensive methods for vaccineproduction that would allow for expanded use of thesevaccines worldwide.PT is composed of five types of subunits, S1-S5, found in

a 1:1:1:2:1 ratio (1). After PT is secreted from the bacterialcell, it interacts with mammalian cells and interrupts signaltransduction in those cells by ADP-ribosylating a family ofGTP-binding regulatory proteins (2, 3). Little is known con-cerning the mechanism by which PT is transported across theinner and outer membranes of B. pertussis. Each PT subunitis synthesized with a signal sequence (4, 5), suggesting thateach subunit may begin the secretion process by entering thegeneral export pathway as exemplified by the sec pathway (6)of Escherichia coli. To our knowledge, the existence ofaccessory proteins involved in the secretion of PT has notbeen demonstrated previously.

In this study, we identified by mutagenesis a region of theB. pertussis chromosome located directly downstream fromthe PT structural genes which appears to encode proteinsnecessary for transport ofPT. Nucleotide sequence analysis§of this region reveals eight open reading frames (orfs). Sevenof the predicted proteins show striking homology with certainof the VirB proteins of Agrobacterium tumefaciens which

have been proposed to be involved in transport ofthe T-DNAmolecule across bacterial and plant membranes (7-9).

MATERIALS AND METHODS

Cloning and Mapping of the pt Operon. A cosmid (pUWli-11) containing pHC79 (10) and a fragment of BP338 chromo-somalDNA encoding all ofPT as well as 20 kb ofdownstreamsequences was subcloned into pBluescript vectors (Strata-gene) containing a gene cassette encoding the P-incompati-bility group origin of transfer and a gentamicin-resistancedeterminant. These plasmids were introduced into B. per-tussis by triparental matings (11). Southern hybridizationverified that all transconjugants contained the plasmid se-quences integrated by homologous recombination (data notshown).

Secretion of PT. Two methods were used to identifymutants defective in secretion of PT: quantitation of PTactivity and a colony blot technique.PT activity was quantified as follows. B. pertussis strains

were grown in Stainer-Scholte medium (static culture) at37°C with appropriate antibiotics included in the medium.For example, the medium for BPM3171 was supplementedwith kanamycin (25 ,ug/ml). Samples (10 ml) were taken atvarious times to monitor PT secretion. Cells were separatedfrom culture medium by centrifugation (12,000 x g for 10min). The supernatant was filtered through sterile Millex-GVfilters (0.22-gm nominal pore size; Millipore). The pellet wassuspended in 1 ml of 50 mM Tris HCl (pH 8.0) containinglysozyme (2 mg/ml) and ethylenediaminetetraacetic acid (50mM). After incubation for 30 min at 35°C, 9 ml of phosphate-buffered saline (pH 7.2) containing Tween 20 (0.05%) wasadded. The preparation containing the cell lysate was thencentrifuged (12,000 x g for 10 min) and the supernatant wascollected and filtered through a sterile Millex-GV filter.Biologically active PT in the culture medium and in the celllysate was quantified by measuring the ability of thesefractions to alter the morphology of Chinese hamster ovarycells (12). Purified PT obtained from Institut Merieux, Lyon,France, was used as the standard for this assay.

In some experiments, the secretion phenotype was deter-mined by colony blots. Bacterial strains were grown onBordet-Gengou agar for 3 days. Nitrocellulose filters (BA85;Schleicher & Schuell) were placed on the colonies, andextracellular material was allowed to bind to the filtersovernight at room temperature. The filters were blocked,washed, and processed by standard immunoblot techniques(13). Monoclonal antibody 3CX4 (14), directed against aconformational epitope on the S1 subunit of PT, was used.Secretion mutants could be clearly distinguished from thewild-type strains.

Abbreviations: PT, pertussis toxin; orf, open reading frame.fTo whom reprint requests should be addressed.§The nucleotide sequence reported in this paper has been depositedin the GenBank data base (accession no. L10720).

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Nucleotide Sequence Analysis. The DNA sequence ofthe ptloperon was determined by Lark Sequencing Technologies(Houston, TX) using clones pPTL2, pPTL4, and pPTL9.Sequence analysis was performed with Genetics ComputerGroup programs (15).

RESULTS AND DISCUSSIONIdentification of an Operon Required for PT Secretion.

Previously (13), mutant BPM3171 was generated by insertionof TnS lac transposon into BP338 -3.2 kb downstream fromthe end of the PT structural genes (Fig. 1). While growth ofBPM3171 was similar to that of the parent strain (data notshown), the level of PT in the culture supernatant of themutant strain was much lower than that found in the culturesupernatant of the parent strain (Fig. 2). PT levels were lowerin the culture supernatant of the mutant strain as comparedwith that of the parent strain when either biologically activePT was measured (Fig. 2) or when the amount of the S1subunit of PT present in the supernatants was determined byimmunoblot analysis (data not shown). In contrast, levels ofcell-associated PT were slightly higher in the mutant strainthan in the parent (Fig. 2). Preliminary results indicate thatthe PT which associates with both BP338 and BP3171 cells ismembrane-associated (S. Hausman and D.L.B., unpublishedobservations). Levels of two other exported proteins, fida-mentous hemagglutinin, which is normally secreted from thebacterium (16), and pertactin, which is normally exported tothe outer membrane (17, 18), were found to be similar in theparent and mutant strains as determined by immunoblotanalysis (ref. 13; data not shown). Moreover, the mutantstrain was hemolytic, suggesting that adenylate cyclase toxinis secreted from the mutant. Thus the defect in BPM3171appears to affect only the export of PT and not othervirulence factors. The gene defined by BPM3171 was origi-nally designated ptl for pertussis toxin-linked, but consider-ing its role in secretion, we propose pertussis toxin liberation.

Genetic Mapping of the ptl Operon. Mutagenesis was usedto define the region essential for PIT secretion. Suicidevectors were constructed which contained a gentamicin-resistance determinant and fragments of B. pertussis DNAwhich either included the end of the operon encoding the PTsubunits or were from immediately downstream of thatoperon. Vectors containing a 3652-bp Bgl II fragment(pPTL7), a 4523-bp Bgl II-Sal I fragment (pPTL1), a 5914-bpEcoRI fragment (pPTL2), a 1125-bp EcoRI-Kpn I fragment(pPTL4), a 1062-bp Kpn I-Pst I fragment (pPTL9), a 1.6-kbpKpn I-BamHI fragment (pPTL8), a 2.6-kbp Kpn I-Sph Ifragment (pPTL5), or a 7-kbp Kpn I-Sst I fragment (pPTL6)were introduced into BP338. Since these plasmids cannot

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FIG. 2. Comparison of the amounts of active PT produced byBP338 (parent) and BPM3171 (mutant) cells. Bacteria were grown forthe indicated times. Cells which had been separated from culturesupernatant were lysed and the concentration of active PT in theculture supernatant (A) and cell lysate (B) was determined for bothBP338 (o) and BPM3171 (e).

replicate in B. pertussis, gentamicin resistance is conferredonly if homologous recombination occurs between the chro-mosome and the B. pertussis sequences on the vector so thatthe entire plasmid is integrated into the chromosome. Thecloned sequences are present twice in the genome but areinterrupted by the vector. Mutations can result if the clonedregion is internal to an operon, in which case the vector will

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FIG. 1. Map of the ptl operon. (Upper) Regions encoding the structural subunits (S1-S5) of PT in the ptx operon and potential orfs (B-H)in the ptl operon are indicated by solid blocks. Stippled blocks indicate regions encoding the signal sequences of the PT structural subunits.The approximate location of the Tn5 lac insertion of BPM3171 is indicated. (Lower) Schematic diagram ofptl region of mutant strains. BPPTL7,BPPTL2, BPPTL4, and BPPTL9 are strains in which pPTL7, pPTL2, pPTL4, and pPTL9, respectively, have integrated into the chromosome.When the plasmid integrates into the chromosome, the cloned sequences are present twice in the genome but interrupted by the vector (seetext). Solid bars represent the integrated plasmid and the homologous sequences on the chromosome. BPPTL7 and BPPTL9 were positive forPT secretion. BPPTL2 and BPPTL4 were negative for PT secretion as measured by both PT activity assays and the colony blot technique.Restriction sites are indicated: B, Bgl II; E, EcoRI; K, Kpn I; P, Pst I.

Microbiology: Weiss et al.

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2972 Microbiology: Weiss et al. Proc. Natl. Acad. Sci. USA 90 (1993)

ATGAATCCCTTOAA.AGACTTO%AGAGCATCG CTACCGCGCCTGGCCTTCATGGCASCCTOC ACCCTGTTGTCCGCCACGCTGCCCGACCTC GCCCAGGCCGGCGGCGGGCTGCAGCGCGTC 3803

AACCACTTCATGGCOAGCATCGTGGTCGTA CTGCOCGGCGCGTCAGTGGCCACGGTGACC ATCGCCATAATCTGGGCGGGCTACAAGCTG CTGTTCCGGCACGCCGATGTGCTOGACGTG 3923

GTGCGAGTGGTGCTGGCGGGACTGCTSATC GGCGCATCGGCCGAAATCGCTCGTTATCTG CTGACCTGAATCCTGGACGTATCGAACATG CGTGATCCGCTTTTCAAGGGCTGCACCCGG 4043M R8 D F K 0 C:tk:

OATOCTMTfltfl~~T ACCCOCCACO ~0C TGGCCGTGTGCAGCGGCACCATTOCC C TGCTi~Or.rCATCTGGT TCAGCAZCGCCTTT CTGGCV I OTCrTOCITCOC 4163MLM:.:I~~~~sPAT: .A V C SOGT I A LL:*:I WF S:A LA:>:F PA~~ A

ATOCQOATCATGATCCGQCAQ-r. TGACCAG CAGTTCCGCCTGATCT&QmCT.TTACC-T.CGC ATGCGTTGGCTGAGrAOCCCCOA...GCGTt.CT.GGCAAAGTACCGTCTATGC6GCGO 4283MR*IM I R5:Xw::::;W.D Q QF RLIW Y:t~.MRWLS D R T FHF Q S T VYV A%0CTrITAGCA CV5OET Or~AOACSOOOOCAACOATSOCATTGCGCGCAACGAGC GCGCCATCGCTGCGTTCATCCCCTACAGCA 4403

MN 88OOQT AFAAi A RN*!R ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ....A I A A FIYSS&CASC.GGACACGACGCTGATCACCC ATGGCGCGGACCTGGTCCGCACCTSGCGCG TACAGjGGAT.C_CTQAAGOCO AAOTOTTOACCCTACGC42

~~~TQT~~~~~T L I T H GOAD L VRTR*T QW V ~S A PjLvSQ HEQL

T.CAAC OOCCIOT.TOPCGCGCCAICTCGTGCG AGCA61ICGCGCTTTGGATCCATTGCATCC ...OOGCGCAAGACGCAGGCCGGGTTGGATGC$6C...OGTACGAAAATCC XTICTGCCGCGCGCT CG 4643*1 LiftS CE~~~~~~~~~~~~Q~:::A L W I4UC I*i R K T Q A OLG i ENPF R AL

ACGCCTCGTACAAC5C.CrSGCTGAACGCOC GQC.4GGCAATGAC.O.AACGAATTCTACCT.CA..CCCTSGGTATATCGGECTOOTGCCACGCQSCCCGC .TQSSrCAAGCGTGCGCATCACGGCCAGGCCG 4763S VN ~~~~~~ARLN A R .QA M T E F VY ::X.wtV: V OGH A At:W:X: RA HHGOQAE

AGGCCqJ.OGCA.iT.GTGCCCATOTA .,GACGCAIOGACGAAATCGGATCCCTGATCG AAACGACrpjITCGCAGCCAIOCAAACAOOCOCATA lCqOOT

AOTCOC$OAAIOTL H V D EI0SL R EET N8H4E Q AlIT

AGACGGACAGC$&CCSGCCGGCGATACTCCC GOACGCTOACCCTGCT.CGAATTCCTGCT.CA. C CC%GCCACT5GCAG4CrGGTACGT.GT.GCCGG C.CGGOCCGOTOOACACGTATCTCGGTTCOA 5003DS AG088YVS 8 T L T L: E F L L IHWQ :VY:A: R:iV P A iP VDAYLOGSS

GECCOOATCCTTGCIpOGOCOAAOTO TGjO.ICGACCTGOCCGCCGCTACO CGCAGTTCATCGATITCAAOGAAOOACCAACA& 0t-A0IAICJ52fI LA *A EM M.jL:i T C 88 VYA Q FIK0G T4H

TQJOACAGATTACOAATATfiT.GAT.CA..COCAT..TC4T. CAGCGCGTCGGCAAGCGAC-.AGGCGCTOOCr.CTACZ OCOGCOCC CAGCTGGCCAACGTGCAGGACOCCOCOT54

YfAK*-3 AA QRt~~It PRVQ8ACTCTCAOTTCGQCCAT OAUCGQOCTCCO ACGCOQCETTTTCCOCACOCCOOTTACOTG T%OCTOATCjCTTCTAOOM JTCMTOCT9AO99A58AOCCTTTACA AAA ICOC OA TOOACICIA1CAO( O.CGAGTOCAIOACCCGTGATGATCCC TCGCGCOACCTCTOCCGGCTTCc50

AACCTTCACTCAGATT.CAT.CCATCCTCOOCTGCTTACOATTOCCACOOCCGTACATO TGGCCTGGOAArCACOCOCTACOCAT.C$OAQGCT.VN

OOATACOCOACGOCOAAC,rCATCCCTCAG ACCCTCGTCAGACACCA ATTAQTTCTCTCTATGCTGCA TGCTCOCAOCCTGGCATOAC6TCOfCCOCTA 5963

vSAQHA K:A AE %VTRPASTL LDif A RM AATCCCWCCCTCOAOCO 6083AACGTAAaCSGGCCTTCAAGAAAOaCC.GCACTGGACOJTCCQATA.CTOOACGGOOAA GCAGCCTTOTOOTCTTCACAACCOAATGGGCATT .CCCTGCCATTCTTTCOCAGCCOOOCACTAC.O 56203L V 8 3 L 0 #~~~~t~#~~i*4~ 0 0 5 A if 0 T 0 M L 9 N P N 1 C L 0F 5 8~~~~~~~N P Pf:: 0 NL :V.;.

T.C.COTTCTCCTTOCAOATT.CATCACATCTCT.GTCTCGACGAAGCATTGCAGCGGCAGATCGA TGCOCAGCAACTCACGCGCTCCOCATOOAO AAATCCATQCPGCGGCATSTCCGTOCAAGCSCCAATT 6683.i YS F~:A NE 0F H LS G L DV E DQ 8R A~ QM M L:*:: R I50 'j Q0 0 E L -*1#t I::T .V:X.' N H:::8.

TCCACGCT.TTC$CTOIICAGCAAATTCGATO CAGCGTOACOCOATCGCGCCT CAGACGATCI ..OATOOOAICATCOCTCACO.ACACCSCTAOACAC60

ATOOCCOOCCTOTCACOAATCCIOCTOTCC TOCACOCTTOCAIOCCTOCTCOCCOOOCAO GCSCCCAOOCCTCCOTOOACOACCCCACCrCOOOCTOOCSOCOACAATCOOOTACOOOCO 692MAO LS RI L~~~~K RS CT LA L AQ A QAVPTIAIODNIL V RA

L RADQA 8 8DV L LTAC ROOP 0 HR ROE PMLVA ARAQA QQQAAAMTSVD~~~:~L:.A~jfZ: SOLA OALR N~~::::F:A.. RMEALLRLT QLQ

LV,... .....LVL..T ......:LA.8D.....WHKi:LL~ACIGTCAGCTOCIGCACCGACCOVIATGCCTOCCG ACTGTCGCAOCGGTGCGCATGACTCGGCGT CGCTOpCTGAACOOOCCAGCTGOCAACAC GACATCACCCAGGCCACGTCCGAGCTGCGGG 74083

S AL SQPKA AZ SA TR~ 0VRMPQDA OW LSOPWIT A P IT:Q:AAT 80 LR*E

AACTGGTCCAACGAG.CCTOOCOCCAC.!SGCCOGCACGGAATC TGCGAAGGGTCOTTCCAACAACOOCAA.GCCTGCTCACSCGCTTTCTOCOCGCAIATCOTOOCCG 75203

CACCTOCCCGCGT.CT.OCTQGOCAACG CTOTACGCGCAC.6CTGATCTTCGATOO.GCTO OOCACOCOCCTOOCT$GCCAIGA&pCTGCTOOC.OCOCZOCO76

CCOCCCTGGATGGACGCGAAC6QOCGCCAIT. CCCAGTTCIGCAGCGAOCCAGCAGCAACCA OTAOCOCOCCAOTACTGGGCCCCTCTCOOC OCOOCCOAATCCAGCCCOAGCCTOCACTCAOC 8003888 LW DP PEFiAGLift3 i AAA P P A A AAV AM::P:A A OP GH

OTOCGACCGACOACOOCCCCOCCOOO CCS_CCGACOOOITCTATCTp;CCCCAAACCC.ACCIACAOAATCAAC CCASATATTOCTOOOCATCCIOCA84TCOCCCCCOICACGCTCOGCOCCTOCOACA.pCMjCGCAAGCCCGCTGCACCOAOCACCOICCTCAACACCOp.TCAAACrCCCBOAAOATCGCATCAA56P~~*4YPLI P DQT H 0 8OHAEAYA::::::A-:::V OWER S 8k: LA Q EAW::K:

TCaIC.CGCTC-CT.GGCG4CQAE-COCATTTT.T.CGT.CAOCATCAACACGCAGGCTAGGCAGTCG TC GCATCAACTAC.CTTCGCAOACOCCGOAT CACGAA.GCCATO.CGT.GAAACTTCCAACOCC 86843

CAIAACCAITGOICAAQCIOCAACGGI FV GCGCCACCOCAACGOACI..GACAGCGGAICCjCOATCGCTTCGACTCl.AICOTA!R OCICCZ.OA86OCCOCOCGCGCTGACCCAOOCCCA~GCGCTACTG.ACGCGC5OCTTCa ACAAJACCIOAO QT*CAC TATGCCGC CCTCACGAGATGACAACCAGC 98083

AL H A L R R S~~~~~HHA GA 8 HR..QCTACTCACC..A... TATCGAC.A CAOGAACTOQ1QCOOO CCOCOIOATCOCOA...... ....T.. IOATI~ACAATC 92

ATGGCCGCCTGTCACGAATCOCTOAGCTGTCCGGAGCACGCTTCATGCTGCTCGAACCGGTCTO GCTCGCTCAAAACCTACCCGTCGACGACCACC ACCTCGTGCTGGCGCGCAATCGCGTACGGGC 69443M'AAGQLOS PAQLSC APEP LLW TN L LVA%TQDR8 D.: P RGDFD L MLAR V.R A

CTCCGCCGACC~AGGCGCGACGAGCGTT C ATGGCTCATCCOCCTGCCCGATCGCCTCCCGG CACCGGCGCCGTCAGCCGGCATCCGCGCCAAC GCCGACGCGCCCAGGGCTGTCCAGCCGTGCGC 9563

TTGGCAGC0CGCCCCATGACACGGAT.CACGCCOICTCGATTTGCATGGCOCA CAICGC AT GCGTACACGCGGCOOCATGAGGCTGOCGC ACTCAGTACCGGCTACACGCCAGCTICCAGOCCG 91683L AiAAfTR!A Q V 0 N OSODGIA A*4I A :A:: A8HA:W LIT V 'R LP0 Q PL 4' A

CTGTTTATCCCATGAT.GDOA-GT.CTGGCTA ACCTOOTTGC.ATCTOCATTCAACCATCTOTAACCGGA0 OCACTGAI.TGCTX'GQOICOOCAGGC.GTCCTCG 92803A V A~~~~~~~~~~ p ifif3 1 0~~~~~~~~~~~~~~~~~N RQ DL R PI H RH: P V AM L~ ~if:I

COMIC-T.GACAATCCIGCCAGGGVTCOTCCA..GMTCCCCCGATCCGCAGGCCGCAA. CGACGCTCTAATCCCGGGTAGCGCGGATCOCCTGCCCOACGATCC928 f V *4 QNG *4 5 PAFE~~~.CGGGCGCCACTHAETG-TCGCCATACGGCC72

TGTTTTCGCCAATGCCGAGCATGCGCATCG GCGTCCGCTGCGCTGGATGTTCGCCCTTGT CGCCGTGGCCCTGTCATGCCTGCTGGCAAC GGGGATATGGCGCAGCCGCGCCGCGCCGCC 10043M FA L A V .i C k L A I. I W R S R A A P P

FIG. 3. (Figure continues on the opposite page.)

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SCACSCCGCSACSCASACCST.CSGCCGGC SGGGCAAGCCCTGCC. SSGGAlATT CACGSTCCACCCACGCGAACCCW.AACGGSC GCCGCTTQCCGGACATGCCGGCQQr%TCCCGA

C.ACCTGCCGGCCC AAIG CGCCC GTACGACTACGiATGAACCGGCGCCCCG(~C G. CGACA(~CGCGGCGCTCAAGTCCGGCCCGSC~~ P V P P P ~~.::P R::P: VX::PA:R! A A L K S G P A

CATGATGGTCGCCACGGCCGCGCGrCCT.TGG ACAAAC-CGAGCGGGC.GGGCATGGCGGACGA CGGCGTGTCCGCGGATGCGGCCAC.CCT.CAT CGGCAGGAACGT.CAGzCCGC.GCAACCCGCTCN M V A T A A R ~~~~~~~~ S Q T ~~~ R A :~~~~$ M A .:.I G V S A D A A T I G R H V R S

GGR~~~~5~~Y R L L P~~G RT .. VSCAGGGGGCAACqCATAQCCGGCGCTGC1CT.G GTTT..AIGAGrGCATCSGCA.CGCCGGTGAC~ACA~TCCTGAC.GTTCTCGCGG G GGCAYR CL GLCPST AAAGCCGC6IACCGTCT~ACACAA.CGTCGCCr,rGCTA~CGA TCTGATGGGAGCACGG

CTCAC%~~~~~~AIASAI~~~~~~CI A~~~~~::~~~:::~~~~:::~~~AVV:r::: L T TDf ' Q-:: I SL GSCGATGCGGCAAGCCxCG.TACATCT.GGCGCGGCC.CCTG AC-GAr-CT.GCTGCGCAGCGAATGAACTTACCGCGCGTG.CAGCGGCTGCAACSAGAATCTACCT GCGCCAT.GAC.GGC.TTAACAA.C.CGATCCGGS~~ S VMR :~V APHi Y N V N T A STQ IN SY A S L~i: F M SG~i$I

~~ . .C~~~~~AG~~~TCGG~~~~CCT~~~ ~ ~ TCC ~~~~ ~~~CTCCATA.... ..........

GCSTCGACT.TCACAACCGAC CGTGG~TAACCR GCGCAGAAAGGAA.TTCGTGAACCGTACCAAGIGCAGCTCSGCAGAGACCTCAGCTGACGCGVD~~~~HQALHP;SRQDIA.i;~~~~~~~~5~ C V ~~~~ LWY ESRNGWNRQET L~~~~~~~~~ ACCTG T ACCCACSCC VTCDCACKAATG C G

TCGO.GATG

S S A L T AL~~~~~~~~~~~........~. A TA.R.F...R.....C.....

GCGiTCCGGGCCACTGCACAAGCGC.GCGCGCA CACCGATG~CGCGACCA-GCGTGGAACGT~GA CATTGACCGACCGCGGAAGCTGAATTCGCT AGGCGCCGCGCTAACCAACICGTCGATCAACV P GSYQ A R PDT P L R H V NR N L TA GTDP:R LA S LAQ A SK N N

CATJ CC..CJCAC ACGACCATGAGCGGGGAATCT.5T.CGC PCCQ AAGACTAGTGAGSCGGAATGAT.T TGCGGCTGGCCGGACAACCAGTCACTTACATCtTACS A E R T T&T~GTACG ICTCCATAAAI~CGGGG

E ~~A~:i F D F 4~~Z~~ Q AC A ~~ S :$::G.::i:::~~~ir AS A S H A L Q~ R D T QHAPCGAGTGCAGATA TCTATCC ..ASGGTSTGACG.C6AATGCT~GT.C. GCGCCAGCTCTGGGC ATGAGACGCAGGTGACCCCGGGACGGTGG.CCA-TCCCGGCGCTCATAC CGCT.GCCGGCCTGCACGCATATAGCGAT-TTC TGCAGTAATCCCTCATTCCCGACCGCGAACACACT

TAATCGGCGCAGCTCAGCTGGTTCGAGTCACCA.GC TCGSGCSTCAAACCGTCTCSACGCTTGTCTCCGAGCCGGCCCSATCCCTTGAAGCCGCCCTGGCAGTCCGGGGCGCCAGCGCSC GAACCCAGCCGACTTGCCCAGCTGACA GCGCAGCCCGCGCATTGCC.CTrGCAGCG:TC.CA .GCATACpCCGCTCGACGAAQ~CGTGGAAATGGT

GCSCCGCAG.TCATGTSCAGGTCGT.,-CCGCGGAACGCGAAGGCACGCTrTC.GCCTCrCATrCGTGCC CGC-CAGCACGGCGATCAGCSGCCACTAGTC-a.AACTGTAGGCGTTGCACGCTCGASCCTGCCAGGA

GCGCGGCGGCGCTCAGCCAGGCGGCCGAAGCATCTTCCCGGT GGAASCACCGGAGGCGTCCCAG CATGGGCCAGCTCCCATGCCCGAGCTGGCgGCC AGGCCTGGGCGCGGCGCATGGCCGTTGCGGCC

AGCTCAAT.TTCGCAGCA .CCATGCTGCCA CACGCAT.TGATCGCGGGGAG.A..CGCCCAGCGTAAAGCATCGTCrGAAACGGGGACGTGCCACC.AC T.TGCCAACTGCAT.CCG13025 A.CC.

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FIG. 3. Nucleotide sequence of the ptl operon. The DNA sequence of the ptl operon is shown along with the predicted amino acid sequenceof each orf. Nucleotides which may represent a transcriptional terminator are underlined. Nucleotides were numbered by using the system ofNicosia et al. (4) for the ptx operon. The sequence of nucleotides 3684-4692 is taken from that study (4). Amino acid sequences in orfs B, C,D, E, F, G, and H which are identical to sequences in VirB3 (8, 19), VirB4 (8, 19), VirB6 (19), VirB8 (20, 21), VirB9 (20, 21), VirBlO (19), andVirBll (8, 19), respectively, are shaded.

disrupt the end of the operon in the first copy and the secondcopy will lack the promoter sequences.Recombination of the BP338 chromosome with pPTL7,

pPTL8, pPTL5, pPTL6, and pPTL9 yields strains BPPTL7,BPPTL8, BPPTL5, BPPTL6, and BPPTL9, respectively,which retain the ability to secrete PT. In contrast, recombi-nation of the B. pertussis chromosome with pPTL2, pPTL1,and pPTL4 yields strains BPPTL2, BPPTL1, and BPPTL4,respectively, which are unable to secrete PT. The mutantsdefining the boundaries of the operons are shown in Fig. 1.Since mutations result only when the cloned region is internalto an operon, the cloned B. pertussis fragments present inpPTL2 and pPTL4 are internal to the operon whereas thecloned fragments in pPTL7 and pPTL9 must each extendacross a boundary of the operon. The cloned region ofpPTL7contains sequences that encode PT structural subunits andBPPTL7 retains the ability to secrete PT, suggesting that theoperon which controls secretion of PT (ptl operon) is distinctfrom the operon which encodes the PT structural subunits(ptx operon). The ptl operon may therefore span up to 9.5 kb.

In addition, two mutants, BPPTL3-1 and BPPTL3-2, wereconstructed by allelic exchange (double crossovers). The437-bp BamHI fragment in orf C was deleted and replacedwith a 4.0-kbp BamHI cassette containing the gentamicinresistance gene and the P-incompatibility origin-of-transferregion in both orientations. These mutants did not contain the437-bp BamHI fragment or plasmid sequences. Both mutantswere negative for PT secretion as measured either by PTactivity assays or by the colony blot technique.

Information obtained by analyzing BPM3171 allows us todeduce the direction of transcription of the ptl operon. SinceTnS lac makes transcriptional fusions, we could determinethe direction of transcription by mapping the orientation ofthe insertion in BPM3171. The BamHI site at the beginningof TnS lac was determined to be proximal to the PT operon

by Southern blot analysis, suggesting that the gene inacti-vated by the insertion in BPM3171 is transcribed in the samedirection as the PT operon. Previous work has demonstratedthat the gene into which TnS lac is inserted is vir-regulated(13).

Nucleotide Sequence Analysis of the ptl Operon. The regionencoding the ptl operon was sequenced (Fig. 3). The DNAsequence contains eight orfs. An inverted repeat beginning atnucleotide 12,134 may represent a transcriptional terminationsite. We propose that the first orf between the end of S3 andorf B is not a trans-acting gene but rather a cis-actingpromoter since it contains several transcriptional signalswhich have loose homology to the ptx -10, -35, andribosome binding sites, as well as a potential binding site forthe positive regulatory element Act (22). A search of theSwiss-Prot data base (Release 23.0) for proteins homologousto the protein predicted by the orfs was conducted. Theproteins predicted by orfs B, C, D, E, F, G, and H were foundto be homologous to the VirB3, VirB4, VirB6, VirB8, VirB9,VirB10, and VirB1l proteins, respectively, from the plantpathogen A. tumefaciens (Fig. 3). The VirB proteins havebeen implicated in the transfer of a piece of DNA, T-DNA,across the bacterial membranes and ultimately across plantcell membranes (7-9). The T-DNA integrates into the plantcell genome and codes for biosynthesis of plant growthhormones. Overexpression of these hormones leads to a lossof division control and thus to tumors (23).The ptl mutants are deficient in the secretion but not the

production of functional PT. Each of the PT subunits issynthesized with a secretion signal sequence (4, 5) whichshould target it to the periplasm where the subunits may thenassociate with membrane proteins. The ptl pathway appearsto be necessary to complete the secretion process. Thesequence homology with the protein complex required totransfer the T-DNA of A. tumefaciens is provocative. The

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T-DNA passes through the two bacterial membranes andthrough plant membranes. PT presumably only needs assis-tance through the outer membrane of B. pertussis, perhapsexplaining why only 7 of the 11 virB genes have a ptlhomologue; however, we cannot exclude the possibility thatgenes mapping outside ofthe ptl operon may also be required.Chemotherapy against bacterial infections requires selec-

tive toxicity against the microorganism. An antimicrobialagent targeted against only pathogenic bacteria would be animportant development. The ptl secretion pathway will allowus to screen for antimicrobial agents targeted against a subsetofbacteria, Gram-negative bacteria that secrete toxic factors.Since ptl mutants are reduced in virulence (24), it seems likelythat a therapeutic or vaccine directed against this pathwaywould also result in less severe infection.

This work was supported by Public Health Service Grant A123695from the National Institute ofAllergy and Infectious Diseases and bya Pew Foundation Fellowship to A.A.W.

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2974 Microbiology: Weiss et al.

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