Transfer of a pertussis toxin expression locus to isogenicbvg-positive andbvg-negative strains ofBordetella bronchisepticausing anin vivotechnique

Microbial Pathogenesis 1996; 20: 263–273

Transfer of a pertussis toxin expression locus to isogenic bvg-positive and bvg-negative strains of Bordetella bronchiseptica using an in vivo technique Adam M. Smith and Mark J. Walker∗ Department of Biological Sciences, University of Wollongong, NSW, Australia. (Received September 12, 1995; accepted in revised form December 4, 1995)

Smith, A. M. (Department of Biological Sciences, University of Wollongong, NSW, Australia) and H. J. Walker. Transfer of a pertussis toxin expression locus to isogenic bvg-positive and bvg-negative strains of Bordetella bronchiseptica using an in vivo technique. Microbial Pathogenesis 1996; 20: 263–273. Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease. Increasing public concern over the safety of current whole-cell vaccines has led to decreased immunization rates and a subsequent increase in the incidence of the disease. The preparation of safer vaccines is at present concentrated on the production of detoxified virulence factors such as pertussis toxin (PT) for inclusion in acellular vaccine preparations. A permanently avirulent Bordetella bronchiseptica strain was previously engineered to constitutively produce PT.1 An in vivo cloning technique, based on the principles of conjugal mating and chromosome transfer was employed to transfer the PT expression locus of this strain to virulent and avirulent strains of B. bronchiseptica. This transfer was confirmed by Southern hybridization. An analysis of PT secretion in isogenic virulent and avirulent strains of B. bronchiseptica revealed that the PT produced was cell-associated, and not secreted to the growth medium. This evidence suggests that B. bronchiseptica does not possess functional PT secretion (ptl) genes. Therefore, to achieve a PT expression and secretion system suitable for vaccine purposes in Bordetella bronchiseptica, functional ptl genes of B. pertussis are also required.  1996 Academic Press Limited

Key words: pertussis toxin; Bordetella bronchiseptica; Bordetella pertussis.

Introduction Whooping cough, the severe childhood respiratory disease, is caused by the obligate human pathogen, Bordetella pertussis.2 The traditional whole-cell vaccine used for the prevention of the disease is currently under scrutiny due to reported side effects. The subsequent decline in vaccine usage has inevitably led to a worldwide increase in the occurrence of whooping cough.3,4 The preparation of safer vaccines is at present concentrated on the production of purified recombinant antigens for inclusion in acellular vaccine preparations. The present study is focused on pertussis toxin, a five subunit, hexameric protein arranged in the classic A protomer, B oligomer structure of bacterial toxins.5 The A protomer or S1 subunit is the enzymatically active moiety which causes damage to surrounding epithelial and immune system cells by breaking ∗ Author to whom correspondence should be addressed: Dr. M. J. Walker, Department of Biological Sciences, University of Wollongong, NSW 2522, Australia. 0882–4010/96/050263+11 $18.00/0

 1996 Academic Press Limited

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down G-protein interactions.6 The remaining subunits, S2, S3, S4 and S5 make up the B oligomer in the ratio 1:1:2:1. This region of the protein facilitates attachment of the toxin to host cells.5 The genes encoding the pertussis toxin subunits are clustered together in an operon. DNA sequence analysis has revealed that each subunit is translated separately with an amino-terminal signal sequence which is cleaved during transport to the periplasm where the holotoxin is then assembled and secreted.7,8 The incompatibility of PT gene expression signals with respect to Escherichia coli make the production of PT in this organism difficult to achieve.9,10 There are also problems associated with obtaining high yields of purified PT from virulent B. pertussis. These include the slow growth rate, fastidious nutritional requirements, and the inherent low PT production levels of the bacterium. There is also the risk that the co-purification of low levels of other virulence regulated toxins may occur in vaccine preparations. Of the Bordetella species, only B. pertussis produces PT. A cryptic PT operon is present in B. parapertussis and B. bronchiseptica, however, base pair mutations in the promoter region leave this operon transcriptionally silent.11 Lee and associates12 have used recombinant plasmids to express PT in Bordetella parapertussis and Bordetella bronchiseptica. Yields were comparable to those of B. pertussis and although the majority of the PT produced was localized in the periplasm, a significant amount of PT was detected in the culture supernatant. B. parapertussis and B. bronchiseptica have also been used to produce genetically detoxified pertussis toxin.13 Overexpression and secretion of genetically detoxified pertussis toxin has been achieved in B. pertussis14 by using allelic exchange techniques to introduce multiple copies of a genetically altered PT operon into the chromosome. Expression of PT was achieved in a permanently avirulent strain of B. bronchiseptica using mini-transposons to clone a promoterless PT operon in front of a strong constitutive B. bronchiseptica promoter (designated PBp) in strain ATCC 10580::TnfusPT1.1 Unfortunately, the toxin was not secreted to the growth medium, making ATCC 10580::TnfusPT1 less than suitable as a vaccine expression system. Likely explanations for this lack of secretion may include (i) the secretion of PT in B. bronchiseptica, as it is in B. pertussis,15 is regulated by the bvg-locus resulting in only virulent strains being capable of PT secretion, (ii) the strain of B. bronchiseptica used does not possess the entire genetic machinery to secrete PT, or (iii) that it does possess PT secretion genes, but the genes are cryptic or the gene products serve an alternate function. If the necessary PT secretion genes are intact, the coordinate regulation by the bvg-locus may result in only virulent phase B. bronchiseptica being able to secrete the toxin. Weiss et al.16 have cloned and sequenced the entire B. pertussis ptl (pertussis toxin liberation) operon, which is required for PT secretion in B. pertussis. Eight open reading frames (ORFs) were detected and insertional mutagenesis was used to determine that ORFs B through H of the operon were necessary for the secretion of PT. Fusion proteins expressed in E. coli have been used to raise polyclonal antibodies against the predicted PtlA, B, C, E, F, G and H proteins.17 Immunoreactive bands in western blots of B. pertussis whole cell extracts were only detected using polyclonal antibodies against PtlE, F and G. The same antibodies raised against PtlE and F failed to detect immunoreactive bands in B. bronchiseptica whole cell extracts.17 Kotob et al.18 have shown that replacement of the B. bronchiseptica PT promoter with the B. pertussis PT promoter results in detectable expression of PtlF in B. bronchiseptica in western blots. These results suggest that B. bronchiseptica either contains cryptic ptl genes or that the gene products of ptlE and ptlF are expressed at a lower level in

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Fig. 1. Genetic analysis of pertussis toxin secretion genes. (A) Diagrammatic representation of the ptl operon and amplification of the ptl gene fragment (ptlH) which is essential for PT secretion. Specific oligonucleotide primers (arrowheads) were designed for the amplification of the 512 bp fragment (shaded box) using PCR technology. (B) Southern hybridization of EcoRI digested chromosomal DNA of three B. bronchiseptica strains, ATCC 10580 (lane 1), 5377 (lane 2), BB7866 (lane 3) and B. pertussis Tohama I (lane 4) using the 0.5 kb ptlH fragment as a probe (lane 5). Molecular weight markers (kb) are indicated.

B. bronchiseptica. In this work, we report that B. bronchiseptica possesses sequences homologous to the 3′ end of the ptl operon (ptlH gene), as determined by Southern hybridization analysis. We also describe the development of a system specifically designed for the transfer of the pertussis toxin expression locus from ATCC 10580::TnfusPT1 to other B. bronchiseptica strains. The PT expression locus was transferred to isogenic bvg-positive and bvg-negative strains of B. bronchiseptica and the PT secretion levels of these strains was monitored. It was found that neither virulent nor avirulent B. bronchiseptica exhibited the ability to secrete PT, confirming that the B. bronchiseptica ptl genes are non-functional.

Results Genetic analysis of B. bronchiseptica for the presence of PT secretion genes With the failure of B. bronchiseptica ATCC 10580::TnfusPT1 to secrete PT in an earlier study,1 and the recent characterization of the ptl genes of B. pertussis which are required for pertussis toxin secretion16 it was important to determine whether B. bronchiseptica possesses an intact ptl operon. Thus, B. bronchiseptica chromosomal DNA was probed for the presence of the 3′ ptlH gene. The EcoRI restricted DNA of three strains of B. bronchiseptica and one strain of B. pertussis underwent Southern hybridization analysis (Fig. 1). The PCR amplified 0.5 kb ptlH

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fragment was radiolabelled and used as a probe. It was found that all strains tested exhibited equal binding, suggesting that the genome of B. bronchiseptica contains sequences homologous to the ptlH gene of B. pertussis.

Transfer of the PT expression locus to B. bronchiseptica strains The cloning of the PT expression locus of ATCC 10580::TnfusPT1, encompassing the PT operon, the B. bronchiseptica promoter (PBp) and the kanamycin resistance gene was first attempted using cosmid cloning techniques. A number of kanamycin resistant clones were obtained, however, whilst all contained the kanamycin resistance gene, none of these contained sequences homologous to the recombinant PT operon as found in Southern hybridization analyses (results not shown). Possibly the genetic locus containing the PT operon controlled by PBp is either unstable in E. coli or produces gene products which are lethal to E. coli. With the failure of cosmid cloning to isolate the PT expression locus for transfer to B. bronchiseptica strains, an in vivo technique was developed. Escherichia coli Q358 (pR751::Tn813)19 harbours the trimethoprim resistant, broad host range plasmid pR751,20 containing the cointegrate-forming transposon, Tn813.21 Transposon Tn813, is a transposase positive (tnpA+), resolvase negative mutant (tnpR−) derivative of the mercury resistant transposon Tn21.21 Plasmid pR751 possesses transfer genes (tra) and an origin of transfer (oriT), allowing for the conjugal transfer of the plasmid DNA between donor and recipient strains. The entire plasmid is then integrated into the chromosome via the transposase of Tn813. Subsequent to the transposition of pR751::Tn813 into the genome the plasmid is capable of promoting chromosome transfer. Bordetella bronchiseptica ATCC 10580::TnfusPT1 was mated with Escherichia coli Q358 pR751::Tn813 (Fig. 2). Of the resultant kanamycin and trimethoprim resistant transconjugants, one was selected and designated ATCC 10580:: TnfusPT1 (pR751::Tn813). The frequency of transfer of pR751::Tn813 was 3.2×10−1 transconjugants per recipient cell. The plasmid is now capable of cointegrative transposition into the bacterial chromosome, mediated by the transposase enzyme of Tn813. The transfer of the PT expression locus from ATCC 10580::TnfusPT1 into recipient strains was achieved using integrated pR751::Tn813 to promote chromosome transfer. Virulent and avirulent B. bronchiseptica strains (BB7865 Rifr Nalr, BB7866 Rifr Nalr, 5376 Rifr Nalr and 5377 Rifr Nalr) were separately mated with ATCC 10580::TnfusPT1 (pR751:: Tn813) and the resultant growth was plated onto SS agar with kanamycin and rifampicin selection (Fig. 2). As would be expected for this mechanistically difficult chromosome transfer event, transconjugants arose at a frequency in the order of 10−6 to 10−8 kanamycin resistant transconjugants per recipient cell (Table 1). Due to the promiscuous nature of the conjugative plasmid, the transfer of trimethoprim resistance occurred at a much higher rate, in the order of 10−1 transconjugants per recipient cell (Table 1). To avoid the possibility of selecting a rifampicin resistant spontaneous mutant of the donor strain, Kanr Rifr single colonies were replica plated onto SS agar with nalidixic acid selection. The Kanr Rifr Nalr transconjugants identified were also found to be trimethoprim resistant (results not shown). Selected Kanr Rifr Nalr transconjugants were designated, for example, 5377 Rifr Nalr::TnfusPT1. Virulent derivatives containing the mobilized expression locus retained the virulent phenotype as determined by haemolytic activity on BG agar plates (results not shown).

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Q358 pR751::Tn813

ATCC 10580::TnfusPT1 CONJUGATION

pR751:: Tn813

KANAMYCIN and TRIMETHOPRIM SELECTION

ATCC 10580::TnfusPT1 (pR751::Tn813)

pR751:: Tn813

TRANSPOSITION ATCC 10580::TnfusPTI (pR751::Tn813)

BB7866 Rifr Nalr CONJUGATION

KANAMYCIN and RIFAMPICIN SELECTION BB7866 Rif r Nal r::TnfusPTI

Fig. 2. In vivo transfer of the PT expression locus of Bordetella bronchiseptica ATCC 10580::TnfusPT1. The Escherichia coli strain, Q358 (pR751::Tn813) contains the conjugative plasmid pR571, harbouring the cointegrate forming transposon Tn813 (shaded box). When this strain is mated with ATCC 10580:: TnfusPT1, the tra genes of pR751 induce the transfer of the plasmid to the B. bronchiseptica strain. The transposase enzyme encoded by Tn813 catalyses cointegrate formation of the plasmid with the chromosome of ATCC 10580::TnfusPT1. This forms a permanent genomic cointegrate due to the defective resolvase gene of Tn813. This newly formed strain, designated ATCC 10580::TnfusPT1 (pR751::Tn813) is then conjugally mated with the B. bronchiseptica strains (eg. BB7866 Rifr Nalr). The tra genes of pR751, once within the chromosome, can promote chomosome transfer. The PT expression locus is thus transferred to the recipient strain.

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Table 1 Frequency of transfer data of the PT expression locus (Kanr) and the conjugative plasmid pR751::Tn813 (Tpr) from ATCC 10580::TnfusPT1 (pR751:: Tn813) to each of the B. bronchiseptica recipient strains Recipient strain

Frequency of transfer of Kanr (transconjugants/recipient)

Frequency of transfer of Tpr (transconjugants/recipient)

5376 Rifr Nalr 5377 Rifr Nalr BB7865 Rifr Nalr BB7866 Rifr Nalr

1.04×10−6 7.92×10−8 1.52×10−6 8.25×10−9

1.60×10−2 8.71×10−1 2.50×10−2 6.60×10−1

A EcoRI

EcoRI

EcoRI

PT Subunits S1

S2

S4

S5

Kanr

S3

PBb

B 1

2

3

4

5

6

7

8

9

10

11

12

23.1 PBb + TnfusPT

9.4 6.6

PT operon 4.4

Kanr

2.3 2.0

Fig. 3. Diagrammatic representation of the PT expression locus (A). PT subunit genes S1 to S5 (open boxes), the kanamycin resistance gene (Kanr; open box) and the constitutive B. bronchiseptica promoter (PBp; filled arrow) are indicated. Only EcoRI restriction enzyme sites are shown. (B) Southern hybridization analysis of B. bronchiseptica strains before and after the transfer of the PT expression locus. TnfusPT (incorporating the PT operon and kanamycin resistance gene) harboured on plasmid pMW 127 (1) was used to probe the EcoRI digested genomic DNA of ATCC 10580 (lane 1), ATCC 10580::TnfusPT1 (lane 2), 5377 Rifr Nalr (lane 3), 5377 Rifr Nalr::TnfusPT1 (lane 4), BB7866 Rifr Nalr (lane 5), BB7866 Rifr Nalr::TnfusPT1 (lane 6), 5376 Rifr Nalr (lane 7), 5376 Rifr Nalr::TnfusPT1 (lane 8), BB7865 Rifr Nalr (lane 9), and BB7865 Rifr Nalr::TnfusPT1 (lane 10). Lanes 11 and 12 contain EcoRI digested B. pertussis Tohama I and pMW127 DNA respectively. Sizes of markers (kilobases) are indicated.

Southern hybridization analysis of B. bronchiseptica strains containing the PT expression locus To confirm the success of the in vivo cloning of the PT expression locus, Southern hybridization analysis was performed. The chromosomal DNA of recombinant strains and their respective parental strains was digested with EcoRI restriction endonuclease and subjected to agarose gel electrophoresis. The subsequent restriction profile was then Southern transferred, and probed with 32P labelled pMW127 DNA.1 This plasmid contains the recombinant mini-transposon TnfusPT (Fig. 3A). The results presented in Fig. 3B suggest that the PT expression locus,

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OD560 nm

2

1

0

A

B

C

D

E

F

G

H

I

J

K

Fig. 4. Analysis of pertussis toxin secretion by recombinant B. bronchiseptica strains. PT levels in cell-associated (Ε) and supernatant fractions (Φ) of B. bronchiseptica strains ATCC 10580 (A), ATCC 10580::TnfusPT1 (B), 5377 Rifr Nalr (C), 5377 Rifr Nalr::TnfusPT1 (D), 5376 Rifr Nalr (E), 5376 Rifr Nalr:: TnfusPT1 (F), BB7866 Rifr Nalr (G), BB7866 Rifr Nalr::TnfusPT1 (H), BB7865 Rifr Nalr (I) and BB7865 Rifr Nalr::TnfusPT1 (J) were compared to equivalent fractions of B. pertussis Tohama I (K) using a PTspecific ELISA assay. In contrast to B. pertussis Tohama I which secreted a large proportion of the PT it produced to the growth medium, the PT produced by the recombinant B. bronchiseptica strains was cell-associated.

(PBb+TnfusPT, 10.5 kb band) was transferred from ATCC 10580::TnfusPT1 to each of the two virulent and two avirulent strains of B. bronchiseptica. The 2.2 kb kanamycin resistance gene, and the cryptic PT operon which is present in all B. bronchiseptica strains11, were also detected. The plasmid pMW127 contains an additional reactive band (Fig. 3B, Lane 12) which represents the suicide plasmid into which the promoterless PT operon was cloned (pUT::miniTn5/Km)22 to form pMW127.

Analysis of B. bronchiseptica strains for PT secretion by ELISA Supernatant and cellular fractions of Bordetella cultures were assayed for the presence of PT using ELISA techniques. Virulent and avirulent B. bronchiseptica strains and their PT producing derivatives were tested, as was B. pertussis Tohama I (Fig. 4). Expression of PT by all B. bronchiseptica strains containing the PT expression locus was cell-associated, whereas the B. pertussis strain was capable of secreting large quantities of PT to the culture supernatant. All strains of B. bronchiseptica containing the PT expression locus expressed similar amounts of cell-associated PT. As expected, the parental strains of B. bronchiseptica which did not contain the expression locus did not produce PT.

Discussion Expression and secretion of PT in organisms other than virulent phase B. pertussis has been attempted by a number of laboratories. Lee et al.12 introduced plasmids containing a B. pertussis PT operon into B. bronchiseptica and found that the

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levels of PT expression were similar to those of B. pertussis. When the location of expressed PT was examined in B. bronchiseptica, significant amounts of PT were detected in the culture medium. The pertussis toxin expression locus, including the constitituve promoter, PBb of B. bronchiseptica ATCC 10580::TnfusPT1, was transferred into virulent and avirulent strains of B. bronchiseptica, using an in vivo cloning technique. The tra genes of the plasmid pR751, when integrated into a chromosome, have the ability to induce conjugal transfer to a recipient bacterium. Transposon Tn813 contains a defective resolvase gene, therefore once it transposes into the chromosome it cannot resolve the cointegrate formed. E. coli Q358 (pR751::Tn813) was conjugally mated with the original avirulent B. bronchiseptica PT expressing strain, ATCC 10580::TnfusPT1 to permit pR751::Tn813 to transpose into the chromosome of ATCC 10580::TnfusPT1, thereby facilitating chromosome transfer in subsequent conjugal mating experiments. Using this technique, the PT expression locus was successfully cloned in vivo into derivatives of virulent and avirulent B. bronchiseptica strains. The levels of PT expression in strains of B. bronchiseptica containing the PT expression locus were similar and all of the B. bronchiseptica strains examined in this study failed to secrete PT into the culture medium, whereas B. pertussis Tohama I was found to secrete significant amounts of PT. It has been established that B. bronchiseptica contains genes homologous to B. pertussis ptlA, ptlB and part of ptlC.11 Significantly, none of the base pair differences detected in the sequenced region of the B. bronchiseptica ptl operon result in the introduction of a stop codon thereby truncating the putative open reading frames. Our Southern blot analysis demonstrated that B. bronchiseptica also contains sequences homologous to the 3′ end of the ptl operon (ptlH gene). Two recent studies18,23 have provided evidence that the PT and ptl operons of B. pertussis are both transcribed from the PT promoter. Replacement of the B. bronchiseptica PT promoter with the B. pertussis PT promoter results in expression in B. bronchiseptica of PtlF at levels similar to those found in B. pertussis.18 It seems that B. bronchiseptica contains essentially intact genes encoding at least PtlA, B, C, F, and H proteins. Mutations in the promoter region prevent transcription of the B. bronchiseptica ptl operon taking place. Our results confirm those of Kotob et al.18 and strongly suggest that the ptl genes of B. bronchiseptica are cryptic. Introduction of the necessary B. pertussis ptl genes under the control of appropriate promoters, into the strains used in this study, may result in the production and secretion of PT in B. bronchiseptica. The cloning technique developed in this study may also be utilized to obtain expression of pertussis toxin in both virulent and avirulent strains of other members of the genus Bordetella so that a wider comparison of PT expression and secretion systems may be conducted. Furthermore, this in vivo chromosome transfer system could be used in other transposon-tagged loci of Bordetella spp. to analyse transposon induced gene mutations across species and genera.

Materials and methods Bacterial strains, plasmids, and media. All bacterial strains and plasmids used in this study are listed in Table 2. Escherichia coli was grown on Z agar27 and Bordetella spp. were grown using Bordet-Gengou agar or SS medium.28 Liquid cultures of Bordetella strains assayed for PT secretion were grown in SS medium supplemented with 2% heptakis2,6-dimethyl-b-cyclodextrin. Antibiotic concentrations used were as follows: cephalexin,

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Table 2 Bacterial strains and plasmids used in this study Bacterial Strain/plasmid Strains E. coli Q358 (pR751::Tn813) B. pertussis Tohama I 5376 ATCC 10580 BB7865 BB7866 ATCC 10580::TnfusPT1 5377 ATCC 10580::TnfusPT1 (pR751::Tn813) 5377 Rifr Nalr BB7866 Rifr Nalr 5377 Rifr Nalr::TnfusPT1 5376 Rifr Nalr::TnfusPT1 BB7866 Rifr Nalr::TnfusPT1 BB7865 Rifr::TnfusPT1 Plasmids pR751::Tn813 pMW132 pMW127 pHC79

Description∗

Reference/source

Tpr, Hgr serotype 2 fim+, fha+ B. bronchiseptica fim+, fha+ fim−, fha− hly+ hly− fim−. fha−, PT+ hly− hly−, PT+

(19) J. Pemberton (24) R. Brownlie

hly− hly− hly−, PT+ hly+, PT+ hly−, PT+ hly+, PT+

tra+, tnpA+, tnpR− bvg+, Kanr TnfusPT, Kanr Apr, Tcr

R. Brownlie M. Ho¨fle (25) R. Rappuoli (25) R. Rappuoli (1) This study This study This This This This This This

study study study study study study

(19, 20, 21) (1) (1) (26)

∗Tpr, trimethoprim resistant; Hgr, mercury resistant; Apr, ampicillin resistant; Tcr, tetracycline resistant; Kanr, kanamycin resistant; fim, fimbriae production; fha, filamentous haemagglutinin production; hly, haemolytic activity; TnfusPT, recombinant PT expression locus; bvg, Bordetella virulence gene locus; tra, conjugative transfer genes; tnpA, transposase; and tnpR, resolvase.

50 lg/ml; kanamycin, 50 lg/ml; nalidixic acid, 50 lg/ml; rifampicin, 100 lg/ml and trimethoprim, 50 lg/ml. All bacterial strains were grown at 37°C. Liquid cultures were aerated by shaking in a BioLine Shaking Incubator.

Isolation of spontaneous mutants. Initially, a non-haemolytic derivative of the virulent B. bronchiseptica strain 5376 was detected after continued subculture on Bordet Gengou agar. This strain, designated B. bronchiseptica 5377, was found to be avirulent as the addition to this bacterium of the Bordetella virulence gene containing plasmid pMW1321 converted the phenotype of this strain from non-haemolytic to haemolytic (results not shown). To aid in the selection of transconjugants in subsequent mating experiments, all four B. bronchiseptica recipient strains (5376, 5377, BB7865 and BB7866) were subjected to passaging on plates containing various antibiotics until spontaneous resistant mutant colonies arose. A rifampicin/nalidixic acid double resistance mutant of each strain was isolated. These strains, designated BB7865 Rifr Nalr, BB7866 Rifr Nalr, 5376 Rifr Nalr and 5377 Rifr Nalr were used as recipients in subsequent conjugation experiments. The donor strain, B. bronchiseptica ATCC 10580::TnfusPT1 was found to be sensitive to both of these antibiotics. Genetic manipulations. Chromosomal DNA was obtained using the phenol extraction method of Priefer et al.29 Plasmids and the cosmid pHC7926 were isolated using a method modified from Naumovski and Friedberg.30 Restriction endonucleases and T4 DNA ligase (Boehringer Mannheim) were used essentially as described by Sambrook et al.31 The polymerase chain reaction was carried out using Pfu DNA polymerase (Stratagene) in accordance with manufacturers instructions in a PCR thermal reactor (Hybaid). The primers PTSEC5′ (5′-GGCCAGACCGGTTCGGGCAAGACCACA-3′) and PTSEC3′ (5′TCATGGCGCCGGGAGGCCATCCCGGTA-3′) were used to amplify a region of the ptl operon (ORF H) from B. pertussis Tohama I genomic DNA. The primer sequences were taken from the sequence published by Weiss et al.16 Electro-elution of the ptl fragment for radiolabelling and subsequent Southern hybridization was carried out by first electrophoresing the fragment on a 0.7% agarose

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gel, then excising the fragment and electro-eluting from the agarose onto the membrane of a centricon 100 microconcentrator tube (Amicon). The centricon tube was then used to purify and concentrate the DNA down to a total volume of 50 ll in aqueous solution. Southern hybridization analysis was performed on chromosomal DNA from Bordetella strains digested with EcoRI restriction enzyme then electrophoresed on a 0.7% agarose gel. The DNA was transferred to a positively charged nylon membrane (Boehringer Mannheim) using a method modified from Southern.32 The samples were bound to the membrane using a UV Stratalinker 1800 (Stratagene), and the blot was blocked with a citrate buffered solution of 5X Denhardt’s solution31 and salmon sperm DNA to 200 lg/ml. Plasmid or electro-eluted fragment DNA was labelled (Gibco BRL, nick translation kit) with a-32P-dATP (Bresatec) and used to probe the membranes. Hybridization was carried out at 65°C in a Hybaid mini oven MkII.

Conjugation and chromosome transfer. Strains to be mated were grown on selective medium and resuspended together in 0.5 ml of 0.7% NaCl. The suspension was plated out onto agar plates at 37°C for 24 h allowing conjugation to take place. The resultant growth was 10-fold serial diluted in 0.7% NaCl and plated out onto medium supplemented with appropriate antibiotics to allow for the selection of transconjugants. Enzyme linked immunosorbent assay (ELISA). Bordetella culture samples were taken at the end of log-phase growth (approximately 1010 cells/ml). The cells were separated from the medium by centrifugation at 10 000 g for 10 min and heat killed at 65°C for 15 min. An appropriate dilution (1/5) of each preparation was used for the ELISA assay. Supernatant and cellular fractions were tested for the presence of PT using a non-competitive, simultaneous sandwich assay (pre-coated ELISA plates supplied by CSL Ltd. Australia). ELISA plate results were analysed at 450 nm using a Bio-Rad Model 3550 Microplate Reader. The authors would like to thank J. Pemberton for the kind gift of E. coli strain Q358 pR751:: Tn813. B. pertussis Tohama I and B. bronchiseptica 5376 were obtained from R. Brownlie; B. bronchiseptica ATCC 10580 was obtained from M. Ho¨fle. Isogenic B. bronchiseptica strains BB7865 and BB7866 were obtained from R. Rappuoli. The electroelution system used was provided by Mr. H. S. Rothenfluh. We also extend thanks to J. Bennet and D. Drane of CSL Ltd. Australia and K. Timmis of the German National Centre For Biotechnology for helpful discussions, and for the supply of reagents. AMS was supported by an Australian postgraduate award (Industry) in conjunction with CSL Ltd. This work was initially supported by a grant from the German National Centre for Biotechnology and subsequently by the National Health and Medical Research Council (930123).

References 1. Walker MJ, Rhode M, Wheland J, Timmis KN. Construction of mini-transposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica. Infect Immun 1991; 59: 4238–48. 2. Bordet J, Gengou O. Le microbe de la coqueluche. Ann Inst Pasteur (Paris) 1906; 20: 731–41. 3. Robinson A, Irons LI, Ashworth LAE. Pertussis vaccine: present status and future prospects. Vaccine 1985; 3: 11–22. 4. Romanus V, Jonsell R, Bergquist S-O. Pertussis in Sweden after the cessation of general immunization in 1979. Pediatry Infect Dis 1987; 6: 364–71. 5. Tamura M, Nogimori I, Murai S et al. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 1982; 21: 5516–22. 6. Katada T, Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci USA 1982; 79: 3129–33. 7. Locht C, Keith JM. Pertussis toxin gene: nucleotide sequence and genetic organization. Science 1986; 232: 1258–64. 8. Nicosia A, Perugini M, Franzini C et al. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci USA 1986; 83: 4631–5. 9. Burnette WN, Mar VL, Cieplak W et al. Direct expression of Bordetella pertussis toxin subunits to high levels in Escherichia coli. Bio/Technology 1988; 6: 699–706. 10. Nicosia A, Bartoloni A, Perugini M, Rappuoli R. Expression and immunological properties of the five subunits of pertussis toxin. Infect Immun 1987; 55: 963–7.

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