Vol. 57, No. 12INFECTION AND IMMUNITY, Dec. 1989, p. 3907-39130019-9567/89/123907-07$02.00/0Copyright © 1989, American Society for Microbiology

Cloning and Expression of the Pasteurella multocida Toxin Gene,toxA, in Escherichia coli

SVEND K. PETERSEN'* AND NIELS T. FOGED2Genetic Engineering Group, The Technical University ofDenmark, Building 227, DK-2800 Lyngby,1 and

National Veterinary Laboratory, 27 Bulowsvej, DK-1503 Copenhagen V,2 Denmark

Received 21 April 1989/Accepted 18 July 1989

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida wasestablished in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using amonoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clonesshowed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids,pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 whichcould be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in thecytoplasm of E. coHl, was purified by affinity chromatography with immobilized monoclonal antibody and wasshown to react in a manner identical to that ofPMT in a quantitative structural test using a series of monoclonalantibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouselethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activitywas named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. Aprobe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophicrhinitis in pigs.

Pasteurella multocida can be isolated from the upperrespiratory tract of a variety of animals and from humans. Inpigs, progressive atrophic rhinitis is caused by infection withtoxigenic P. multocida and is characterized by deformationof the snout, sneezing, nasal discharge, epistaxis, and reduc-tion in the rate of body weight increase (23). Patho-anatom-ically, atrophy of the nasal turbinates is evident (22, 23).

P. multocida toxin (PMT), also called dermonecrotictoxin, osteolytic toxin, or turbinate atrophy toxin, has beenpurified by several groups (3, 4, 18, 24). It is one largepolypeptide, with an apparent molecular mass of 143 kilo-daltons (kDa) (4), located in the cytoplasm of P. multocida(16). Nakai and Kume showed that it can be dissociated, bypartial trypsin digestion, into three polypeptides intercon-nected by at least two disulfide bridges, suggesting a three-domain structure of the mature protein (15). Upon reductionof disulfide bridges, the functional toxin could be reconsti-tuted from these three polypeptides.The in vivo effects of purified PMT include turbinate

atrophy (in pigs and rats), death (guinea pigs, mice, rabbits,turkeys, and swine), atrophy of the spleen (mice), andenhancement of swine herpesvirus 1 replication (6, 7, 17,24), as well as dermonecrosis in guinea pigs (M. F. de Jong,H. L. Oie, and C. J. Tetenburg, Proc. Int. Pig Vet. Soc. 1980Congress, p. 211, 1980).

In vitro, PMT stimulates osteoclastic bone resorption,which is apparently mediated by a stimulation of preosteo-clast proliferation (9). Furthermore, cytopathic effects onembryonic bovine lung (EBL) cells and enhancement ofswine herpesvirus 1 replication have been demonstrated (7,25).The functions of PMT at the molecular level are still

unknown, however. To conduct molecular studies and todescribe the structure of the protein by using recombinantDNA techniques, we have cloned the PMT gene in Esche-richia coli.

* Corresponding author.

In this paper, we report the isolation and preliminarycharacterization of the PMT gene from P. multocida subsp.multocida; we have named the gene toxA. We also demon-strate the production in and isolation from E. coli of thecorresponding protein. This recombinant protein was struc-turally and functionally compared to native PMT by enzyme-linked immunosorbent assay (ELISA) and by tests for cyto-pathic, dermonecrotic, and lethal effects.The present report also describes the use of the cloned

DNA fragment in hybridization experiments to demonstratethe localization and distribution of the toxA gene amongreference strains of P. multocida.

MATERIALS AND METHODSBacterial strains, media, and growth conditions. E. coli

MT102 [araD139 A(ara-leu)7697 AlacX74 galU galK rpsL r-m+], constructed and kindly supplied by Mogens TrierHansen, NOVO Industri A/S, Bagsvaerd, Denmark, wasused throughout this work. Toxigenic type D P. multocidasubsp. multocida NCTC 12178 was used as a DNA donorstrain for the gene library. P. multocida subsp. multocidareference strains ATCC 12945 (type A, nontoxigenic),NCTC 12177 (type A, toxigenic), ATCC 7707 (type D,nontoxigenic), and NCTC 12178 (type D, toxigenic) wereused in the Southern blot analysis. All strains were culti-vated at 37°C. E. coli strains were cultivated in L broth (14).P. multocida strains were grown in Trypticase soy broth(BBL Microbiology Systems, Cockeysville, Md.).

General procedures. Plasmid DNA was isolated by themethod of Bimboim and Doly (2). Restriction endonucleasesand T4 DNA ligase were purchased from New EnglandBioLabs, Inc., Beverly, Mass., and used as specified by thesupplier. E. coli MT102 was transformed by the method ofHanahan (8).DNA extraction. To isolate chromosomal DNA from P.

multocida, cells from a 250-ml stationary culture were sus-pended in 10 ml of 50 mM Tris hydrochloride (pH 8.0)-100mM EDTA and incubated with 25 mg of lysozyme for 20 min

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at 37°C. Two milliliters of 10% (wt/vol) sodium dodecylsulfate (SDS) was added, and the mixture was put on ice for10 min. Following the addition of 15 ml of phenol saturatedwith TE buffer (10 mM Tris hydrochloride [pH 8.0], 1 mMEDTA), the lysate was heated to 65°C, gently mixed, andcooled on ice. After centrifugation, extraction with ether,and precipitation with ethanol, the pellet was suspended inTE buffer. The DNA was further purified by banding in aCsCl-density gradient (13).To isolate large-plasmid DNA from P. multocida, a mod-

ification of the SDS lysis method (13) was used in which 50mg of lysozyme was added.

Cloning procedure. Chromosomal DNA prepared as de-scribed above was digested partially by incubation for dif-ferent times with the restriction endonuclease Sau3A. Thereactions were stopped by the addition of 1/20 volume of0.25 M EDTA. A fraction containing 4- to 22-kilobase-pair(kb) fragments was identified and further fractionated on an8-ml sucrose gradient (40 to 10%) (13).DNA fragments prepared as described above were in-

serted into BclI-restricted pUN121 (20). The resulting plas-mids were transformed into competent E. coli MT102 cells.Because insertion of a DNA fragment into the BclI site ofpUN121 renders the vector tet+, positive selection forclones with plasmid inserts was achieved by the addition oftetracycline (10 ,ug/ml) to the medium.Immunoblotting procedures. In the colony blot procedure

(28), bacterial colonies were transferred to nitrocellulosefilters, which were subsequently incubated as follows: (i) 15min in 50 mM Tris (pH 9.6)-150 mM NaCl-0.05% Tween 20(washing buffer)-2 ,ug of DNase I per ml; (ii) two 10-minincubations in washing buffer without DNase I; (iii) 30 min inwashing buffer plus 3% gelatin; (iv) two 10-min incubationsin washing buffer plus 1% Triton X-100; (v) 60 min in a10-fold dilution (in washing buffer) of a previously describedhybridoma supernatant, P3F51 (4); (vi) three 5-min incuba-tions in washing buffer; (vii) 60 min in washing buffer plushorseradish peroxidase-linked anti-mouse immunoglobulinwhole antibodies from Amersham (NA.931), diluted 1:1,000;(viii) three 5-min incubations in washing buffer; (ix) 1 min in10 mM Na2HPO4-10 mM citric acid (pH 5.0) (C/P buffer);and (x) approximately 5 min in a staining solution mixedimmediately prior to use consisting of 80 mg of dioctylsodi-umsulfosuccinate (DONS), 24 mg of 3,3',5,5'-tetramethyl-benzidine, 10 ml of ethanol, 30 ml of C/P buffer, and 20 ,ul ofH202. The enzyme reaction was terminated by incubation in100 mg of DONS in 12.5 ml of ethanol plus 37.5 ml of H2O.

In the Western blotting (immunoblotting) procedure, 1-mlstationary-phase cultures were pelleted and suspended in 0.5M Tris hydrochloride (pH 6.8)-3% SDS-15% glycerol-5%mercaptoethanol-bromphenol blue. The samples wereboiled for 5 min prior to loading on a gel. Proteins wereseparated by SDS-polyacrylamide gel electrophoresis(PAGE) (11); the separating gel consisted of 7% (wt/vol)acrylamide (acrylamide/bisacrylamide ratio of 40:1) in 0.4 MTris (pH 6.8)-0.1% SDS-0.05% glycerol. Subsequent trans-fer to nitrocellulose filters was performed in a semi-dryelectroblotter as described by Kyhse-Andersen (10). Furtherhandling of the filters was as described above for the colonyblot procedure.DNA hybridization procedure. DNA was transferred to

Hybond-N filters (Amersham International) by the methodof Southern (27). Probe DNA was extracted from agarose gelby the Gene-Clean procedure (BIO101) and labeled by nicktranslation with [a-32P]dATP (13).

Prehybridization and hybridization were performed in 6x

SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate,pH 7)-0.5% SDS-5 x Denhardt solution for 2 h and over-night, respectively, at 65°C. After hybridization, the filterswere washed at 25°C in 2x SSC-0.5% SDS twice for 15 minand in 0.2x SSC-0.5% SDS twice for 1 h at 65°C andsubjected to autoradiography overnight.DNA quantification procedure. To determine the relative

amount of the toxA-carrying DNA region in DNA preparedby the chromosomal DNA preparation procedure and by thelarge-plasmid DNA preparation procedure, samples contain-ing comparable amounts of chromosomal DNA were sub-jected to Southern blot analysis. Photographic negatives ofthe ethidium bromide-stained agarose gel and autoradio-grams of the Southern blot were scanned in an LKB 2202ULTRASCAN laser densitometer (LKB), and the relativeamount of toxA-carrying DNA isolated by these procedureswas calculated as the relative scan values of the respectivefilms (P-32 scan value/ethidium bromide scan value).

Cell fractionation and toxin purification procedures. Frac-tionation of stationary-phase cultures of E. coli was achievedby a modified spheroplast method (12, 19). One milliliter ofculture was pelleted by centrifugation, the medium fractionwas removed, and the pellet was suspended in 100 ,ul of 30mM Tris hydrochloride buffer (pH 8.0)-20% (wt/vol) su-crose-10 mM EDTA-70 pug of lysozyme per ml. This sus-pension was subjected to cold osmotic shock for 30 min at0C, followed by centrifugation (10 min, 7,000 x g), yieldinga periplasmic and a cell-associated material fraction. Thefractions thus obtained were analyzed by SDS-PAGE, fol-lowed by Coomassie blue staining, by Western blotting asdescribed above using anti-PMT monoclonal antibody(MAb) P3F51, in the PMT ELISA as described below (PMTassays), and by enzyme assay for P-lactamase using amodified nitrocefin assay (1) to verify the validity of thefractionation procedure.

In the toxin purification procedure, cells harvested from a1-liter stationary-phase culture of a positive E. coli clonewere suspended in 10 ml of H20 and sonicated several timesfor 0.5 min each time at 0°C by using a Branson sonifier 250(Branson Sonic Power Company, Danbury, Conn.). Thesonic extract was diluted to 50 ml in 0.1 M Tris hydrochlo-ride (pH 7.8) containing 0.5 M NaCl before application to theaffinity column, which was prepared by immobilizing theanti-PMT MAb P3F51 as previously described (4). Afterrepeated washings of the affinity column, recombinant PMT(rPMT) was eluted with 0.1 M glycine hydrochloride (pH2.8) as described earlier for the affinity purification of PMTfrom extracts of toxigenic P. multocida (4). All fractionswere immediately neutralized with 1 M K2HPO4.PMT assays. Quantification of rPMT was done in a sand-

wich ELISA procedure as previously described for PMT (4),by using (as the capture antibody) anti-PMT MAb P3F51 and(for detection) the biotinylated anti-PMT MAb P3F37. Thisprocedure is based on the same technique that is explainedbelow for the study of epitopes on rPMT and PMT. Resultsof quantification by this method were compared to resultsobtained in a modified Coomassie brilliant blue dye-bindingmicroassay previously used for the determinations of proteinconcentrations (26) and dye-binding ability of PMT com-pared with that of bovine serum albumin (4).

Epitopes on rPMT and PMT were compared in sandwichELISAs based on 10 anti-PMT MAbs (4) purified fromhybridoma supernatants on protein A-agarose columns.These MAbs have previously been shown to react withdifferent epitopes on PMT (4). The sandwich ELISAs wereinitiated by coating wells of microdilution plates (96-well

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VOL.57,1989CLONING OF THE PASTEURELLA MULTOCIDA TOXIN GENE 3909

Immuno Plate II; Nunc, Roskilde, Denmark) with, per well,50 ,u1 of a 2-,ug/ml solution of purified anti-PMT MAb in 0.05M carbonate buffer, pH 9.6, for 16 h at 4°C and for 1 h at20°C. The microdilution plates were emptied, and each wellwas incubated for 1 h with 200 ,ul of phosphate-bufferedsaline (PBS) containing 0.05% Tween 20 and 1% bovineserum albumin. After two washings with 200 ,ul of PBScontaining 0.05% Tween 20 (PBS-T), each well was incu-bated with 50 ,ul of a 100-ng/ml mixture of either rPMT orPMT in PBS-T-1% bovine serum albumin for 1 h at 20°C.After three PBS-T washings, each well was incubated with50 p,l of biotin-conjugated MAb (0.5 ,ug/ml) for 1 h at 20°C,followed by another three PBS-T washings and incubationwith 50 ,ul of a 0.5-,ug/ml solution of horseradish peroxidase-conjugated avidin (Kem-En-Tec) per well for 45 min at 20°C.Finally, 50 ,ul of an o-phenylenediamine-H202 substratesolution was added per well. The reaction was stopped with2 M H2SO4 after 5 min, and the A492 was determined in aKontron SLT-210 photometer (SLT) (reference wavelength,620 nm). Two determinations were performed for bothantigens in all 100 combinations of the 10 catching MAbs andthe same biotinylated, detecting MAbs. Combinations ofMAbs resulting in absorbances below 0.3 were consideredcompetitive. For the noncompetitive combinations, the re-sults were described as the mean of dual determinations ofthe absorbance obtained for rPMT relative to the mean ofdual determinations of the absorbance for PMT.

Sonic extracts of E. coli and P. multocida, prepared asdescribed above, were tested for cytopathic effect in theembryonic bovine lung (EBL) cell test (25). A row of fivefolddilutions was prepared for each sonic extract, and 30 p.l ofeach sample was applied to 1.8 x 104 EBL cells in 120 ,ul ofculture medium. The mixture was incubated for 3 days at37°C before fixation and staining. Samples which resulted inmonolayers of EBL cells morphologically discernible fromthe epithelium-like swirling patterns of a negative controlculture were scored as cytopathic. The cytopathic effects foraffinity-purified rPMT and PMT in the EBL cell test weredetermined in the same way. The minimal cytopathic dose(MCD) for the samples was calculated as the smallestamount of rPMT or PMT, determined by the quantitativePMT ELISA, causing a cytopathic effect.

Neutralization of the cytopathic effect of recombinant E.coli sonic extract by anti-PMT MAbs was compared toneutralization of pure PMT as described previously (4).Samples (30 ,ul) containing approximately 1 ,ug of MAb andvarious amounts of sonic extract or PMT were incubated for15 min at 20°C before application to EBL cells. The resultswere recorded as the number of MCDs neutralized by eachMAb and as the ratio of the number of neutralized MCDs ofthe sonic extract to that of pure PMT for each MAb.The dermonecrotic effects of rPMT and PMT were deter-

mined by injecting 200 ,ul of dilutions of the ELISA-quan-tified samples intradermally into guinea pigs. Samples result-ing in a dermal lesion with a diameter of 10 mm or more at 48h after intradermal injection were scored as dermonecrotic.All results were based on at least two determinations.The lethal effects of rPMT and PMT were determined by

injecting 200-,u samples intraperitoneally into BALB/c mice(21). Samples that killed mice less than 5 days after intra-peritoneal injection were scored as lethal. All results werebased on at least two determinations.

RESULTSCloning and characterization of the P. multocida toxA gene.

A genomic library of P. multocida NCTC 12178 DNA was

A B C D E F

_200

.92.5

.70

FIG. 1. Expression of rPMT in E. coli clones detected by West-ern blotting using an anti-PMT MAb. Lanes: A, P. multocida NCTC12178; B and C, E. coli MT102 carrying plasmid pUN121 containinga randomly chosen P. multocida DNA insert and containing noinsert, respectively; D and E, E. coli MT102 carrying pSPE312 andpSPE308, respectively; F, molecular weight marker.

constructed by cloning 9- to 16-kb fragments and 15- to 22-kbfragments, generated by partial Sau3A restriction, into theplasmid vector pUN121. The library consisted of 3,332clones with an average insert size of 14 to 15 kb.The entire library was screened for toxin-producing clones

by the colony blot method. Colonies were transferred tonitrocellulose, and positive clones were detected by usingMAb P3F51 directed against native PMT. Two positiveclones, SPE308 and SPE312, were identified.To prove that these clones produced PMT, the protein

profiles and biological activities of extracts of SPE308 andSPE312 were examined. Sonic extracts of both clones weretested by Western blotting and in the EBL cell test. TheWestern blot shown in Fig. 1 demonstrates the production inthe recombinant clones of an approximately 143-kDa pro-tein, which is specifically recognized by MAb P3F51. Signif-icant degradation of this protein in E. coli is also obviousfrom Fig. 1 (lanes D and E), as is a similar degradationpattern ofPMT in P. multocida (lane A). Furthermore, sonicextracts of both clones expressing this protein were shownto cause morphological changes in EBL cells identical tochanges caused by sonic extracts of toxigenic strains of P.multocida (Fig. 2) (data for SPE308 not shown). As wasobserved for pure PMT, the cytopathic effect of the sonicextract of E. coli clone SPE312 could be neutralized byincubation with anti-PMT MAbs. Between 5 and 125 MCDsof the sonic extract could be neutralized by various anti-PMT MAbs, whereas between 3 and 125 MCDs of the purePMT were neutralized. The overall mean + standard devia-tion for the 10 calculated values of the relative numbers ofneutralized MCDs of E. coli SPE312 sonic extract comparedwith the value for PMT was 98 + 32%. A non-PMT-relatedMAb used as a control did not neutralize the effects of thetwo cytopathic preparations. This demonstrated the cloningof the PMT gene, in SPE308 and SPE312. We have namedthis gene toxA.The P. multocida DNA inserts of pSPE308 and pSPE312

were found to be approximately 17.8 and 9.8 kb, respec-tively. By restriction enzyme analysis, a 6.5-kb overlapbetween the inserts was established (Fig. 3). The clonedregion therefore contained the entire toxA gene, with a

coding region size of approximately 3.7 kb, as estimatedfrom the apparent molecular weight of PMT.

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In order to confirm the conservation of the structure of thecloned toxA region, the restriction map of the pSPE312insert was compared with that of the toxA region of P.multocida. The three P. multocida DNA-encompassing frag-ments indicated in Fig. 3 (black bars) were radioactivelylabeled and used as DNA probes in Southern blot analysis.lbThis analysis of restricted chromosomal donor DNA and

$* pSPE312 DNA is shown in Fig. 4.When the EcoRI probes were used, these data were in

accordance with the restriction map in Fig. 3 (in Fig. 4B,_ @ only data obtained by using the 4.5-kb EcoRI probe are

shown). Note that the 200-bp overlap between this probe andthe 4.4-kb HindIII fragment results in only a vague hybrid-

*e) ization signal (lanes 2 and 3*). The 6.4-kb HindIII fragment}} in lane 2 and the 3.9-kb EcoRV fragment in lane 5 are vector

DNA-encompassing fragments. Moreover, this EcoRV frag-4,A ment comigrates with the 3.9-kb EcoRV fragment of P.

multocida DNA (lane 6). Longer exposure times of lanes 5' and 6 show the 0.7- and 0.2-kb EcoRV fragments, and the

9.0-kb fragment in lane 6 furthermore indicates the positionof the leftmost EcoRV fragment in Fig. 3.When the HindIll probe was used (Fig. 4A), four HindlIl

fragments, as well as four EcoRV fragments, were recog-P8 nized, which cannot be accounted for by the restriction map

in Fig. 3. Also, the rightmost EcoRV restriction site (to theright of the EcoRV restriction site at position 16300 in Fig. 3)could not be mapped by this analysis, since the probestrongly hybridized to both a 5.0- and a 9.7-kb fragment ofP.

* 3 multocida DNA (lane 6). These bands in lanes 3 and 6 areI caused by repetition of the region around the BglII restric-

tion site indicated in Fig. 3 elsewhere in the P. multocidagenome (Lars Ole Andresen, personal communication).Since these repeated sequences are not recognized by theEcoRI probes, they are located outside the overlappingregion between pSPE312 and pSPE308 and therefore cannotresult from a repetition of the toxA gene. Note that the3.9-kb EcoRV fragment recognized by the EcoRI probe inFig. 4B is recognized here as well (lanes 5 and 6).To determine the basis for the difference in toxin produc-

tion among strains of P. multocida, we performed a South-ern blot analysis of DNA from type A and type D reference

bW|- strains showing toxin production (PMT+ strains) or lack oftoxin production (PMT- strains) (5). The probe used wasradioactively labeled pSPE308 DNA. As shown in Fig. 5, the17.8 kb of P. multocida DNA contained in this probe had no

' homology to DNA from PMT- strains of P. multocida,whereas positive signals were obtained for DNA from PMT+strains. Lack of PMT production, therefore, appears to bethe result of a lack of the region spanning the toxA gene,

, rather than of the existence of a mutation, minor deletion, ornegative regulatory function in PMT- strains.To test the possibility of a chromosomal location of the

toxA gene, DNA from P. multocida NCTC 12178 was alsoisolated by the SDS lysis method for isolation of large

_st ~plasmids described in reference 13. This DNA was used inthe Southern blot of Fig. 5 (lane 1*) along with chromosomalDNA from the same strain (lane 1). Amounts of each samplecorresponding to similar amounts of chromosomal DNA

it - FIG. 2. Determination of toxic activities of sonic extracts ofisq_ recombinant E. coli clones. E. coli MT102 carrying pUN121 had no

cytopathic effect on EBL cells when diluted 1/25 in PBS (a). Sonic'%\ extracts of E. coli SPE312 (b) and toxigenic P. multocida (NCTCC,C 12178) (c) diluted 1/3,125 showed significant and identical cytopathic

effects. Magnification, x80.

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CLONING OF THE PASTEURELLA MULTOCIDA TOXIN GENE 3911

E EH R A CI EH HHpRPE R H B

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pSPE308

pSPE312HindIII EcoRV

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FIG. 3. Restriction enzyme cleavage map of the cloned 20.3-kb toxA region (top row). The middle and bottom rows indicate the extensionof the DNA contained in pSPE308 and pSPE312, respectively. M Vector DNA; _, DNA fragments used as probes in the Southern blotanalysis of Fig. 4. Restriction sites: A, AvrII; B, BglII; C, ClaI; E, EcoRV; H, Hindlll; Hp, HpaI; P, PvuII; and R, EcoRI.

were applied to each lane of the agarose gel (Fig. 5, rightpanel), and the relative gene dosage of toxA, described asP-32 scan value/ethidium bromide scan value, was 1.0 (lane1), 1.4 (lane 1*), and 0.7 (lane 2). These results suggest achromosomal location for toxA.Recombinant toxin purification and characterization. The

localization of the toxin in the E. coli cells was determinedby analysis of the cell-associated-material, periplasmic, andgrowth medium fractions of a stationary-phase culture ofSPE312. By Coomassie blue staining of polyacrylamide gels,Western blotting, and quantitative ELISA, the toxin wasshown to reside in the cell-associated-material fraction. Thevalidity of the fractionation procedure was confirmed byassay for P-lactamase (Fig. 6).

Quantitative ELISA results with sonic extracts of SPE308and SPE312 indicated a production of 1 to 3 ,ug of rPMT perml per optical density unit at 450 nm. A diluted bacterialsonic extract containing approximately 82 ,ug of rPMT wasapplied to a 1-ml affinity column to which was coupledapproximately 5 mg of anti-PMT MAb P3F51. No rPMTcould be detected in the effluent from the column. Uponelution, approximately 75 ,ug of rPMT was obtained in thetwo main fractions, each of which was 1.4 ml. This corre-sponds to a recovery of 91% of the applied rPMT.

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FIG. 4. Confirmation of the structure of the cloned toxA-carryingP. multocida DNA of pSPE312 by Southern blot analysis. Theradiolabeled probes used were a 4.4-kb HindIII fragment (A), a4.5-kb EcoRI fragment (B), and a 2.9-kb EcoRI fragment (data notshown) shown in Fig. 3. Lanes: 1 and 4, vector DNA; 2 and 5,pSPE312 DNA; 3 and 6, P. multocida NCTC 12178 chromosomalDNA. Lanes 1 to 3, HindIII-restricted DNA; lanes 4 to 6, EcoRV-restricted DNA. Lanes marked with an asterisk were exposedlonger.

To compare the protein structures of the toxins producedin E. coli and in P. multocida, affinity-purified rPMT andPMT were tested for recognition by a variety of anti-PMTMAbs. Affinity-purified rPMT and PMT had very similarreaction patterns in the structural ELISA test based on 10combinations of 10 different anti-PMT MAbs and 100 ng ofaffinity-purified antigen per ml. For PMT, 25 combinations ofMAbs resulted in competitive reactions, defined by absorb-ance values (A492) below 0.3. The same 25 pairs showedcompetitive reactions when the antigen was rPMT at 100ng/ml. The remaining 75 noncompetitive pairs resulted inA492 values above 0.3 when either PMT or rPMT was used.The overall mean + standard deviation for the 75 calculatedvalues of the relative absorbances of rPMT compared withthat of PMT was 112 + 8%. Only minor differences from theoverall mean were observed for the mean values for the 10capture MAbs and the 10 biotinylated detector MAbs.PMT and rPMT reacted very similarly when tested for

cytopathic effect on EBL cells, for dermonecrotic activity inguinea pigs, and for lethality in mice (Table 1). Furthermore,

123 4

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FIG. 5. Demonstration of the lack of the toxA-carrying DNAregion in PMT- strains of P. multocida types A and D anddemonstration of the location of the toxA gene in the chromosome oftoxigenic P. multocida NCTC 12178. Left panel, Southern blotanalysis of DNA from four reference strains of P. multocida; rightpanel, ethidium bromide-stained agarose gel before Southern blot-ting. Lanes 1 to 4, chromosomal DNA; lane 1*, DNA prepared bythe procedure for isolation of large plasmids. Lane 1, NCTC 12178;lane 1*, NCTC 12178; lane 2, NCTC 12177; lane 3, ATCC 7707; lane4, ATCC 12945. The probe used was radiolabeled pSPE308. LanesMW, Molecular weight marker.

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3912 PETERSEN AND FOGED

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FIG. 6. Localization of rPMT in E. coli. Western blot analysisand SDS-PAGE of cellular fractions. The gel was stained withCoomassie blue. MW, Molecular weight marker. Lanes C, P, andM, Cell-associated-material, periplasmic, and growth medium frac-tion of SPE312 stationary-phase culture, respectively. Also shownare values describing the amounts (per milliliter) of rPMT (inmicrograms per milliliter) and P-lactamase (in units per milliliter) ineach fraction, determined by quantitative ELISA and enzyme assay(1), respectively.

the abilities of PMT and rPMT to bind Coomassie brilliantblue were equal and approximately 2.5 times weaker thanthe dye-binding ability of bovine serum albumin (Table 1),confirming accordance between the results of the two quan-titation methods used, on the basis of antibody recognitionand Coomassie blue binding, respectively.

DISCUSSIONWe have isolated the PMT gene from P. multocida and

demonstrated its expression in E. coli. The gene, which wasnamed toxA, is the first gene from P. multocida to be cloned.

TABLE 1. Cytopathic, dermonecrotic, lethal, and dye-bindingeffects of PMT and rPMT

MCD Minimal Minimal DyeSample dermonecrotic lethal binding

(pg) dose (ng) dose (ng) (%)a

PMT 20-40 15-45 25-50 4045rPMT 20-40 35 30 35-45

a Concentration of bovine serum albumin relative to the concentration ofsample resulting in equal color formation in the Coomassie brilliant blue dyebinding microassay.

The toxA gene was cloned in both orientations in pUN121(pSPE308 and pSPE312, respectively). The only knownpromoter of this plasmid directing transcription into theinsert region is PRM from phage lambda (Fig. 3). Since therecombinant gene in both orientations directed the expres-sion of an intact functional toxin, this suggests that P.multocida transcription- and translation-directing nucleotidesequences seem to be functional in E. coli. Moreover,comparable amounts of toxin were produced in SPE308 andSPE312.A number of degradation intermediates were observed

when PMT was produced in E. coli. Interestingly, theseintermediates, although more prominent in sonic extracts ofE. coli, were also detectable in sonic extracts of P. multo-cida (Fig. 1), as well as in affinity-purified preparations ofPMT, after prolonged incubation (data not shown). A 70-kDaband was especially prominent in SDS-PAGE gels of allthree PMT preparations. This spontaneous degradationproduct was also observed by Chanter et al. (3), whohypothesized that a subunit structure of PMT was impli-cated. Nakai and Kume (15) also suggested involvement of asubunit structure of PMT on the basis of their trypsindigests. In this context, it would be of interest to determinewhether the 70-kDa polypeptide observed in this study isidentical to one of the partial trypsin digestion products of 74and 67 kDa described by Nakai and Kume.A preliminary study of the PMT content of different

cellular compartments (data not shown) indicated that 5 to10% of the PMT was contained in the membrane fraction, asjudged by visual inspection of Coomassie blue-stained SDS-PAGE gels. However, the protein seen was not detected inthe quantitative ELISA. The reason for this discrepancy isnot clear, but shielding of the relevant PMT epitopes of amembrane fraction ofPMT molecules by membrane materialcould potentially result in failure of MAbs to detect them.We are presently investigating the possible existence of asmall subfraction of membrane-bound PMT molecules. Nev-ertheless, the localization ofrPMT in E. coli determined hereis well in keeping with the cellular localization ofPMT in P.multocida (16).No significant differences between purified PMT and

rPMT could be detected in the variety of structural andfunctional tests used. The rPMT produced in E. coli, there-fore, provides a basis for the study of the structure andfunction of PMT. Also, a modification of rPMT by DNAtechniques could lead to the production of a modified atoxicprotein with potential as a component of a vaccine againstprogressive atrophic rhinitis in pigs.

Strains of P. multocida subsp. multocida are either toxi-genic (PMT+) or nontoxigenic (PMT-) (5). By using fourreference strains, we have shown that the toxA gene, as wellas a large part of the neighboring DNA, is found only intoxigenic strains of P. multocida. However, our resultssuggest that the gene is not plasmid borne, since similaramounts of toxA were isolated by plasmid DNA and chro-mosomal DNA preparation techniques. This conclusion hasrecently been confirmed by Lars Ole Andresen (personalcommunication), who isolated recombinant plasmids con-taining DNA partially homologous with the P. multocidaDNA of pSPE308 and pSPE312. These plasmids, whichwere isolated by "chromosome walking" by using the genelibrary described here, showed extensive homology to chro-mosomal DNA of both PMT+ and PMT- strains. Theconsiderable extent of a toxA-carrying, PMT+-specific chro-mosomal DNA region could indicate a gene localization on a

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CLONING OF THE PASTEURELLA MULTOCIDA TOXIN GENE 3913

bacteriophage or transposon, in analogy with certain othertoxin genes.Two newly developed methods are available for the rapid

detection of PMT+ strains. These are a PMT ELISA basedon monoclonal antibodies (5) and the EBL cell overlaymethod based on the specific toxic effect of PMT on thesecells (25). A toxA-encompassing DNA fragment with theshown specificity for DNA from PMT+ strains could be analternative tool for diagnosis and surveillance of progressiveatrophic rhinitis.

ACKNOWLEDGMENTS

We are grateful to Annemette Jorgensen, Henriette Lou, andHelle Petersen for excellent technical assistance; to Per LinaJorgensen for help in portions of this project; to Mette Misciattellifor typing; and to Francesco Blasi, Poul Andersson, Ole F. Rasmus-sen, and Claus Christiansen for critical reading of the manuscript.

This work was supported financially by The Joint Committee forAgricultural Research and Experiments and by Northern Drugs andChemicals Ltd.

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