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INFECTION AND IMMUNITY,0019-9567/00/$04.0010
Mar. 2000, p. 1164–1170 Vol. 68, No. 3
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Actinobacillus pleuropneumoniae Iron Transport: a Set of exbBDGenes Is Transcriptionally Linked to the tbpB Gene and
Required for Utilization of Transferrin-Bound IronWALAIPORN TONPITAK, SVENJA THIEDE, WINFRIED OSWALD, NINA BALTES,
AND GERALD-F. GERLACH*
Institut fur Mikrobiologie und Tierseuchen, Tierarztliche Hochschule Hannover, 30173 Hannover, Germany
Received 3 September 1999/Returned for modification 3 November 1999/Accepted 29 November 1999
Upon iron restriction, Actinobacillus pleuropneumoniae has been shown to express the transferrin-bindingproteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order toidentify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrin-binding protein genes in an A. pleuropneumoniae serotype 7 strain. We located immediately upstream of thetbpB gene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. Byraising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, andRT-PCR analysis revealed that the exbBD genes and the tbpB gene are transcribed on a single polycistronicmRNA. By constructing an isogenic and nonpolar exbBD mutant, we showed that the exbBD genes are requiredby A. pleuropneumoniae for utilization of transferrin-bound iron. Using PCR and Western blotting, we showedthat the genetic organization found in A. pleuropneumoniae serotype 7 is similar in all 12 A. pleuropneumoniaeserotype reference strains.
Iron is an essential factor for the growth of bacterial patho-gens, as it plays an irreplaceable role in most oxidation andreduction processes. In the environment, the vast majority ofiron occurs in the ferric state, which has an extremely lowsolubility. To overcome this problem, bacteria are able to takeup iron in a variety of chelated and therefore more solubleforms (18, 30, 36). This acquisition is mediated by an energy-coupled process involving essentially an outer membrane re-ceptor, the TonB protein, and inner membrane protein com-plexes, such as ExbBD, serving as energy couplers (1, 6, 11, 20,22).
Actinobacillus pleuropneumoniae is the etiologic agent ofporcine pleuropneumonia, a highly infectious disease of fat-tening pigs occurring worldwide (12). A. pleuropneumoniae,like other members of the families Neisseriaceae and Pasteu-rellaceae, has developed for iron assimilation a highly sophis-ticated system which allows the specific utilization of the trans-ferrin-bound iron of the respective hosts (8, 18, 33, 34). Theouter membrane proteins involved in transferrin binding havebeen designated TbpB and TbpA; they are highly immuno-genic antigens able to induce a protective immune response (2,23, 31, 38). In addition, it has been shown for Neisseria gonor-rhoeae that transferrin-binding proteins are required for exper-imental infection (9). The encoding genes (tbpB and tbpA)have been cloned and characterized for N. meningitidis (21),Haemophilus influenzae (17), A. pleuropneumoniae (14, 15, 16,38), Pasteurella haemolytica (26), and Moraxella catarrhalis(23). Both genes appear to be located in one operon withputative promoter and regulatory regions preceding the tbpBgene (4, 16, 17, 21). The acquisition of protein-bound iron hasbeen shown to be tonB dependent in N. meningitidis (35) andN. gonorrhoeae (5), and in both organisms, the tonB gene islocated immediately upstream of a set of exbBD genes. The
exbBD genes appear not to be linked to the tbpBA operon, andiron-regulated expression of the ExbBD proteins has not beenobserved in members of the families Pasteurellaceae and Neis-seriaceae. A functional requirement of both genes for the up-take of transferrin-bound iron has been shown for N. gonor-rhoeae (5), whereas for N. meningitidis the possibility offunctional complementation by Tol proteins is being discussed(35). For members of the family Pasteurellaceae, no experimen-tal evidence on the function of ExbBD proteins is available.
In the present communication, we show that in A. pleuro-pneumoniae a set of functional exbB and exbD genes is tran-scriptionally linked to the tbpB gene. By constructing an iso-genic and nonpolar A. pleuropneumoniae serotype 7 exbBDdeletion mutant, we show that the exbBD genes are essentialfor the utilization of transferrin-bound iron.
MATERIALS AND METHODS
Bacterial strains, plasmids, and primers. The strains, plasmids, and primersused in this work are listed in Table 1.
Preparation of antisera and of porcine transferrin. The serum raised againstthe A. pleuropneumoniae TbpB protein has been described previously (15). Theserum directed against the ExbB protein was raised in rabbits by an initialintracutaneous injection and two subcutaneous boost injections of 100 mg ofdissolved recombinant glutathione S-transferase (GST)–ExbB fusion protein inEmulsigen-Plus (MVP Inc., Ralston, Nebr.). Porcine transferrin was prepared byammonium sulfate precipitation and subsequent column chromatography usingDEAE–Sepharose CL-6B (Pharmacia, Freiburg, Germany) (24).
Media and growth conditions. Escherichia coli strains were cultured in Luria-Bertani (LB) medium supplemented with the appropriate antibiotics (ampicillin,100 mg/ml; kanamycin, 50 mg/ml); for cultivation of E. coli b2155 (DdapA),diaminopimelic acid (1 mM [Sigma Chemical Company, Deisenhofen, Germa-ny]) was added. A. pleuropneumoniae strains were cultured in PPLO medium(Difco GmbH, Augsburg, Germany) supplemented with NAD (10 mg/ml [E.Merck AG, Darmstadt, Germany]), L-glutamine (100 mg/ml [Serva, Heidelberg,Germany]), L-cysteine hydrochloride (260 mg/ml [Sigma]), L-cystine dihydrochlo-ride (10 mg/ml [Sigma]), dextrose (1 mg/ml), and Tween 80 (0.1%). Sucrosecounterselection was performed as described previously (28). For the selection ofA. pleuropneumoniae transconjugants, kanamycin (25 mg/ml) was added, and ironrestriction was induced by the addition of 2,2-dipyridyl (Sigma) to a final con-centration of 100 mM.
Plate bioassay testing of the utilization of transferrin-bound iron. Brain heartinfusion agar (Difco) was supplemented with 200 mM diethylenetriamine–penta-acetic acid calcium trisodium salt hydrate (Na3CaDTPA; Fluka Chemika and Bio-
* Corresponding author. Mailing address: Institut fur Mikrobiologieund Tierseuchen, Tierarztliche Hochschule Hannover, BischofsholerDamm 15, 30173 Hannover, Germany. Phone: 49-511-856 7598. Fax:49-511-856 7697. E-mail: [email protected].
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Chemika, Deisenhofen, Germany) and NAD (10 mg/ml). A. pleuropneumoniae over-night broth cultures (supplemented PPLO medium) were diluted 1:10 in NaCl (150mM), and 100 ml was evenly spread on a plate. Sterile filter paper disks were placedon the agar and loaded with 75 ml of porcine transferrin (500 mM) or ferric citrate
(500 mM ferric nitrate, 1 mM sodium citrate), both in HEPES-NaCl-bicarbonatebuffer (10 mM HEPES, 150 mM NaCl, 10 mM sodium bicarbonate [pH 7.4]);HEPES-NaCl-bicarbonate buffer alone was used as a negative control. Plates wereincubated overnight at 37°C in a 5% CO2 atmosphere.
FIG. 1. Organization of the A. pleuropneumoniae exbBD-tbpBA operon. The wide arrows indicate the relative locations, sizes, and directions of translation of therespective ORF. The zigzag line at the beginning of the tonB arrow indicates that the start of the ORF is not on the clones characterized to date. The gaps within thetbpB and tbpA arrows indicate that they are not drawn to scale. The vertical arrows marked with SD indicate putative Shine-Dalgarno sequences. The positions of restrictionendonuclease sites used in this study are indicated by dotted lines, with the position of the first base (GenBank accession no. Y17916) given in parentheses. The dotted lineunderneath the exbB gene indicates the DNA region missing in the isogenic A. pleuropneumoniae mutant AP76Dexb. The small horizontal arrows indicate the relativepositions of the primers used in the study; the number in parentheses indicates the position of the nucleotide at the 39 end. The solid line on the bottom gives the scalein base pairs; the numbers indicate the position of the first base of the start and stop codons of the ORF with respect to the BamHI site at position 1.
TABLE 1. Characteristics of bacterial strains, plasmids, and primers used in this study
Strain, plasmid,or primer Characteristics Reference
or source
StrainsE. coli DH5aF9 F9/endA1 hsdR17 (rK
2 mK1) supE44 thi-1 recA1 gyrA (Nalr) relA1 D(lacZYA-argF)U169 deoR
[f80dlacD(lacZ)M15]29
E. coli HB101 F2 D(gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1 D(mcrC-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA13 32E. coli b2155 thrB1004 pro thi strA hsdS lacZDM15 (F9 lacZDM15 lacIq traD36 proA1 proB1) DdapA::erm (Ermr) recA::RP4-2-tet
(Tcr)::Mu-km (Kmr) l pir10
A. pleuropneumoniaeAP76
A. pleuropneumoniae serotype 7 strain kindly provided by the Western College of Veterinary Medicine,Saskatoon, Canada
3
A. pleuropneumoniaeAP76Dexb
Unmarked ExbB-negative knockout mutant of A. pleuropneumoniae AP76 This work
PlasmidspBluescript SK E. coli cloning vector carrying an ampicillin resistance determinant Stratagenea
pGEX3x-5 E. coli cloning vector carrying an ampicillin resistance determinant and devised to construct GST fusion proteins PharmaciapGH432 E. coli cloning vector carrying an ampicillin resistance determinant and a tac promoter 14pJFF224 E. coli-A. pleuropneumoniae shuttle vector 13pBMK1 Transconjugation vector based on pBluescript SK with mobRP4, polycloning site, Kmr, and transcriptional
fusion of the omlA promoter with the sacB gene28
pTF401 A. pleuropneumoniae serotype 7 DNA from a l2001 library subcloned into pGH432 This workpEXB1 pGEX3x5 containing a BamHI-EcoRV fragment from a PCR product derived from pTF401 with primers BA9
and RE1 and expressing a GST-ExbB fusion proteinThis work
pEXB10 pBluescript SK carrying a BamHI-NsiI fragment from pTF401 This workpEXB10K pBMK1 carrying the pEXB10 insert This workpEXB10Dexb pEXB10 with a ScaI-PacI deletion abolishing the exbBD genes This workpEXB10DexbK pBMK1 carrying the pEXB10Dexb insert This workpFOKE2 pJFF224 carrying the BamHI fragment from pEXB10K This workpFOKE5 pJFF224 carrying the BamHI fragment from pEXB10K controlled by the vector-derived T4 promoter This work
PrimersBA6 AGT GGG ATC CTG AAA GTT ACT ATT GGA G; primer (internal BamHI site) upstream of the exbB gene This workBA7 CAA TGG ATC CAT TTT ATC TTC TTC AGG C; primer (internal BamHI site) upstream of the exbB gene This workBA9 CAA GGG ATC CTA CTC TCG GTA TCT TTC T; primer (internal BamHI site) on 59 end of the exbB gene This workBA23 CAA CGG ATC CTG AAA GTC CTT GAA TTA G; primer (internal BamHI site) upstream of the tbpB gene This workRE1 AAG TTT AAA ATG CAT ATT GC; primer overlapping the start codon of the tbpB gene This workTF6 AGA ACT CAC CGG AAC T; primer ending at position 212 of the tbpB gene This workOF1f CAA GAG ATC TTT CTT ATG CAT CTT ACT; primer overlapping the ATG codon of the rho gene S. Thiedeb
OF1r CAG AGT CCA TGG ATT TTT TAT GAA CGT TTC; primer overlapping the stop codon of the rho gene S. Thiedeb
a Stratagene Europe, Amsterdam, The Netherlands.b EMBL accession no. Y17915.
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Manipulation of DNA. DNA-modifying enzymes were purchased from NewEngland Biolabs (Bad Schwalbach, Germany) and used according to the manu-facturer’s instructions. Taq polymerase was purchased from GIBCO-BRL LifeTechnologies (Karlsruhe, Germany). DNA for PCR and Southern blotting aswell as plasmid DNA was prepared by standard protocols (32). Transformations,gel electrophoresis, PCR, and Southern blotting were done by standard proce-dures (32), and pulsed-field gel electrophoresis (PFGE) of A. pleuropneumoniaeDNA was performed as described previously (27).
Cloning of the A. pleuropneumoniae tbpB upstream region. A. pleuropneumo-niae serotype 7 DNA was partially digested, and a l2001 library was constructed.The library was screened using the tbpB gene (NsiI-KpnI-fragment) (15) as a probe,and a hybridizing clone was isolated. A BamHI-BglII-fragment was cloned intopGH432 cut with BamHI (resulting in plasmid pTF401), and a BamHI-EcoRVfragment was subcloned into M13mp18 and mp19 for DNA sequencing analysis.
Construction of recombinant plasmids. To construct plasmid pEXB1 express-ing a GST-ExbB fusion protein, a PCR fragment was obtained using primers
FIG. 2. Comparison of the ExbB (top) and ExbD (bottom) proteins of A. pleuropneumoniae (ACTPL), N. gonorrhoeae (neigo), and N. meningitidis (neime) as wellas the TolQ and TolR proteins of E. coli (ecoli). Boxes indicate identity; dashes indicate differences.
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BA9 and RE1 (Table 1 and Fig. 1). The fragment was restricted with BamHI andEcoRV and ligated into pGEX5x3 (Pharmacia) restricted with BamHI and SmaI.To construct plasmid pEXB10DexbK, used for transconjugation, several inter-mediate steps were required. First, the BamHI-NsiI fragment from pTF401 wascloned into pBluescript SK cut with BamHI and PstI, resulting in pEXB10.Plasmid pEXB10 was linearized with ScaI, completely digested with PacI, treatedwith E. coli DNA polymerase, and religated, resulting in pEXB10Dexb. Thedeletion obtained was characterized by nucleotide sequencing (Fig. 1). To con-struct pEXB10DexbK, the insert from pEXB10Dexb was removed with XbaI andSalI and ligated into the transconjugation vector pBMK1 (28) cut with XbaI andSalI. Plasmids pFOKE2 and pFOKE5 were constructed by ligating the XbaI-SalIfragment from pEXB10 into pBMK1. From here, a BamHI fragment was re-moved and ligated in either orientation into pJFF224 (13) with the vector-derived T4 promoter controlling exbBD transcription in pFOKE5.
Electroporation, transconjugation, and analysis of transconjugants and dele-tion mutants. Electroporation and transconjugation were performed as de-scribed previously (28). Kanamycin-resistant colonies were analyzed by colonyblotting using a 32P-dATP-labeled kanamycin resistance (Kmr) determinant.Counterselection to obtain unmarked deletion mutants was performed as previ-ously described (28), and colonies were tested by PCR analysis using primersBA7 and RE1 (Table 1 and Fig. 1). Colonies with the correct PCR profile wereconfirmed by Southern blot analysis using the BamHI-EcoRV fragment ofpEXB10 as a probe, by PFGE, by nucleotide sequence analysis, and by Westernblotting.
Preparation of protein aggregates, electrophoresis, and Western blotting.Protein aggregates were prepared as previously described (14). A. pleuropneu-moniae whole-cell lysates and protein aggregates were analyzed by discontinuoussodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blottingas described earlier (15).
RNA isolation and RT-PCR. RNA was prepared using the RNeasy MiniprepKit (Qiagen, Hilden, Germany). RNA was treated for 30 min with RNase-freeDNase (100 U; GIBCO-BRL) in the presence of RNase inhibitor (40 U;GIBCO-BRL), followed by phenol extraction and ethanol precipitation. RNAwas resuspended in double-distilled water and stored at 270°C. For the reversetranscriptase (RT) PCR, primer TF6 (Table 1 and Fig. 1) was annealed to 20 mgof A. pleuropneumoniae RNA in a volume of 10 ml; the RT reaction was carriedout in a total volume of 20 ml with Superscript II (GIBCO-BRL) according to themanufacturer’s instructions. The resulting cDNA was treated with DNase-freeRNase (2 U; Boehringer GmbH, Mannheim, Germany) for 20 min at 37°C,phenol extracted, ethanol precipitated, and resuspended in 10 ml of double-distilled water. The PCR was performed using the cDNA in a 1:1,000 dilution.
Nucleotide sequence accession number. The DNA sequence containing the A.pleuropneumoniae exbBD genes has been assigned GenBank accession no. Y17916.
RESULTS
Cloning and transcriptional organization of the A. pleuro-pneumoniae exbBD genes. In order to identify additional iron-regulated genes, we isolated a clone from a l2001 librarycontaining a 1,477-bp region upstream of the tbpB gene (Fig.1). Sequence analysis of this region revealed the presence oftwo open reading frames (ORF) encoding products of 222 and136 amino acids, the first one showing significant homologiesof approximately 43% with the N. gonorrhoeae and N. menin-gitidis ExbB proteins and 29% with the E. coli TolQ protein
FIG. 3. RT-PCR analysis of RNA from A. pleuropneumoniae grown understandard (lanes a) and iron-restricted (lanes b) conditions. Lanes C, A. pleuro-pneumoniae chromosomal DNA; lanes d, negative controls with no templateDNA. Primer TF6 was paired with primers BA23 (panel 1), BA9 (panel 2), andBA6 (panel 3). In order to ensure the absence of chromosomal DNA in the RNApreparations, a pair of primers specific for the A. pleuropneumoniae rho gene wasused as a control (panel 4).
FIG. 4. Analysis of A. pleuropneumoniae AP76 (lanes 1) and mutant strain AP76Dexb (lanes 2) and a negative control for PCR (lane N). Lanes M, size markers.(A) PCR using primers RE1 and BA7. (B) Southern blot analysis with EcoRV- and PacI-digested DNA and the exbB gene as a probe. (C) PFGE of ApaI-, AscI, andNotI-digested DNA. (D) Coomassie blue-stained gel (top) and Western blots developed with serum directed against the TbpB protein (middle) and the ExbB protein(bottom). Growth under iron-restricted conditions was achieved by the addition of dipyridyl (lanes 1D and 2D). The open arrowhead indicates the position of the TbpBprotein; the solid arrowhead indicates the position of the ExbB protein.
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and the second one showing similarities with the respectiveExbD and TolR proteins (Fig. 2). Based on the very high de-gree of homology to the more recently accessible genes ofNeisseria spp., the first gene was renamed from tolQ (27) toexbB, and the second one was designated exbD. The exbB ORFis preceded by a Shine-Dalgarno consensus sequence. The exbDORF directly follows the exbB ORF, with the methioninecodon of exbD overlapping the exbB stop codon, and the pu-tative exbD ORF ends only 27 bp upstream of the tbpB methi-onine codon (Fig. 1). The transcriptional organization of theexbBD and tbpB genes, as assessed by RT-PCR analysis, re-vealed that all three ORF are located on a single polycistronicmRNA (Fig. 3).
Functional analysis of the exbBD genes. In order to investi-gate translation of the putative exbBD genes in A. pleuropneu-moniae, an GST-ExbB fusion protein was constructed, andantibodies raised against the fusion protein were used to de-termine expression in A. pleuropneumoniae grown under reg-ular and iron-restricted conditions. A protein of the predictedsize was expressed under iron-restricted growth conditions only(Fig. 4). In order to investigate a possible role of the ExbBDproteins in the utilization of transferrin-bound iron, an iso-genic and nonpolar deletion mutant, designated AP76Dexb,was constructed (Fig. 1) and confirmed by PCR analysis, South-ern blotting, PFGE, and Western blotting (Fig. 4) as well as bynucleotide sequence analysis of a PCR product obtained fromAP76Dexb. Then, the A. pleuropneumoniae wild-type strain AP76,AP76Dexb, and AP76Dexb transformed with plasmid-carriedexbBD genes were grown on porcine transferrin as the sole sourceof iron. The deletion mutant was unable to utilize transferrin-bound iron but could be complemented in trans by exbBD-carrying recombinant plasmids pFOKE2 and pFOKE5 (Fig. 5).
Localization of the exbBD genes in A. pleuropneumoniae se-rotype reference strains. In order to investigate whether thelinkage of the exbBD and tbpB genes was unique to A. pleuro-pneumoniae serotype 7, DNAs of the 12 A. pleuropneumoniaeserotype reference strains were investigated by PCR usingexbB- and tbpB-specific primers BA7 and RE1; in addition,whole-cell lysates of these strains grown under iron-limitingconditions were tested in a Western blot for the presence ofExbB protein. The close linkage of the exbBD and tbpB geneswas present in all A. pleuropneumoniae serotype referencestrains, and all strains expressed the ExbB protein upon ironrestriction (Fig. 6).
DISCUSSION
In this report, we describe the cloning and molecular anal-ysis of a 1.5-kb region upstream of the tbpB gene of A. pleuro-pneumoniae serotype 7. We show that in A. pleuropneumoniae,two ORF whose products have more than 40% homology toneisserial ExbBD proteins (5, 35) are located immediately up-stream of the tbpBA genes. This localization of exbBD genes ina single operon with the tbpBA genes has not been observed forother bacterial species. Thus, in Neisseria spp., in H. influenzae,and in P. haemolytica, a putative promoter region is locatedimmediately in front of the tbpB gene (4, 17, 21); this arrange-ment had also been proposed for A. pleuropneumoniae (16). InH. influenzae and in P. haemolytica, the accessible genomicsequence data reveal no homology of DNA upstream of tbpBto exbBD sequences. In Neisseria spp., the exbBD genes havebeen cloned (5, 35), and no genetic linkage to the tbpBA geneshas been observed. These results further support previous ob-servations that despite significant homology between single
FIG. 5. ExbBD function is required for the utilization of transferrin-bound iron and can be complemented in trans. (A) Plate bioassay with strains cultured oniron-depleted brain heart infusion agar supplemented via paper disks with porcine transferrin (top), dilution buffer (middle), and ferric citrate (bottom). (B) Coomassieblue-stained gel (top) and Western blots developed with serum directed against the TbpB protein (middle) and the ExbB protein (bottom) of A. pleuropneumoniae AP76(lane 1), AP76Dexb (lane 2), and AP76Dexb transformed with pFOKE2 (lane 3) or pFOKE5 (lane 4) in the absence and presence of dipyridyl (D). The openarrowhead indicates the position of the TbpB protein; the solid arrowhead indicates the position of the ExbB protein.
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genes, genomic organization significantly differs among themembers of the family Pasteurellaceae (23, 27).
In order to investigate whether the exbBD ORF were trans-lated and coregulated with the tbpBA genes, a specific anti-serum was raised against a GST-ExbB fusion protein and usedin Western blot analyses. This investigation proved a clearlyincreased expression of ExbB protein under iron-restrictedconditions (Fig. 4D). Due to the close linkage and coordinatedexpression of exbBD and tbpBA genes, it was hypothesized thatthe ExbBD proteins of A. pleuropneumoniae might be requiredas energy couplers for the utilization of transferrin-bound iron,as has been described for N. gonorrhoeae (5). This hypothesiswas further supported by a recently reported linkage of a set ofexbBD genes to heme transport genes of Vibrio cholerae (25)and, in addition, by the observation that the exbBD genes areessential for the uptake of ferric iron in Xanthomonas campes-tris (37).
In order to prove this hypothesis, a newly developed methodof constructing nonpolar deletion mutants of A. pleuropneu-moniae (28) was used to interrupt the exbB ORF withoutinterfering with the expression of the transferrin-binding pro-teins (Fig. 4). Growth experiments with wild-type and isogenicmutant strains showed that the exbBD genes were in fact re-quired for the utilization of transferrin-bound iron in A. pleu-ropneumoniae. This result was further confirmed by restoring
the wild-type phenotype of the mutant in trans by transformingit with exbBD-expressing plasmids. This dependence on exbBshowed that the A. pleuropneumoniae uptake of transferrin-bound iron cannot be supplemented by a backup system, suchas tolQR. However, the unaffected growth in vitro as well as thecontinuous utilization of ferric citrate supports the presenceof a second locus involved in the uptake of ferric iron byA. pleuropneumoniae, as has been described previously (7).
Since a putative promoter sequence upstream of the tbpBgene of A. pleuropneumoniae had been reported previously(16), the transcriptional organization of the exbBD and tbpBgenes was investigated; these genes are located on a singlepolycistronic mRNA. In addition, the Western blot investiga-tion of A. pleuropneumoniae AP76Dexb transformed withpFOKE2 and pFOKE5 implied that the iron-regulated pro-moter of the operon is located upstream of the DNA fragmentinvestigated in this study. Thus, no increase of ExbB expressioncould be demonstrated in A. pleuropneumoniae pFOKE2 trans-formants containing the exbBD genes, although these genes arenot controlled by the vector-derived T4 promoter (Fig. 5).Further, this finding suggests that an incomplete ORF whoseproduct has a significant degree of homology to the carboxy-terminal end of the neisserial TonB protein and which is lo-cated immediately upstream of the exbB ORF might also belocated on the same transcript driven by a single iron-regulatedpromoter even further upstream.
An investigation of the A. pleuropneumoniae serotype refer-ence strains further revealed that this genomic organizationwas not unique to the A. pleuropneumoniae serotype 7 strainused in this study (Fig. 6). Thus, a PCR fragment of identicalsize was amplified from all A. pleuropneumoniae serotype ref-erence strains with primers located upstream of the exbB geneand at the beginning of the tbpB gene. In addition, the antibodydirected against the ExbB protein detected a protein of iden-tical size in all lysates. The variable intensities seen could havebeen due to the presence of various amounts of protein; alter-natively, slight antigenic differences might have been respon-sible.
In conclusion, the results described here imply that a non-polar deletion of exbBD might present a suitable way to atten-uate A. pleuropneumoniae strains for use as a live vaccine.Thus, this mutation does not interfere with routine in vitroculturing or with the expression of protective iron-regulatedproteins and, at the same time, completely blocks an ironuptake mechanism considered to be of prime importance dur-ing infection (19). In addition, the possibility of restoring theability of using transferrin-bound iron in trans might allow theuse of the exbBD genes as a nonantibiotic selection marker formaintaining recombinant plasmids in A. pleuropneumoniae.
ACKNOWLEDGMENTS
This work was supported by grant GE522/3-1 from the DeutscheForschungsgemeinschaft, Bonn, Germany. W.T. is a fellow of the Ma-hanakorn University of Technology, Bangkok, Thailand. S.T. is a fel-low of the Graduiertenkolleg Zell- und Molekularbiologie in der Tier-medizin of the Deutsche Forschungsgemeinschaft, Bonn, Germany.
REFERENCES
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2. Ala’Aldeen, D. A. 1996. Transferrin receptors of Neisseria meningitidis: prom-ising candidates for a broadly cross-protective vaccine. J. Med. Microbiol. 44:237–243.
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FIG. 6. ExbB expression and localization of the exbBD genes in A. pleuro-pneumoniae serotype reference strains 1 to 12 (lanes 1 to 12), AP76 (lane 13),and AP76Dexb (lane 14). (A) Coomassie blue-stained gel (top) and Western blotdeveloped with serum directed against the ExbB protein (bottom) of A. pleuro-pneumoniae strains grown under iron-restricted conditions. The arrowhead in-dicates the position of ExbB. (B) Ethidium bromide-stained gel of a PCR anal-ysis using primers RE1 and BA7.
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