INTRODUCTION

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Bordetella pertussis is a gram-negative pathogen causing whooping cough in children and increasingly being implicated in respiratory infections in adults (9, 21, 24, 27). Bordetella parapertussis is also recognized as a cause of whooping cough in children (14, 47) and also infects ovine species (10, 29-31, 46). Bordetella bronchiseptica has only rarely been associated with human disease (13, 34, 42) and is more commonly known as a pathogen of a range of species, including rabbits, pigs, dogs, and cats, among others (1, 5, 17, 23, 35, 43, 44, 49). In the search for improved modern vaccines directed against B. pertussis, a large body of work has been generated regarding protein virulence factors and targets for protective immunity (8, 32). This has led to a relative lack of research into the lipopolysaccharide (LPS) molecule found on the bordetellae, which is highly biologically active as an endotoxin, an immunomodulator, and an antigen (3, 7, 48). It is probable that this molecule plays a role in the infection process, a role overlooked for the want of molecular genetic analysis and appropriate animal models.

B. pertussis LPS appears to have the simplest structure of those of the three bordetellae considered in this paper. It consists of a lipid A molecule linked via a single ketodeoxyoctulosonic acid (Kdo) residue to a branched oligosaccharide core structure, containing heptose, glucose, glucuronic acid, glucosamine, and galactosaminuronic acid (6, 18, 19). This structure may be identified on a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel as band B LPS (25). Linked to this core structure is a trisaccharide consisting of N-acetyl-N-methyl-fucosamine (FucNAcMe), 2,3-dideoxy-di-N-acetylmannosaminuronic acid (2,3-diNAcManA), and N-acetylglucosamine (GlcNAc). The complete band B LPS plus this trisaccharide forms band A LPS on SDS-PAGE gels (25). B. bronchiseptica LPS also has band B and band A LPS, but in addition, it synthesizes an O-antigen structure consisting of a polymer of the single sugar residue 2,3-dideoxy-di-N-acetyl-galactosaminuronic acid (12). B. parapertussis LPS is somewhat different from either of the other two molecules described. It lacks band A and has a truncated band B, the structure of which has not been published. It does, however, have an O antigen apparently consisting of the same sugar polymer as in the B. bronchiseptica O antigen (12).

The genetics and molecular biology of LPS biosynthesis in the bordetellae have only recently been studied. The wlb locus (previously called bpl) (2, 33) required for the biosynthesis of band A LPS in B. pertussis has been cloned and sequenced, and mutations have been introduced into genes within the locus with consequent loss of band A structures (2). These mutations affect only a distal structure on the LPS and leave the rest of the molecule intact. To study the role of LPS in pathogenicity and immunity, bordetellae with LPS molecules with the deepest possible rough phenotype would be desirable. The deepest rough LPS mutants of Salmonella and Escherichia coli result from lesions in the waaC (rfaC) gene (22), which encodes the glycosyltransferase responsible for the addition of the first heptose residue to Kdo (38). B. pertussis waaC has been identified, but attempts to mutate this gene have been unsuccessful, probably because waaC is immediately upstream of the waaA (previously kdtA) gene (2), which is essential for cell viability. The gene responsible for the next step in enterobacterial LPS biosynthesis is waaF (rfaF) (39). Consequently, we report here the identification, cloning, and sequencing of a DNA locus containing a candidate for B. pertussis waaF and report the construction of deep rough mutants of B. pertussis, B. parapertussis, and B. bronchiseptica. These are the first mutations in these bacteria that result in a deep rough phenotype, and they are the first mutants of any kind constructed that affect LPS in B. parapertussis and B. bronchiseptica.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bordetellae used in this study were B. pertussis BP536, B. parapertussis CN 2591, and B. bronchiseptica CN 7635E from our culture collection. For cloning experiments and maintenance of plasmids, E. coli XL1-Blue (Stratagene) was used. E. coli HU835 was used to package cosmids in vivo. SM10pir was used as the donor strain in conjugation experiments. Salmonella typhimurium SL3789 has a mutation in the waaF gene (rfaF511) and was a kind gift from Brian Robertson, St. Mary's Hospital at Imperial College, London, United Kingdom. S. typhimurium AS68 was a r m⁺ strain carrying the E. coli LamB protein, enabling it to be infected by  phage particles.

Media, chemicals, and reagents. B. pertussis was routinely cultured on Bordet-Gengou medium supplemented with 15% horse blood. E. coli was cultured on Luria broth or agar (36). Media were purchased from Difco Ltd. or Oxoid Ltd. Antibiotics were used where appropriate. For the bordetellae, gentamicin at 10 µg/ml, ampicillin at 100 µg/ml, and streptomycin at 200 µg/ml were used. For E. coli and S. typhimurium, ampicillin was used at 100 µg/ml. SL3789 with its waaF lesion complemented by the BP536 waaF gene was selected on novobiocin at 2.5 µg/ml. All antibiotics and routine chemicals were purchased from Sigma Chemical Company. Restriction and modifying enzymes were purchased from Boehringer Mannheim. DNA ligase was purchased from Gibco-BRL. Sequenase sequencing kits were purchased from Amersham International.

Cloning of LPS genes. A cosmid library was constructed in the vector pHC79 (16) from BP536 chromosomal DNA partially digested with Sau3AI. Size selection of 35- to 45-kb DNA fragments was performed with a 0.8% low-melting-point agarose gel in pulsed-field gel electrophoresis. This DNA was purified from the gel with agarase, then ligated with pHC79, and packaged with Gigapack Gold III packaging mixes (Stratagene). These packaged cosmids were transfected into E. coli XL1-Blue, and 1,000 resultant colonies were maintained as a representative library. The packaged library was also amplified with the in vivo packaging strain E. coli HU835. Before using purified cosmids to infect the S. typhimurium SL3789 waaF selection strain, the cosmids were used to infect S. typhimurium AS68 so that the cosmid DNA became modified for S. typhimurium restriction systems, thus ensuring high efficiency and representative transformation of the library into the selection strain. Selection for AS68-carrying cosmids was with ampicillin. Cosmid DNA from a pool of 4,000 resultant ampicillin-resistant colonies was isolated and used to electroporate SL3789 (0.1-cm cuvette; 1,750 V, 25 µF, and 600 ). Resultant colonies were selected for complementation of the waaF mutation in SL3789 by selection on novobiocin and ampicillin. Analysis of the LPS from the resultant colonies was performed by silver-stained SDS-PAGE and agglutination experiments with anti-O4,5 antiserum (Murex Diagnostics, Dartford, United Kingdom).

LPS preparation and SDS-PAGE. LPS was purified by a modification of the method of Hitchcock and Brown (15). Briefly, B. pertussis was grown on Bordet-Gengou plates for 2 to 3 days and then harvested into phosphate-buffered saline. S. typhimurium was grown in the appropriate antibiotics in Luria broth, and bacteria were pelleted by centrifugation and resuspended in phosphate-buffered saline. Resuspended bacteria were lysed by addition of a one-third volume of lysis solution (0.2 M Tris-HCl [pH 6.8], 3% [wt/vol] SDS, 30% [vol/vol] glycerol), followed by incubation at 100°C for 30 min. After cooling, proteinase K was added to a final concentration of 0.1 mg/ml. This was incubated at 55°C for 60 min. Equal volumes of phenol were added and mixed. This was incubated at 68°C for 15 min for LPS extraction from wild-type bordetellae and salmonellae or at room temperature for 5 min for LPS extraction from mutant bordetellae and salmonellae. The LPS-containing aqueous phase was separated from the phenol phase by centrifugation. LPS was then precipitated by the addition of 0.1 volume of 3 M sodium acetate, pH 5.5, and 4 volumes of ethanol followed by centrifugation. Pelleted LPS was redissolved in lysis solution at one-third of its normal concentration, containing bromophenol blue, and boiled for 5 min prior to electrophoresis. SDS-PAGE gels were run in a Tricine buffer system according to the method of Lesse et al. (20). Silver staining was performed according to the method of Tsai and Frasch (45).

DNA sequencing. Plasmid DNA was sequenced with Sequenase 2 and an ABI automated sequencer. Sequences were assembled and analyzed with the Genetics Computer Group (GCG) package (11) or the Staden programs (40) running on the Oxford University molecular biology VAX computer.

Southern hybridizations. Southern hybridizations were performed according to the method of Sambrook et al. (36) with probes labelled with [³²P]dCTP with a randomly primed labelling kit (Stratagene, Cambridge, United Kingdom).

Mutagenesis of the bordetellae. For allelic replacement mutagenesis, waaF was insertionally inactivated in B. pertussis, B. parapertussis, and B. bronchiseptica by introduction of the vector pSS2141 into the gene (single-crossover mutagenesis). A 439-bp PCR product (bases 1134 to 1572 in the published sequence) generated by using as primers oligonucleotides BpwaaF1 (5'-CAACTGGCGGGCATCGACCGCC-3') and BpwaaF2 (5'-GTCGTGGCACTACCTGACCC-3'), corresponding to the middle of the B. pertussis waaF gene, was amplified with Taq DNA polymerase (Applied Biosystems) and cloned into pT7-Blue. This fragment was then released with XbaI and PaeI and cloned into the equivalent sites in pSS2141 (41). This procedure was performed so that the rpsL gene was removed from the vector, allowing the use of streptomycin to select for transconjugants. Conjugation experiments were performed on Bordet-Gengou plates containing 15% horse blood and 10 mM MgCl₂ with the appropriate bordetellae as recipients and E. coli SM10pir containing the recombinant suicide vector pSS2141 carrying the 439-bp fragment as donor. Single crossovers were selected on gentamicin and ampicillin (resistance to both being encoded by the vector) and streptomycin (to which the recipient bordetellae are resistant). LPS from resultant colonies was purified and analyzed by silver-stained SDS-PAGE. Genomic DNAs from resultant colonies were also analyzed by Southern hybridization for the expected DNA rearrangements and confirmed to be single-crossover mutants with mutated waaF genes (data not shown).

Nucleotide sequence accession number. The DNA sequence described here is deposited with the EMBL database under accession no. Y13475.

	RESULTS AND DISCUSSION

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Identification and cloning of waaF. A cosmid library of B. pertussis BP536 DNA, constructed in the vector pHC79, was amplified and modified as described in Materials and Methods. This cosmid DNA was isolated and used to electroporate S. typhimurium SL3789(rfaF511), which has a deep rough LPS phenotype. Complementation of this genetic lesion would enable the bacteria to synthesize complete, smooth LPS. To select for complementation by recombinant cosmids, bacteria were plated on media containing novobiocin, since this antibiotic selectively kills rough bacteria at much lower concentrations than are needed to kill smooth bacteria (4). Transformants and controls consisting of wild-type S. typhimurium SL3770 (positive) and SL3789 alone (negative) were selected on various concentrations of novobiocin with or without ampicillin. SL3770, being smooth, was capable of growth on novobiocin at 2.5 µg/ml, whereas SL3789 was sensitive to this concentration as a consequence of having rough LPS. Electroporation of SL3789 with the cosmid library produced four colonies resistant to both ampicillin and novobiocin at 2.5 µg/ml. LPS was purified from one of these and analyzed by silver-stained SDS-PAGE, confirming the restoration of the O-antigen phenotype (Fig. 1). The complemented bacteria were also agglutinable with anti-O4,5 antiserum. These data indicate the presence of a functional waaF homolog within the locus. The fact that the deep rough LPS molecule from the S. typhimurium waaF mutant is efficiently restored to the wild-type phenotype by the B. pertussis waaF homolog shows that the bordetella protein can recognize the S. typhimurium waaF mutant LPS as a substrate. This might not be immediately expected, as the inner core structures of Salmonella and Bordetella are different in a number of respects (6, 7, 18, 19, 22). For example, two Kdo residues are present between lipid A and the first heptose in the S. typhimurium core, whereas in the equivalent region of the B. pertussis LPS molecule, only one Kdo residue is observed. This difference does not seem to interfere with the correct functioning of the bordetella enzyme.

View larger version (48K): [in this window] [in a new window]   FIG. 1.   Silver-stained Tris-tricine polyacrylamide gel of S. typhimurium LPS isolated from wild type, waaF mutant, and waaF mutant complemented with B. pertussis waaF. Lane 1, S. typhimurium waaF mutant (SL3789) complemented by the BP536 waaF gene; lane 2, SL3789 alone; lane 3, S. typhimurium wild type (SL3770). The waaF mutant displays the deep rough LPS phenotype expected, while the wild-type control shows the ladder pattern expected for a full-length smooth LPS. The complemented mutant also has the O-antigen ladder, but a rough core molecule is also observed in the LPS preparation, suggesting that the complementation is not completely effective.

View larger version (48K): [in this window] [in a new window]   FIG. 2.   BOXSHADE of a PILEUP performed in the GCG DNA analysis package with MsbA protein sequences from E. coli (Ecoli) and H. influenzae (Hin) and the proposed homolog from B. pertussis (BP536). The black shading surrounds blocks of amino acids which are identical, and the grey shading surrounds blocks with conservative substitutions. The B. pertussis sequence is shown as starting with a leucine residue since it has TTG as a start codon. This sequence is truncated at the position of the SacI site where the DNA sequence published here starts. Only the parts of the E. coli and H. influenzae sequences corresponding to the truncated B. pertussis sequence are shown.

View larger version (127K): [in this window] [in a new window]   FIG. 3.   BOXSHADE of a PILEUP performed in the GCG DNA analysis package with WaaF protein sequences from N. gonorrhoeae (Ngon), Neisseria meningitidis (Nmen), E. coli (Ecoli), S. typhimurium (Salty), H. influenzae (Hin), and Pseudomonas aeruginosa (Psaer) and the proposed B. pertussis (BP536) WaaF protein. See Fig. 2 legend for an explanation of the shading.

Identification of waaF in B. parapertussis and B. bronchiseptica. A 439-bp PCR fragment from the central region of waaF also used to mutagenize the bordetellae (see Materials and Methods) was used to probe Southern blots of restriction digests of B. pertussis, B. parapertussis, and B. bronchiseptica genomic DNA. This identified a single 4-kb SalI fragment in B. parapertussis and B. bronchiseptica, showing that the locus is present in all three bordetellae tested. Further genetic analysis of these waaF loci is ongoing and may allow the differences between B. parapertussis core and the other Bordetella LPS molecules to be addressed at the molecular genetic level.

Construction of waaF mutants in the bordetellae. To confirm that waaF was required for inner core LPS biosynthesis in the bordetellae, allelic exchange mutants were generated. A single-crossover strategy was chosen to ensure the successful mutagenesis of waaF in all three strains. The same 439-bp PCR product was used to mutagenize the three bordetellae (see Materials and Methods). Following mutagenesis, the LPS phenotypes of resultant colonies were analyzed by silver-stained SDS-PAGE. Each of the waaF mutants had single LPS bands that migrated equally with each other and much faster than band B seen in the controls (Fig. 4). This is consistent with the LPS molecule having a deep rough phenotype. In addition, band A was absent from the B. pertussis and B. bronchiseptica mutants and the characteristic O antigen present in wild-type B. parapertussis and B. bronchiseptica controls was also absent from the waaF mutants. The evidence suggests that the waaF mutation leads to each of the bordetellae biosynthesizing the same deep rough LPS molecule, with concomitant loss of expression of distal structures. This is also consistent with the difference in structure of the B. parapertussis core, compared to B. pertussis and B. bronchiseptica, lying at its nonreducing end. The deeply truncated LPS phenotype observed in the three Bordetella mutants may not have been entirely predictable. Lipid A is linked to a single Kdo which in turn is linked to the first heptose residue. This heptose is then substituted by a glucose residue upon which the rest of the core main chain, the band A trisaccharide, and the O antigen are built. The second heptose, whose transfer is catalyzed by WaaF, forms a branch linked to the first heptose, with this second heptose being substituted by glucosamine and glucuronic acid (6, 7, 18, 19). Thus, a mutation in waaF might have been expected to lead to the removal of the branch structure from the core, leaving the rest of the core intact and potentially allowing the addition of distal structures. The fact that the deep rough phenotype was observed in the waaF mutants indicates that the addition of the rest of the core is dependent on prior addition of the branch structure. Another possibility that cannot be excluded at this stage is that the insert into the waaF gene, being large and complex, may have polar effects on genes downstream of waaF in the locus, which may themselves be required for biosynthesis of the rest of the LPS core molecule. These possibilities are currently being investigated.

View larger version (59K): [in this window] [in a new window]   FIG. 4.   Silver-stained Tris-tricine polyacrylamide gel of wild type and waaF mutant allelic exchange mutants from B. pertussis, B. bronchiseptica, and B. parapertussis. Lane 1, B. pertussis (BP536) wild type; lane 2, B. pertussis waaF mutant; lane 3, B. bronchiseptica (CN 7635E) wild type; lane 4, B. bronchiseptica waaF mutant; lane 5, B. parapertussis (CN 2591) wild type; lane 6, B. parapertussis waaF mutant.

Conclusions. In this study we have identified, cloned, and sequenced the waaF gene from Bordetella species and have mutated this gene in B. pertussis, B. parapertussis, and B. bronchiseptica. This has led to these three bordetellae each having a deep rough LPS phenotype. These are the most minimal LPS structures constructed so far in the bordetellae.