Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis

Brownlie, Robert; Coote, J. G.; Parton, R.; Schultz, Joachim E.; Rogel, A; Hanski, Emanuel

doi:10.1016/0882-4010(88)90061-7

Cited by 39 publications

(32 citation statements)

References 19 publications

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“…It also includes the sites of insertion of TnS in the two B. pertussis mutants BP353 and BP354 which had an FHA-deficient phenotype (37). We and others (3,35) have observed that these mutant strains produce much lower but detectable levels of FHA. This FHA is apparently normal in size and retains biological activity.…”

Section: Methodsmentioning

confidence: 99%

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir

1988

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The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of TnS insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the ffa and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only flia or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by TnS mutagenesis in E. coi and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.Bordetella pertussis, the causative agent of whooping cough, coordinately regulates the production of toxins and other factors that are associated with its ability to cause disease (36). These virulent-phase genes include at least four toxins (pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, and hemolysin) and an attachment factor, the filamentous hemagglutinin (FHA).Two forms of regulation control the expression of these genes. The first is a coordinate, reversible repression influenced by the conditions of bacterial growth. B. pertussis does not express the virulent-phase genes when grown at temperatures lower than 37°C or in the presence of high concentrations of magnesium ions or nicotinic acid (15,18). In addition, B. pertussis strains undergo a metastable genetic event (phase variation) such that, at a frequency as high as 10-3 for some strains, variants arise in the population that no longer express any of the virulent-phase genes (20 As a first step in this investigation, we undertook the cloning of the vir locus and other genes that are regulated by the vir locus. These cloning experiments led to the isolation of a region of the B. pertussis chromosome encompassing the vir andfha loci. In E. coli this region directs the synthesis of a product that is immunoreactive with anti-FHA. This synthesis is modulated by environmental conditions in the same way as it is in B. pertussis. Characterization of this region by TnS mutagenesis identified sequences that are essential for the synthesis of FHA in E. coli. The return of these cloned sequences to B. pertussis ...

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Section: Methodsmentioning

confidence: 99%

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir

1988

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“…Plasmid vectors pGEM3Z (Promega, Madison, Wis.), pBluescript SK ϩ (Stratagene, La Jolla, Calif.), pBBR1MCS (29), and pRK415 (28) were used for the construction of recombinant plasmids. The cosmid pCP13-based gene library of B. pertussis UT25 has been described (12). Plasmids pBRM1, pBRM6, and pBRM9 contain the cloned mini-Tn5 lacZ1 chromosomal insertions of mutants BRM1, BRM6, and BRM9, respectively, along with flanking genomic sequences (6).…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes

et al. 1996

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Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-SmaI Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.Iron is a fundamental nutritional requirement for virtually all cells, and its assimilation is considered essential for invading pathogenic bacteria to establish infection in the iron-limiting environment of the host (13, 56). Additionally, iron serves as an environmental modulator of the production of certain virulence factors in a number of bacteria (14,24,31,32,46). Despite host iron sequestration, mediated primarily by the glycoprotein family of iron-binding transferrins, pathogens multiply successfully in vivo because they express efficient ironscavenging systems in response to decreased iron availability. These iron retrieval systems utilize two general strategies: one involving high-affinity iron-chelating soluble siderophores (30,40) and the other using siderophore-independent cell surface receptor mechanisms allowing iron uptake directly from host sources such as transferrin, lactoferrin, and heme compounds (7,35,38,54).Bordetella pertussis, the causative agent of human whooping cough or pertussis, and Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, are bacterial pathogens that infect the respiratory epithelial mucosae of their hosts. Early reports described the production of putative siderophores by both B. pertussis and B. bronchiseptica in response to iron deficiency (1, 23). Armstrong and Clements isolated and characterized B. bronchiseptica transposon-induced siderophore-deficient mutants; DNA hybridization studies using sequences flanking those transposon insertions confirmed the existence of homologs o...

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“…Plasmid pRK/3.2P, carrying the speC constitutive ornithine decarboxylase gene of E. coli, was constructed by subcloning the 3.2-kb E. coli PstI insert DNA fragment of plasmid pODC1 (8) into the broad-host-range plasmid vector pRK415 (26). DH5␣ (pRK2013) was used as the source of plasmid-encoded mobilization functions (19) in triparental matings to transfer the cosmid pCP13-based gene bank of B. pertussis UT25 (11) as well as plasmid vector pRK415 derivatives to Bordetella strains. E. coli K38 (pGP1-2) (46) was used as the host strain in Bordetella protein expression studies.…”

Section: Methodsmentioning

confidence: 99%

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin

Brickman

Armstrong

1996

J Bacteriol

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Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.Nutritional iron limitation, mediated primarily by specific host iron-binding glycoproteins, is a front-line host defense against disease-causing infectious agents. Strategies aimed at defeating host iron restriction may involve the action of lowmolecular-mass, high-affinity, ferric iron-specific chelators of microbial origin, termed siderophores, that are synthesized coordinately with their cognate surface receptors and transport machinery in response to iron starvation (28). The role of siderophores in microbial pathogenesis is well established (29,53,54).Bordetella pertussis, the causative agent of human whooping cough or pertussis, and Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, are mucosal pathogens that colonize the upper respiratory tracts of their mammalian hosts. In the first reported molecular genetic studies of Bordetella iron acquisition systems, Armstrong and Clements (3) described the isolation of B. bronchiseptica mutants deficient in siderophore activity following transposon mutagenesis. DNA hybridization analysis using DNA probe sequences flanking the transposon insertions established the existence of homologs of B. bronchiseptica siderophore genes in B. pertussis. Reciprocal cross-feeding ex...

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Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis

Cited by 39 publications

References 19 publications

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir

Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin

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