Theodor-Boveri-Institut für Biowissenschaften, Lehrstuhl für Mikrobiologie, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
Correspondence
Roy Gross
roy{at}biozentrum.uni-wuerzburg.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ748854, AJ748855 and AJ48856.
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INTRODUCTION |
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Within the past few years, four new species have been added to the genus Bordetella. Bordetella holmesii can cause disease in man and has been isolated from blood cultures of patients with underlying disease and from patients with whooping cough-like symptoms (Weyant et al., 1995; Tang et al., 1998
; Yih et al., 1999
; Mazengia et al., 2000
; Shepard et al., 2004
). Bordetella hinzii appears to be a commensal of birds, although it has been isolated occasionally also from the blood of immunocompromised patients (Cookson et al., 1994
; Kattar et al., 2000
). Bordetella trematum was isolated from patients suffering ear or wound infections, but was not found in the respiratory tract (Vandamme et al., 1996
); at present its pathogenic potential is not known. Finally, B. petrii was the first member of the genus to be isolated from the environment (von Wintzingerode et al., 2001
). Little is known about the biology and the pathogenic potential of these new Bordetella species, although B. holmesii is increasingly recognized as being associated with human disease (Yih et al., 1999
).
During the course of the development of new vaccines against whooping cough, much has been learnt about the pathogenicity and the virulence-associated factors of B. pertussis, B. bronchiseptica and B. parapertussis. Interestingly, these three species share several virulence-relevant factors (Hewlett & Cowell, 1989; Weiss, 1992
; Parton, 1999
), including adhesion or colonization factors, such as the filamentous haemagglutinin, fimbriae of several serotypes and several autotransporter proteins. Moreover, these bacteria produce highly related toxic factors such as the tracheal cytotoxin, the dermonecrotic toxin and the adenylate cyclase toxin. Pertussis toxin is expressed exclusively by B. pertussis, although the ptx genes are also present in B. parapertussis and B. bronchiseptica (Gross & Rappuoli, 1988
). It is well known that the three closely related Bordetella species B. pertussis, B. bronchiseptica and B. parapertussis, which are also referred to as members of the B. bronchiseptica cluster (Gerlach et al., 2001
), coordinately regulate the expression of these virulence factors via a highly conserved two-component system encoded by the bvgAS locus (Weiss et al., 1983
; Arico et al., 1989
; Cotter & DiRita, 2000
; Bock & Gross, 2001
).
BvgAS mediates the transition between the virulent and the avirulent phenotype by two phenomena called antigenic modulation and phase variation. Antigenic modulation describes a reversible on-and-off switch in the expression of pathogenicity factors in response to environmental signals, such as temperature, magnesium sulphate or nicotinic acid, and is caused by changes in the phosphorylation state of the BvgAS two-component system (Lacey, 1960; Uhl & Miller, 1996
; Zu et al., 1996
; Perraud et al., 1998
). In contrast, phase variation, which occurs in vitro at a strain-dependent frequency of 102106, is characterized by the irreversible loss of expression of the virulence-associated genes irrespective of the growth conditions (Leslie & Gardner, 1931
; Peppler, 1982
; Peppler & Schrumpf, 1984
). It has been shown in a B. pertussis isolate that phase variation was due to a frameshift mutation in the bvgS gene (Stibitz et al., 1989
), while in B. bronchiseptica, phase variation is frequently caused by small deletions within the bvgS gene (Monack et al., 1989
).
In contrast to the classical species, virtually nothing is known about virulence-relevant features of the new Bordetella species. Since the BvgAS system is the master regulator of virulence in the classical species, we characterized the orthologous genes from the new species in order to use the BvgAS system as a starting point for the identification of possible virulence factors in these species. In the present paper we describe the properties of the BvgAS systems of the new species, with the main emphasis on the human pathogen B. holmesii.
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METHODS |
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Characterization of the bvgAS loci of B. holmesii, B. trematum and B. hinzii.
PCR reactions with degenerate oligonucleotide primers were performed essentially as described by Morel-Deville et al. (1997) using chromosomal DNA of B. holmesii G7702, B. trematum and B. hinzii as templates. The degenerate primers were deduced from highly conserved regions of the receiver domain and from the output domain of BvgABP from B. pertussis. Plasmids and oligonucleotides used in this study are listed in Table 1
and Table 2
, respectively.
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The PCR products were cloned into pGEM-T vector DNA, giving rise to plasmids pGEM-T-deg1BH, pGEM-T-deg1BHZ, pGEM-T-deg2BH and pGEM-T-deg2BT, and sequenced. Sequence analysis demonstrated that the cloned DNA fragments were indeed derived from the bvgA orthologues of the new Bordetella species. Interestingly, primer deg2-reverse turned out to have annealed to a sequence motif within bvgABH and bvgABT, which is located further upstream than the motif from which the degenerate primer sequence was derived, therefore yielding PCR fragments of unexpected length.
Partial genomic libraries of B. holmesii were screened using PCR fragment deg1BH as probe. The DNA inserts of positive clones were subsequently sequenced to assemble the complete nucleotide sequence of bvgABH including its flanking DNA regions. The nucleotide sequences of deg1BHZ and deg2BT were extended by a combination of different techniques including inverse PCR, genome walking [using the Universal Genome Walker Kit (Clontech)] and the screening of partial genomic libraries of the respective organisms.
Cloning of the bvgASBH locus of B. holmesii ATCC 51541 (bvgASBH ATCC 51541) and B. holmesii G7702 (bvgASBH G7702).
In order to construct plasmids pSL-bvgASBH G7702 and pSL-bvgASBH ATCC 51541 the 4·7 kbp DNA region comprising the orfX-bvgABH intergenic region and the entire bvgASBH locus, from B. holmesii G7702 and ATCC 51541, respectively, was assembled by stepwise cloning from three individual PCR fragments (bvgASBHF1, bvgASBHF2 and bvgASBHF3) amplified from chromosomal DNA of the corresponding B. holmesii strain. The following steps were repeated for both B. holmesii strains. PCR fragment bvgASBHF1, which includes the orfX-bvgASBH intergenic region and extends to the SphI site of the bvgSBH gene, was synthesized using primer pair bvgASBHF1-SpeI/bvgASBHF1-SphI. PCR using primer pairs bvgASBHF2-SphI/bvgASBHF2-KpnI and bvgASBHF3-KpnI/bvgASBHF3-EcoRI, respectively, yielded DNA fragment bvgASBHF2, containing the DNA segment located between the SphI and KpnI site of the bvgSBH gene, and PCR fragment bvgASBHF3, containing the sequence flanked by the KpnI site and the stop codon of the bvgSBH gene. Fragments bvgASBHF1 and bvgASBHF2 were first ligated into SpeI-and KpnI-digested pSK-Bluescript DNA, yielding plasmid pSK-bvgASBHF1/F2. Fragment bvgASBHF1/F2 was excised by SpeI and KpnI digestion of pSK-bvgASBHF1/F2 and ligated, together with fragment bvgASBHF3, into SpeI- and EcoRI-linearized plasmid pSL1180, generating pSL-bvgASBH.
For the construction of plasmids pRK-bvgASBH G7702 and pRK-bvgASBH ATCC 51541, fragment bvgABHFI, including the orfX-bvgASBH intergenic region and extending to the HindIII restriction site of the bvgABH gene, was PCR amplified using primer pair bvgABHFI-XbaI/bvgASBHFI-HindIII and genomic DNA from the corresponding strain of B. holmesii. A DNA fragment, ranging from the HindIII site of the bvgABH gene to the bvgSBH stop codon, was excised by HindIII/EcoRI digestion from pSL-bvgASBH G7702 and pSL-bvgASBH ATCC 51541 and subsequently inserted, together with PCR fragment bvgABHFI, into XbaI- and EcoRI-digested pUC18 vector DNA. The resulting plasmids, pUC-bvgASBH G7702 and pUC-bvgASBH ATCC 51541, were digested with XbaI and EcoRI and the resulting 4·7 kbp DNA fragments were finally inserted into plasmid pRK415, giving rise to plasmids pRK-bvgASBH G7702 and pRK-bvgASBH ATCC 51541, respectively.
Construction of a bvgABH mutant of B. holmesii G7702.
For the construction of the suicide plasmid pSS1129-bvgABH-kanR, a 1·8 kbp DNA fragment comprising the bvgABH gene and part of its 5' and 3' flanking regions was PCR amplified from genomic DNA of B. holmesii G7702 using primer pair bvgABH-kanR5/bvgABH-kanR3, which provided BamHI restriction sites at the 5' and 3' termini. This PCR fragment was cloned into pACYC184, and the resulting plasmid was digested with PstI, thereby deleting 354 bp from bvgABH because of the presence of three PstI restriction sites (at positions 206, 467 and 560 with respect to the bvgABH start codon) in the bvgABH gene. Subsequently, a kanamycin-resistance cassette, excised by PstI cleavage from pUC4K, was inserted into the PstI-digested plasmid pACYC-bvgABH, yielding plasmid pACYC-bvgABH-kanR. The bvgABH-kanR fragment was excised with BamHI and ligated into the suicide vector pSS1129, giving rise to plasmid pSS1129-bvgABH-kanR, which was subsequently transformed into E. coli SM10. pSS1129-bvgABH-kanR was then conjugated into B. holmesii G7702 using E. coli SM10 as donor strain. Allelic exchange resulted in the B. holmesii strain G7702/bvgA : : kan. The correct insertion of the kanamycin-resistance cassette into the bvgABH gene of the kanamycin-resistant exconjugants was proven by PCR reactions with primers flanking the integration site and by Southern blot analysis.
Construction of B. holmesii and B. pertussis strains containing a plasmid with a fusion of the upstream region of bvgABH to the gfp reporter gene.
A DNA fragment containing the promoterless gfp-mut2 gene (Cormack et al., 1996) was excised with XbaI and PstI from plasmid pKEN and ligated into plasmid pUC18, giving rise to plasmid pUC-gfp. A 455 bp DNA fragment containing the upstream region of the bvgASBH locus was PCR amplified from genomic DNA of B. holmesii G7702 using primer pair bvgASBHup-5/bvgASBHup-3, thereby introducing BamHI and XbaI restriction sites at the 5' and 3' termini, respectively, and the fragment was cloned into pUC-gfp, resulting in plasmid pUC-bvgASBHup-gfp. The bvgASBHup-gfp fragment was then excised by BamHI- and PstI-digestion and was subsequently ligated into plasmid pMMB208. In the resulting plasmid pMMB-bvgASBHup-gfp, the bvgASBHup-gfp promoter fusion is located in the opposite orientation to the plasmid-borne tac promoter. pMMB-bvgASBHup-gfp was subsequently transformed into E. coli SM10 and transferred by conjugation into various B. holmesii and B. pertussis strains.
Construction of plasmids expressing recombinant BvgABP, BvgSBP and BvgABH proteins, and protein purification.
The construction of pQE-BvgSTRO and pCYT-BvgABP has been described by Perraud et al. (1998). For the generation of plasmid pQE-BvgABH, a DNA fragment including the complete bvgABH gene (621 bp) was amplified from genomic DNA of B. holmesii G7702 using primer pair bvgABH-BamHI/bvgABH-KpnI, thus introducing BamHI and KpnI sites at its 5' and 3' ends respectively. This PCR fragment was ligated into BamHI/KpnI-digested pQE30 vector DNA, creating an N-terminal His6-tag. Recombinant His6-BvgABH and His6-BvgSBP, encoded on plasmids pQE-BvgABH and pQE-BvgSTRO, respectively, were expressed in E. coli M15 cells and the proteins were purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose (Qiagen) essentially as described by Perraud et al. (1998)
. Native BvgABP encoded on plasmid pCYT-BvgABP was overexpressed in E. coli DH5
cells and the protein was purified by affinity chromatography on heparin-Sepharose CL-6B (Pharmacia) as described by Perraud et al. (1998)
. In vitro-phosphorylation assays with the recombinant proteins were performed as described by Bock & Gross (2002)
.
Primer extension experiments.
Total RNA was prepared from bacteria grown in liquid culture as described previously (Gross & Rappuoli, 1989). Primer extension experiments were carried out essentially as described by Scarlato et al. (1991)
with primer oligonucleotides fhaB-PE, bvgABH-PE and gfp-PE (Table 2
). Sequencing reaction mixtures, with plasmids pSL-bvgASBH G7702 and pUC-bvgASBHup-gfp as template DNA and the appropriate oligonucleotide primer, were analysed on 6 % urea-polyacrylamide gels and used as standards for determination of the transcription initiation sites.
Gel retardation experiments.
A 77 bp DNA fragment encompassing the BvgABP-P binding site of the fhaB promoter was amplified by PCR using primer pair fhaBup5/fhaBup3 and plasmid pProm67 as template. The PCR fragment was 5'-end labelled with [-32P]ATP using T4 polynucleotide kinase (MBI) and purified using the QIAquick Nucleotide Removal Kit (Qiagen). The recombinant BvgA proteins were diluted in 1x dilution buffer (2 mM MgCl2, 50 mM KCl, 0·1 % Igepal CA 630, 10 mM DTT) and were phosphorylated by incubation with 50 mM acetyl phosphate (Sigma) for 20 min at room temperature. Increasing amounts of the proteins were added to approximately 15 000 c.p.m. of the labelled DNA probe in 20 µl of 1x binding buffer (10 mM Tris/HCl, pH 8, 10 mM KCl, 5 mM EDTA, 1 mM DTT, 10 % glycerol, v/v). The samples were incubated for 20 min at room temperature and were then loaded onto a non-denaturing 4 % polyacrylamide gel (Sambrook & Russell, 2000). Gels were run for 2·5 h at 150 V and subsequently the dried gels were autoradiographed.
DNase I footprinting.
DNase I footprint experiments were performed essentially as described by Dickneite et al. (1998). A 253 bp DNA fragment containing part of the upstream region of the bvgASBH locus was PCR amplified from chromosomal DNA of B. holmesii G7702 using primer pair FP1-3/FP1-5. The purified promoter fragment was cloned into plasmid pGEM-T. The resulting plasmid pGEM-T-FP was digested with BamHI and 5'-end labelled with [
-32P]ATP using T4 polynucleotide kinase. The labelled promoter fragment was excised from the plasmid by KpnI digestion, purified by gel electrophoresis and eluted in 3 ml elution buffer (10 mM Tris/HCl, pH 8·0, 1 mM EDTA, 300 mM sodium acetate, 0·2 % SDS). The eluted probe was then extracted with phenol/chloroform (1 : 1, v/v) and ethanol precipitated. Binding reaction mixtures contained various concentrations of protein and approximately 100 000 c.p.m. of labelled DNA probe in 50 µl of 1x binding buffer (10 mM Tris/HCl, pH 8, 2 mM MgCl2, 0·1 mM CaCl2, 1 mM DTT, 10 % glycerol, v/v). The samples were incubated for 20 min at room temperature and then the nucleolytic reactions were initiated by the addition of 1 U DNase I in 1x binding buffer. After 1 min, digestions were terminated by the addition of 140 µl stop buffer (192 mM sodium acetate, 0·14 % SDS, 62 µg ml1 yeast tRNA). The samples were extracted with phenol/chloroform (1 : 1, v/v), ethanol precipitated and run on a 6 % polyacrylamide-urea sequencing gel. A G+C sequence reaction was also conducted in parallel with the labelled DNA probe (Maxam & Gilbert, 1977
) and electrophoresed on the same gel.
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RESULTS |
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The genome organization of the bvgAS loci of the new Bordetella species differs from that of the members of the B. bronchiseptica cluster. In B. holmesii, B. hinzii and B. trematum, a gene (orfX) encoding a putative response regulator was detected at a distance of about 400 bp upstream from bvgA in the same transcriptional direction, while in the members of the B. bronchiseptica cluster the fhaB gene encoding filamentous haemagglutinin (FhaB) is located upstream of bvgA (Parkhill et al., 2003). Also, in B. avium, an ORF encoding a homologous response regulator was found upstream of bvgA; this ORF was annotated by Spears et al. (2003)
as an orthologue of the vieA gene of V. cholerae. Furthermore, no orthologue of the bvgR gene, which is located downstream of bvgAS in the genomes of the members of the B. bronchiseptica cluster is present within the analysed region downstream of bvgAS in B. holmesii. The lack of a bvgR orthologue in the downstream region has already been described for B. avium (Spears et al., 2003
), therefore suggesting that the genome organization of the bvgAS locus is conserved in B. avium and the new Bordetella species (Fig. 1
).
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Interestingly, DNA sequence analysis of the bvgA gene of the B. holmesii type strain ATCC 51541 (bvgABH ATCC 51541) revealed the insertion of an adenosine residue at nucleotide position 212 which leads to a frameshift mutation that causes the premature termination of the encoded protein. Therefore, we sequenced the bvgABH gene of three additional B. holmesii strains which were isolated independently from each other in different geographical regions and at different times. In one of these strains, B. holmesii No1, we found the identical mutation to that seen in the type strain, while the bvgABH genes of the other two isolates, B. holmesii G7702 and G8341, are intact. This observation is reminiscent of the phenomenon of phase variation in B. pertussis and B. bronchiseptica and, therefore subsequently in this paper B. holmesii isolates with an intact bvgABH gene are considered as wild-type strains, while strains carrying the frameshift mutation are classified as phase variants. However, after the insertion of a kanamycin-resistance cassette into the bvgABH gene of the B. holmesii wild-type strain G7702, the resulting bvgABH mutant strain B. holmesii G7702/bvgABH : : kan displayed growth behaviour and colony morphology indistinguishable from its parent strain and from the other B. holmesii strains mentioned above.
The cytoplasmic signalling domains of BvgSBH and BvgSBP are functionally interchangeable
It has been shown already that the bvgAS loci of B. pertussis and B. bronchiseptica can replace each other in vivo (Monack et al., 1989; Martinez de Tejada et al., 1996
). To investigate whether the bvgASBH genes of B. holmesii are able to functionally complement B. pertussis bvgAS mutants, we introduced plasmid pRK-bvgASBH G7702, containing the orfX-bvgAS intergenic region and the entire bvgAS locus of B. holmesii G7702 (bvgASBH G7702) into the B. pertussis strains 359 and 347 by conjugation. BP 359 contains a polar Tn5 insertion in the bvgABP gene and is therefore unable to express both the BvgABP and the BvgSBP proteins. In BP 347, the bvgSBP gene is disrupted, resulting in the low-level expression of BvgABP due to the presence of a weak constitutive promoter upstream of bvgASBP (Scarlato et al., 1991
); however, BvgABP is present in its non-phosphorylated and therefore inactive state (Fig. 2
a).
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To confirm that the successful complementation in BP 347(pRK-bvgASBH G7702) was due to cross-phosphorylation between BvgSBH and BvgABP, plasmid pRK-bvgASBH ATCC 51541, carrying the bvgASBH locus of the bvgA phase variant B. holmesii ATCC 51541, was introduced into BP 347 by conjugation. As expected, the resulting strain BP 347(pRK-bvgASBH ATCC 51541) was haemolytic on BG blood agar plates and expressed FhaB (data not shown). In keeping with these results, efficient cross-phosphorylation between the purified BvgSBP and BvgABH proteins was also observed in vitro (data not shown).
Since it is well known that high concentrations of MgSO4 inhibit the histidine kinase activity of BvgSBP (Melton & Weiss, 1989), strain BP 347(pRK-bvgASBH ATCC 51541) was analysed for the activity of histidine kinase BvgSBH in the presence of MgSO4. The effect of MgSO4 on the phosphorylation status of BvgSBH ATCC 51541, and consequently on the expression of the virulence genes, was investigated by primer extension experiments using the fhaB-specific primer and RNA extracted from BP 347(pRK-bvgASBH ATCC 51541) cells cultured in the presence or absence of 50 mM MgSO4. As shown in Fig. 2(c)
, addition of MgSO4 to the culture medium completely repressed fhaB transcription in the positive control strain BP 347(pLA57vir), while transcription of fhaB was only slightly reduced under these growth conditions in BP 347(pRK-bvgASBH ATCC 51541). Taken together these data demonstrate that the cytoplasmic signalling domains of BvgSBP and BvgSBH are functionally interchangeable, while differences exist regarding the function of their N-terminal signal-perception domains.
BvgABH binds in vitro to the orfX-bvgASBH intergenic region but not to the BvgABP binding site of the fhaB promoter of B. pertussis
The results of the complementation experiments suggest that the failure of plasmid pRK415-bvgASBH G7702 to restore virulence gene expression in the B. pertussis mutant BP 359 is due to the inability of BvgABH to bind to the fhaB and cya promoters. Therefore, we investigated, by gel retardation experiments, the in vitro binding of BvgABH to a 77 bp DNA fragment containing the well-characterized BvgABP binding site of the promoter region of the B. pertussis fhaB gene. A clear band shift of the fhaB probe was observed with the purified BvgA of B. pertussis, irrespective of whether the protein was in vitro phosphorylated with acetyl phosphate prior to the binding assay, as has been described by Zu et al. (1996) (Fig. 3
b). In contrast, no binding to the fhaB promoter fragment could be detected with either the unphosphorylated or the phosphorylated BvgABH protein (Fig. 3a
), suggesting that BvgA of B. holmesii is unable to recognize canonical BvgABP-dependent promoters. In keeping with this hypothesis, we could not detect binding of BvgABH to the bvgABP promoter region in gel retardation experiments (data not shown).
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BvgABH activates transcription of the bvgASBH locus in B. holmesii
To analyse whether the bvgAS locus of B. holmesii is autoregulated, primer extension experiments were performed with RNA extracted from B. holmesii strain G7702 and the isogenic bvgA knockout mutant G7702/bvgABH : : kan, using a bvgABH-specific oligonucleotide. As shown in Fig. 6(a), two transcriptional start sites could be identified at positions 20 (S1) and 34 (S2) with respect to the translational start codon of bvgABH. Upstream of both start sites, putative 10 regions are present which exhibit one (P2: CATAAT) and two (P1: TAAAAC) mismatches compared to the 10 consensus promoter element of E. coli. These putative 10 elements are located at a distance of 27 and 42 bp, respectively, from the IR1 sequence motif, within the region which is protected from DNase I digestion in the footprint experiments performed with BvgABH (Fig. 5
). The amount of bvgABH-specific transcript was clearly reduced in G7702/bvgABH : : kan, suggesting a positive autoregulatory effect of BvgABH on the transcription of bvgASBH in B. holmesii. To confirm these results, a DNA fragment comprising 455 bp of the 5' region of bvgASBH was fused to a promoterless gfp gene in the broad-host-range vector pMMB208 and the resulting plasmid pMMB-bvgASBHup-gfp was conjugated into B. holmesii G7702 and G7702/bvgABH : : kan. Expression of the reporter gene was investigated by immunoblot analysis using a polyclonal antibody directed against GFP. Consistent with the results of the primer extension experiments, expression of the gfp reporter gene was clearly reduced in the bvgA mutant G7702/bvgABH : : kan as compared to the wild-type strain G7702 (Fig. 6b
), corroborating positive autoregulation by BvgABH.
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DISCUSSION |
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By PCR with degenerate oligonucleotide primers derived from the BvgA sequence of B. pertussis, we obtained bvgA-specific DNA fragments from the three organisms; these fragments were used as probes for the cloning of the complete bvgAS locus from B. holmesii and of partial sequences of bvgAS from B. trematum and B. hinzii. At the DNA level, the bvgAS genes of the three new Bordetella species show only limited sequence similarity with their orthologues from the members of the B. bronchiseptica cluster, explaining our previous failure to prove their presence by DNA hybridization experiments with B. pertussis-specific DNA-probes (Gerlach et al., 2001). However, as far as they were determined, the deduced amino acid sequences of BvgAS were found to be conserved between all species including the bird pathogen B. avium whose bvgAS genes have recently been cloned (Spears et al., 2003
). As expected, sequence similarity is highest in the two-component signalling modules but is less pronounced in the recently characterized linker region of BvgA (Bock et al., 2001
) and the sensory domain of BvgS. Clearly the BvgAS proteins of the new Bordetella species and of B. avium are more closely related to each other than to the orthologous proteins of the members of the B. bronchiseptica cluster (Table 3
).
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Unexpectedly, 16S rDNA sequence analysis had suggested that the closest phylogenetic relationship was between B. pertussis and B. holmesii (Weyant et al., 1995; Gerlach et al., 2001
); however, in agreement with comparisons of the amino acid sequences of conserved proteins (von Wintzingerode et al., 2002
), our characterization of the bvgAS loci clearly classifies B. holmesii as proximate to the species B. avium, B. hinzii and B. trematum.
Interestingly, the bvgA sequences of two out of four independent clinical isolates of B. holmesii contained an identical point mutation causing a frameshift that results in the premature termination of the encoded protein. This is reminiscent of phase variation in B. pertussis and B. bronchiseptica leading to the loss of virulence properties upon in vitro passaging of the respective strains due to mutations in the bvgS genes (Monack et al., 1989; Stibitz et al., 1989
). However, phase variants of these organisms have apparently never been isolated from infected hosts. Since inactivation of the bvgA gene in B. holmesii G7702 had no obvious effect on growth and colony morphology of the mutant, the possibility that frameshift mutations in strains ATCC 51541 and No1 occurred after their isolation from the infected patients can not be ruled out. But, since the site of the mutation is not associated with a homo- or dinucleotide tract which could cause slipped-strand mispairing, it seems rather unlikely that the identical insertion mutation should have occurred in both strains as a consequence of in vitro culture. The apparent isolation of bvgA mutants from infected individuals would clearly argue against a role of the BvgASBH two-component system in the regulation of important virulence traits in B. holmesii, while inactivation of bvgASBA caused a clear attenuation of the virulence of B. avium in turkey poults (Spears et al., 2003
). Interestingly, Njamkepo et al. (2000)
reported differences in the protein hybridization patterns of different B. holmesii isolates, including the type strain, ATCC 51541, and G7702, when the bacterial lysates were probed with sera from infected individuals, and therefore they suggested that the phenomenon of phase variation might exist in B. holmesii. However, the observed differences in the protein patterns do not correlate with the presence or absence of a functional BvgABH protein as predicted by our sequence analysis of the same strains.
The observation that the bvgASBH locus is able to restore virulence gene expression in the B. pertussis bvgS mutant BP 347 but not in the bvgAS mutant BP 359 suggested cross-phosphorylation between the BvgSBH histidine kinase and BvgABP (Fig. 2). Similarly, cross-phosphorylation was also demonstrated to occur between BvgSBP and BvgABH in vitro (data not shown). Therefore, the cytoplasmic signalling domains of BvgSBH and BvgSBP, showing 52·9 % identity, are functionally interchangeable in the recognition of the orthologous BvgA response regulator proteins, whose receiver domains are 76·2 % identical. In the genetic background of BP 347, BvgSBH is only slightly responsive to the presence of 50 mM MgSO4, which causes phenotypic modulation in the wild-type B. pertussis strain TI. Therefore, in accordance with the observation that the periplasmic input domains of BvgSBH and BvgSBP show a far lower degree of similarity (39·5 % identity) than the cytoplasmic domains, signal perception of the BvgS proteins from B. holmesii and B. pertussis seems to be different. Similarly, in the genetic background of B. bronchiseptica, the function of the BvgSBP protein was indistinguishable from BvgSBB when the expression of virulence factors, under either standard in vitro culture conditions or in vivo growth in an animal model, was investigated, but the sensitivity to modulating agents in vitro was different between BvgSBB and BvgSBP (Martinez de Tejada et al., 1996
). BvgSBB and BvgSBP are highly similar (87 % overall identity), but, as seen with BvgSBH and BvgSBP, most of the non-conservative amino acid substitutions occur in the periplasmic domain which has been shown to be solely responsible for the observed differences in the sensitivity to modulating signals (Martinez de Tejada et al., 1996
).
In contrast to the cytoplasmic signalling domains of BvgSBP and BvgSBH, the BvgA response regulators are not functionally interchangeable between B. pertussis and B. holmesii. The observations that the B. pertussis bvgAS mutant BP 359 could not be complemented to virulence gene expression by the presence of the bvgAS locus of B. holmesii in trans (Fig. 2) and that BvgABH did not bind to the BvgABP binding sites of the B. pertussis fhaB (Fig. 3
) and bvgA (data not shown) promoters in vitro, indicate that BvgABH is unable to productively interact with BvgA-dependent promoters of B. pertussis which are characterized by the presence of multiple BvgABP binding sites. The fhaB promoter contains an imperfect heptanucleotide inverted-repeat sequence with abutting half-sites centred at position 88·5 relative to the transcriptional start site, which is the high-affinity binding site of a BvgABP-P dimer, as well as two additional low-affinity binding sites centred at position 67·5 and directly adjacent to the 35 region (Boucher et al., 2001
, 2003
). These secondary binding sites show a very limited degree of similarity to the high-affinity heptanucleotide inverted-repeat motif, but binding of BvgABP-P to the low-affinity binding sites is required for full transcriptional activation of the fhaB promoter (Boucher et al., 1997
, 2001
).
Similarly, the ptx promoter harbours two inverted heptad repeats in the region ranging from position 167 to 123 relative to the transcriptional start site. These repeats match the fhaB high-affinity binding motif in five of seven positions, and cooperative binding of multiple BvgABP-P dimers to the DNA downstream of these primary binding sites is necessary for the activation of ptx transcription (Boucher & Stibitz, 1995; Marques & Carbonetti, 1997
). Differences in the affinity of the BvgABP binding sites in the fhaB and ptx promoters, resulting in the requirement of higher concentrations of BvgABP-P for transcriptional activation of ptx, account for the different temporal pattern of expression of these virulence genes (Scarlato & Rappuoli, 1991
). Here we demonstrate that in B. holmesii transcription of bvgASBH is under control of two overlapping promoters which are positively regulated by BvgABH, since transcription of bvgASBH and expression of a gfp reporter gene which was fused to the bvgASBH promoter region were reduced in the bvgABH mutant G7702/bvgABH : : kan (Fig. 6
). However, basal BvgABH-independent transcription from these promoters was also detected in G7702/bvgABH : : kan, and this might be responsible for low-level expression of BvgASBH under conditions in which the BvgSBH histidine kinase is inactive. The moderate difference in the amount of bvgASBH transcript in strains G7702 and G7702/bvgABH : : kan might indicate that, in contrast to BvgSBP, BvgSBH is not fully activated under the standard in vitro culture conditions tested so far.
Interestingly, the region upstream of the regulated bvgASBH promoters, which is protected in DNase I footprint experiments with in vitro-phosphorylated BvgABH, contains four 14 bp sequence motifs (IR1IR4) in a regular spacing of 2021 bp, which show similarity to the heptanucleotide inverted-repeat sequence that has been deduced as consensus motif for the primary binding site for BvgABP in BvgABP-dependent B. pertussis promoters (Fig. 5). This arrangement of binding sites might suggest end-to-end binding of BvgABH dimers to the same face of the DNA helix. The sequence motifs IR1 and IR2, showing the highest degree of similarity to the BvgABP consensus half-site motif T/A-T-T-C-C/T-T-A, are located proximal to the transcriptional start site of bvgASBH, while IR3 and IR4, which match the sequence of the consensus binding site in four of seven positions, are located further upstream. This architecture of putative high- and low-affinity BvgABH binding sites differs from the fhaB and ptx promoters, but resembles the B. pertussis core bvgR promoter, which, for activity, requires binding of BvgABP-P to a secondary binding site located at a distance of 22 bp upstream from the primary binding site, which is centred at position 53·5 proximal to the transcriptional start site. Interestingly, the BvgABP binding motifs in the bvgR promoter also consist of one half-site with high-scoring similarity to the BvgABP consensus binding motif in combination with a low-scoring half-site (Merkel et al., 2003
). However, DNase I footprint experiments did not provide indications for differences in the binding affinities of sequence motifs IR1IR4 in the bvgASBH promoter since a sharp onset of protection, spanning the complete region from position 70 to 195 relative to the bvgABH start codon, was observed upon addition of low amounts of BvgABH. Considering the striking similarity of the bvgASBH promoter region to BvgA-dependent promoters of B. pertussis and the fact that in vitro binding of BvgABP-P to this promoter is virtually indistinguishable from BvgABH-P, the inability of BvgABH to interact with B. pertussis promoters remains unexplained.
In the B. pertussis bvgAS mutant BP 359 containing the bvgASBHup-gfp promoter fusion, transcription of the gfp reporter gene is directed from a promoter different to the overlapping P1 and P2 promoters in B. holmesii. Transcription from this promoter starts within the region of BvgABH- and BvgABP-binding, unless the detected transcript is the defined processing product of a longer mRNA transcribed from a cryptic promoter located further upstream or in the plasmid backbone (Fig. 5, Fig. 7
). In the B. pertussis wild-type strain TI, BvgABP-P does not stimulate transcription from the overlapping BvgABH-dependent P1 and P2 promoters, but instead binding of BvgABP-P to the upstream region of bvgASBH seems to interfere with transcription from the alternative promoter since no expression of the gfp reporter gene could be detected in TI(pMMB-bvgASBHup-gfp). Although in vitro binding of unphosphorylated BvgABP to the bvgASBH promoter region was not observed, expression of gfp was also strongly reduced in the bvgS mutant BP 347(pMMB-bvgASBHup-gfp) as compared to BP 359(pMMB-bvgASBHup-gfp) (Fig. 7
). It should be noted that despite the inhibitory effect of BvgABP on transcription from the bvgASBH upstream region in B. pertussis, low-level expression of BvgSBH was sufficient to restore virulence gene expression in BP 347(pRK-bvgASBH G7702). We are currently trying to identify the members of the BvgA regulon of B. holmesii. The characterization of additional BvgABH-dependent promoters will help to unravel the molecular basis of the striking differences in promoter recognition observed with the highly homologous BvgA proteins of B. holmesii and B. pertussis.
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ACKNOWLEDGEMENTS |
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Received 24 June 2004;
revised 10 August 2004;
accepted 12 August 2004.
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