Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes

Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica. It is shown here that these two species possess but do not express the complete toxin operon. Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B. parapertussis and B. bronchiseptica are 98.5% and 96% homologous, respectively, to B. pertussis. The changes (mostly base pair substitutions) in many cases are identical in B. parapertussis and B. bronchiseptica, suggesting that these two species derive from a common ancestor. Many of the mutations common to B. parapertussis and B. bronchiseptica involve the promoter region, which becomes very inefficient. The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B. pertussis, indicating that the mutations in the S1 gene described here do not affect its function.     

Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica

Bordetella pertussis and Bordetella bronchiseptica contain nearly identical BvgAS signal-transduction systems that mediate a biphasic transition between virulent (Bvg⁺) and avirulent (Bvg^–) phases. In the Bvg⁺ phase, the two species express a similar set of adhesins and toxins, and in both organisms the transition to the Bvg^– phase occurs in response to the same environmental signals (low temperature or the presence of nicotinic acid or sulphate anion). These two species differ, however, with regard to Bvg^–-phase phenotypes, host specificity, the severity and course of the diseases they cause, and also potentially in their routes of transmission. To investigate the contribution of the virulence-control system to these phenotypic differences, we constructed a chimeric B. bronchiseptica strain containing bvgAS from B. pertussis and compared it with wild-type B. bronchiseptica in vitro and in vivo . The chimeric strain was indistinguishable from the wild type in its ability to express Bvg⁺- and Bvg^–-phase-specific factors. However, although the chimeric strain responded to the same signals as the wild type, it differed dramatically in sensitivity to these signals; significantly more nicotinic acid or MgSO₄ was required to modulate the chimeric strain compared with the wild-type strain. Despite this difference in signal sensitivity, the chimeric strain was indistinguishable from the wild type in its ability to cause respiratory-tract infections in rats, indicating that the bvgAS loci of B. pertussis and B. bronchiseptica are functionally interchangeable in vivo . By exchanging discrete fragments of bvgAS , we found that the periplasmic region of BvgS determines signal sensitivity.     

Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants.

We report the isolation and preliminary phenotypic characterization of manganese-resistant Bordetella bronchiseptica mutants with respect to deregulation of siderophore and iron-regulated protein expression. The fur gene of Bordetella pertussis was cloned by genetic complementation of this deregulated phenotype and confirmed as fur by nucleotide sequence analysis.     

Temporal expression of pertussis toxin and Ptl secretion proteins by Bordetella pertussis

Pertussis toxin is an AB(5) toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia

Infection of the respiratory tract caused by Burkholderia cepacia complex poses a serious risk for cystic fibrosis (CF) patients due to the high morbidity and mortality associated with the chronic infection and the lack of efficacious antimicrobial treatments. A detailed understanding of the pathogenicity of B. cepacia complex infections is hampered in part by the limited availability of genetic tools and the inherent resistance of these isolates to the most common antibiotics used for genetic selection. In this study, we report the construction of an expression vector which uses the rhamnose-regulated P(rhaB) promoter of Escherichia coli. The functionality of the vector was assessed by expressing the enhanced green fluorescent protein (eGFP) gene (e-gfp) and determining the levels of fluorescence emission. These experiments demonstrated that P(rhaB) is responsive to low concentrations of rhamnose and it can be effectively repressed with 0.2% glucose. We also demonstrate that the tight regulation of gene expression by P(rhaB) promoter allows us to extend the capabilities of this vector to the identification of essential genes.     

Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica

The gene coding for a thermostable alpha-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulfurogenes carrying the alpha-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

The effect of pH on the production of pertussis toxin by Bordetella pertussis.

The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields.     

Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.     

Purification and characterization of heat-labile toxin from Bordetella bronchiseptica

The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.     

Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.