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1.
Genome Announc ; 5(45)2017 Nov 09.
Article in English | MEDLINE | ID: mdl-29122874

ABSTRACT

Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives.

2.
Mol Microbiol ; 40(4): 991-9, 2001 May.
Article in English | MEDLINE | ID: mdl-11401705

ABSTRACT

In order to define a consensus binding sequence for the response regulator BvgA, we have undertaken a systematic analysis of contributions made by each nucleotide within the heptad half-sites that are present in an inverted orientation at the promoter for the fha operon. Using in vitro binding assays, we examined the full complement of 21 single point mutations symmetrically arranged in this heptad repeat. Both gel shift and nitrocellulose filter-binding assays provided evidence that nucleotides at positions 3 (thymidine), 4 (cytosine) and 7 (adenine) in the binding heptad contribute substantially to sequence-specific recognition by BvgA. Furthermore, a T to A conversion at position 6 reduced binding. Selected binding site mutations were introduced into a modified fha promoter and examined for their effects on BvgA activation of promoter activity in vivo. Only those substitutions most severely affecting binding in vitro affected promoter activity in vivo. The in vivo effects of substitutions that had a significant effect on binding in vitro but did not severely affect in vivo promoter activity under standard culture conditions could be detected in vivo either in combination with additional substitutions or from their effect on the sensitivity of the mutant promoters to modulation by magnesium sulphate.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/metabolism , Bordetella pertussis/genetics , Fimbriae Proteins , Transcription Factors/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Bordetella pertussis/chemistry , Bordetella pertussis/drug effects , DNA Mutational Analysis , Magnesium Sulfate/pharmacology , Molecular Sequence Data , Oligonucleotides/chemical synthesis , Oligonucleotides/metabolism , Operon , Promoter Regions, Genetic , Transcription Factors/genetics
3.
J Bacteriol ; 183(2): 536-44, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11133947

ABSTRACT

The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA approximately P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.


Subject(s)
Adhesins, Bacterial/genetics , Bacterial Proteins/metabolism , Bordetella pertussis/genetics , Hemagglutinins/genetics , Promoter Regions, Genetic , Transcription Factors/metabolism , Virulence Factors, Bordetella , Base Sequence , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Models, Genetic , Molecular Sequence Data , Mutation , Protein Binding
4.
J Bacteriol ; 181(17): 5234-41, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10464192

ABSTRACT

Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvg regulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed at 10 min following an inducing signal, while ptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those of fha and ptx. We have identified cis-acting sequences necessary for expression of prn in B. pertussis by using prn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate transcription from the promoter. Our genetic data indicate that the region critical for prn activation extends upstream to position -84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, including prn-lac fusions, reverse transcriptase PCR, and 5' rapid amplification of cDNA ends, to localize and identify the bvg-dependent 5' end of the prn transcript to the cytosine at -125 with respect to the published start site.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/metabolism , Bordetella pertussis/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Transcription Factors/metabolism , Virulence Factors, Bordetella , Base Sequence , Binding Sites , DNA, Bacterial , Genes, Bacterial , Kinetics , Molecular Sequence Data , Transcriptional Activation
5.
J Bacteriol ; 181(17): 5512-5, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10464229

ABSTRACT

We determined the genomic organization of 14 clinical strains of Bordetella pertussis isolated over an 18-month period in Alberta, Canada. The maps of these 14 strains, while demonstrating general similarity of gene order, display a number of examples of genomic rearrangements in the form of large chromosomal inversions.


Subject(s)
Bordetella pertussis/genetics , Genome, Bacterial , Bordetella pertussis/isolation & purification , Canada/epidemiology , Chromosomes, Bacterial , Disease Outbreaks , Electrophoresis, Gel, Pulsed-Field , Humans , Whooping Cough/epidemiology , Whooping Cough/microbiology
6.
J Immunol ; 162(4): 2291-8, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-9973506

ABSTRACT

Bacterial DNA containing unmethylated CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-gamma, IL-12, TNF-alpha, and IL-6, which are important in the control of intracellular bacterial infection. Here, we show that bacterial DNA, as well as synthetic oligonucleotides containing CpG motifs, induce protection against large lethal doses of Francisella tularensis live vaccine strain (LVS) and Listeria monocytogenes. Methylation of DNA at CpG dinucleotides or inversion of the motif abolished this protection. Surprisingly, DNA-mediated protection was highly dependent on lymphocytes, particularly B cells, as well as the production of IFN-gamma. Optimal protection was elicited 2-3 days after inoculation with DNA and persisted for up to 2 wk. Further, animals surviving lethal challenge developed pathogen-specific secondary immunity. These findings indicate that host innate immune responses to bacterial DNA may contribute to the induction of protective immunity to bacteria and the subsequent development of memory.


Subject(s)
CpG Islands/immunology , DNA, Bacterial/immunology , Intracellular Fluid/immunology , Listeriosis/prevention & control , Lymphocytes/immunology , Lymphocytes/microbiology , Tularemia/prevention & control , Animals , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , DNA, Bacterial/genetics , Francisella tularensis/genetics , Intracellular Fluid/microbiology , Listeria monocytogenes/genetics , Listeriosis/immunology , Listeriosis/mortality , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, SCID , Oligonucleotides/immunology , Tularemia/immunology , Tularemia/mortality , Vaccines, DNA/immunology
7.
J Bacteriol ; 180(18): 4963-6, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9733704

ABSTRACT

The insertion sequence IS481 and its isoform IS1002 have been observed to transpose into the bvgAS locus of Bordetella pertussis, for which the DNA sequence has previously been determined. Upon insertion of IS481 at three different sites and IS1002 at one site, a 6-bp sequence originally present was found at the junction of bvg and insertion sequence DNA. This indicates that, contrary to prior reports, IS481 and IS1002 do create a duplication upon insertion. In this light, examination of these and other examples of IS481 and IS1002 reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS481 or IS1002, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.


Subject(s)
Bordetella pertussis/genetics , DNA Transposable Elements , Repetitive Sequences, Nucleic Acid , Base Sequence , Humans , Molecular Sequence Data
8.
Infect Immun ; 66(9): 4367-73, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9712789

ABSTRACT

Whooping cough is an acute respiratory disease caused by the small, gram-negative bacterium Bordetella pertussis. B. pertussis expresses several factors that contribute to its ability to cause disease. These factors include surface-associated molecules, which are involved in the adherence of the organism to respiratory epithelial cells, as well as several extracellular toxins that inhibit host defenses and induce damage to host tissues. The expression of virulence factors in B. pertussis is dependent upon the bvg locus, which consists of three genes: bvgA, bvgS, and bvgR. The bvgAS genes encode a two-component regulatory system consisting of a sensor protein, BvgS, and a transcriptional activator, BvgA. Upon modification by BvgS, BvgA binds to the promoter regions of the bvg-activated genes and activates transcription. One of the bvg-activated genes, bvgR, is responsible for the regulation of the bvg-repressed genes, the functions of which are unknown. The fact that these genes are regulated by the bvg locus suggests that they play a role in the pathogenesis of the bacterium. In order to evaluate the contribution of bvg-mediated regulation to the virulence of B. pertussis and determine if expression of the bvg-repressed genes is required for the virulence of B. pertussis, we examined the ability of B. pertussis mutants, defective in their ability to regulate the expression of the bvg-activated and/or the bvg-repressed genes, to cause disease in the mouse aerosol challenge model. Our results indicate that the bvgR-mediated regulation of gene expression contributes to respiratory infection of mice.


Subject(s)
Bacterial Proteins/physiology , Bordetella pertussis/physiology , Plant Proteins/physiology , Transcription Factors/physiology , Whooping Cough/microbiology , Adhesins, Bacterial/metabolism , Aerosols , Animals , Bacterial Proteins/genetics , Bordetella pertussis/genetics , Bordetella pertussis/metabolism , Disease Models, Animal , Gene Expression Regulation, Bacterial , Hemagglutinins/metabolism , Mice , Mice, Inbred BALB C , Plant Proteins/genetics , Rabbits , Transcription Factors/genetics , Virulence/genetics , Virulence Factors, Bordetella/metabolism , Whooping Cough/metabolism
9.
J Bacteriol ; 180(9): 2484-92, 1998 May.
Article in English | MEDLINE | ID: mdl-9573202

ABSTRACT

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg- phenotype. When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the alpha subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the alpha subunit of B. pertussis RNA polymerase were also isolated. Two of these resulted in increased levels of the alpha subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced.


Subject(s)
Bacterial Proteins/metabolism , Bordetella pertussis/metabolism , DNA-Directed RNA Polymerases/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bordetella pertussis/genetics , Bordetella pertussis/growth & development , Bordetella pertussis/pathogenicity , Chromosomes, Bacterial , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Bacterial , Genetic Linkage , Molecular Sequence Data , Mutation , Phenotype , Plasmids/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/genetics
10.
J Bacteriol ; 180(7): 1682-90, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9537363

ABSTRACT

Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors is dependent upon the bvg locus (originally designated the vir locus), which encodes two proteins: BvgA, a 23-kDa cytoplasmic protein, and BvgS, a 135-kDa transmembrane protein. It is proposed that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by the bvg locus. Expression of this class, the bvg-repressed genes (vrgs [for vir-repressed genes]), is reduced under conditions in which expression of the aforementioned bvg-activated virulence factors is maximal; this repression is dependent upon the presence of an intact bvgAS locus. We have previously identified a locus required for regulation of all of the known bvg-repressed genes in B. pertussis. This locus, designated bvgR, maps to a location immediately downstream of bvgAS. We have undertaken deletion and complementation studies, as well as sequence analysis, in order to identify the bvgR open reading frame and identify the cis-acting sequences required for regulated expression of bvgR. Studies utilizing transcriptional fusions of bvgR to the gene encoding alkaline phosphatase have demonstrated that bvgR is activated at the level of transcription and that this activation is dependent upon an intact bvgAS locus.


Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , Genes, Bacterial , Genes, Regulator , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Genetic Complementation Test , Molecular Sequence Data , Open Reading Frames
11.
Gene ; 208(2): 183-9, 1998 Feb 27.
Article in English | MEDLINE | ID: mdl-9524261

ABSTRACT

A plasmid vector was designed, constructed, and used for the repeated retrieval of the bvgA gene from a number of Bordetella pertussis strains that, due to mutations in this gene, exhibited interesting phenotypes regarding the regulation of virulence genes. The vector was used in a scheme called allelic retrieval that exploits two cross-overs between cloned plasmid and native chromosomal sequences flanking the bvgA gene. This scheme is very similar to allelic exchange through the use of plasmid suicide vectors, but in the case presented here, the non-replicating plasmid that has received the chromosomal gene is recovered, rather than being allowed to be lost due to segregation. Incorporation of the counterselectable sacB gene of Bacillus subtilis in place of the plasmid copy of bvgA allows selection, after recovery in Escherichia coli, for only those plasmids that have retrieved the chromosomal bvgA gene. The validity of this approach was demonstrated by the retrieval of bvgA alleles with distinctive physical markers, as well as by the reintroduction of retrieved bvgA alleles to demonstrate that they conferred the expected phenotypes. It is expected that this approach will be applicable to the analysis of other genes in other bacterial species.


Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , Chromosomes, Bacterial , Escherichia coli Proteins , Transcription Factors/genetics , Alleles , Bacterial Proteins/biosynthesis , Bordetella pertussis/pathogenicity , Chromosome Mapping , Cloning, Molecular/methods , Escherichia coli , Gene Expression Regulation, Bacterial , Genetic Techniques , Models, Genetic , Plasmids , Restriction Mapping , Transcription Factors/biosynthesis , Virulence/genetics
12.
J Bacteriol ; 179(18): 5820-6, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9294440

ABSTRACT

The genomic organization of Bordetella pertussis strains has been examined by using a new method. This method does not depend on the prior determination of a restriction map of the bacterial chromosome but is based on the ability to measure directly the distance between two genes. This is accomplished through the integration at each gene of a suicide vector containing a cleavage site for the intron-encoded endonuclease I-SceI, which is not otherwise found in the chromosome. Integration is mediated by homologous recombination between the chromosomal and cloned plasmid copies of a gene of interest. Digestion with I-SceI gives rise to a fragment the size of which represents the distance between the two genes. Multiple pairwise determinations within a set of genes provide sufficient information to derive a map of the relative gene positions. Mapping a set of 11 to 13 genes for five strains of B. pertussis and one strain of B. parapertussis revealed extensive divergence of gene order between B. pertussis Tohama I, B. pertussis 18-323, and B. parapertussis ATCC 15311. Less extensive divergence of gene order was observed between B. pertussis Tohama I and B. pertussis Tohama III, BP165, and Wellcome 28, with most of the observed differences explainable by large inversions.


Subject(s)
Bordetella pertussis/genetics , Bordetella/genetics , Chromosome Mapping/methods , Chromosome Inversion , DNA, Bacterial/genetics , Genes, Bacterial , Plasmids , Species Specificity
13.
J Bacteriol ; 179(5): 1755-63, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9045838

ABSTRACT

The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system. The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Previous studies have demonstrated that these two loci are differentially regulated by BvgA. As an initial step in gaining insight into the mechanism underlying this differential regulation, we initiated DNA binding and in vitro transcription analyses to examine the activities of BvgA and RNA polymerase (RNAP) purified from both B. pertussis and Escherichia coli at the fha promoter. We discovered that unphosphorylated BvgA binds to a single region (-100 to -70, relative to the start of transcription), whereas phosphorylated BvgA binds both this region and another, farther downstream, that extends to the -35 nucleotide. In the absence of BvgA, RNAP binds a region farther upstream than expected (-104 to -35). However, occupation of both sites by BvgA phosphate repositions RNAP to the site used in vivo. The binding of BvgA phosphate to the downstream site correlates with in vitro transcriptional activity at the fha promoter. As the DNA binding and transcription activities of the E. coli-derived RNAP are similar to those observed for the B. pertussis enzyme, we employed several mutant E. coli proteins in in vitro transcription analyses. We observed that polymerases carrying either a deletion of the C-terminal domain of the alpha subunit or substitution of alanine at either of two critical residues within this domain were severely impaired in the ability to mediate BvgA-activated transcription at fha.


Subject(s)
Adhesins, Bacterial/genetics , Bacterial Proteins/metabolism , Bordetella pertussis/genetics , DNA, Bacterial/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins , Hemagglutinins/genetics , Promoter Regions, Genetic , Transcription Factors/metabolism , Virulence Factors, Bordetella , Base Sequence , Bordetella pertussis/enzymology , Bordetella pertussis/metabolism , DNA-Directed RNA Polymerases/chemistry , Escherichia coli/enzymology , Molecular Sequence Data , Phosphorylation , Protein Conformation , Protein Folding , Protein Structure, Secondary , Transcription, Genetic
14.
J Bacteriol ; 177(22): 6486-91, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7592424

ABSTRACT

Regulation of virulence factor expression in Bordetella pertussis is mediated by the BvgAS two-component regulatory system. Although previous studies have demonstrated that the transcriptional regulation of the filamentous hemagglutinin gene (fhaB) involves binding of the BvgA activator directly to the fhaB promoter region, the mechanism of pertussis toxin operon (ptx) regulation by BvgA has remained unclear. We demonstrate in vitro the specific binding of BvgA to a region upstream of the ptx promoter that encompasses a 20-bp directly repeated sequence (positions -157 to -117) previously shown to be critical for BvgA-dependent activation. This binding is strictly dependent on the phosphorylation of BvgA, which can be obtained by incubation of BvgA with acetyl phosphate. By DNase I protection studies, we demonstrate the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the ptx promoter. In the presence of the polymerase holoenzyme, a greatly extended footprint encompassing the region between -163 and the putative polymerase binding site was observed. The implications of these observations for pertussis toxin expression and regulation are discussed.


Subject(s)
Bacterial Proteins/metabolism , Bordetella pertussis/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins , Pertussis Toxin , Promoter Regions, Genetic/genetics , Transcription Factors/metabolism , Virulence Factors, Bordetella/genetics , Base Sequence , Binding Sites , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial/genetics , Molecular Sequence Data , Organophosphates , Phosphates/metabolism , Phosphorylation , Repetitive Sequences, Nucleic Acid/genetics
15.
J Bacteriol ; 177(10): 2727-36, 1995 May.
Article in English | MEDLINE | ID: mdl-7751282

ABSTRACT

In Bordetella pertussis, the coordinate regulation of virulence factor expression is controlled by the products of the bvgAS locus. In the presence of modulating signals such as MgSO4, nicotinic acid, or reduced temperature, the expression of bvg-activated genes is reduced while the expression of bvg-repressed genes is induced. One model for the regulation of bvg-repressed genes predicts the existence of a repressor protein encoded by a bvg-activated gene. Once activated, the product of this bvg-activated gene would bind to and repress transcription from the bvg-repressed genes. We isolated five genetically independent transposon insertion mutants of B. pertussis that have a phenotype consistent with the knockout of a putative bvg-regulated repressor. These mutants constitutively expressed a vrg6-phoA transcriptional fusion but demonstrated normal bvgAS function. Genomic mapping and DNA sequence analysis of the sites of transposon insertion demonstrated that these mutants define a locus downstream of bvgAS. Introduction of an in-frame, 12-bp insertion within this locus also conferred the mutant phenotype, confirming that the phenotype seen in the transposon mutants is the result of disruption of a distinct gene, which we have designated bvgR, and is not a consequence of polar effects on bvgAS.


Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Regulon/genetics , Repressor Proteins/genetics , Alkaline Phosphatase/biosynthesis , Alkaline Phosphatase/genetics , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Base Sequence , Bordetella pertussis/pathogenicity , Chromosome Mapping , DNA Transposable Elements , Genetic Complementation Test , Models, Genetic , Molecular Sequence Data , Mutagenesis, Insertional , Operon/genetics , Reading Frames/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription Factors/genetics , Virulence/genetics
16.
J Bacteriol ; 176(23): 7260-6, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7961497

ABSTRACT

We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis. A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B. pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B. pertussis bacterium has been constructed. This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B. pertussis genome. In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored. In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined. We have used this genetic system to map a newly described locus in B. pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).


Subject(s)
Bordetella pertussis/genetics , Bordetella pertussis/pathogenicity , Chromosome Mapping/methods , Gene Expression Regulation, Bacterial , Point Mutation/genetics , Adenylate Cyclase Toxin , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Chromosomes, Bacterial , Conjugation, Genetic , Crosses, Genetic , Genes, Bacterial/genetics , Molecular Sequence Data , Pertussis Toxin , Plasmids/genetics , Protein Precursors/biosynthesis , Protein Precursors/genetics , Virulence/genetics , Virulence Factors, Bordetella/biosynthesis , Virulence Factors, Bordetella/genetics
17.
J Bacteriol ; 176(18): 5615-21, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8083156

ABSTRACT

By using chemical mutagenesis and genetic mapping, a search was undertaken for previously undescribed genes which may be involved in different regulatory mechanisms governing different virulence factors of Bordetella pertussis. Previous studies have shown that the fha locus encoding filamentous hemagglutinin is regulated directly by the bvgAS two component system, while regulation of ptx encoding pertussis toxin is less direct or occurs by a different mechanism. With a strain containing gene fusions to each of these regulated loci, screening was done for mutations which were defective for ptx expression but maintained normal or nearly normal levels of fha expression. Two mutations which had such a phenotype and were also deficient in adenylate cyclase toxin/hemolysin expression were found and characterized more fully. Both were found to affect residues in the C-terminal portion of the BvgA response regulator protein, a domain which shares sequence similarity with a family of regulatory proteins including FixJ, UhpA, MalT, RcsA, RcsB, and LuxR. The residues affected are within a region which, by extension from studies on the LuxR protein, may be involved in transcriptional activation.


Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , Gene Expression Regulation, Bacterial/genetics , Genes, Regulator/genetics , Mutation/genetics , Transcription Factors/genetics , Adenylate Cyclase Toxin , Amino Acid Sequence , Bacterial Outer Membrane Proteins/analysis , Bacterial Proteins/analysis , Base Sequence , Bordetella pertussis/pathogenicity , DNA Mutational Analysis , Hemagglutinins/genetics , Molecular Sequence Data , Pertussis Toxin , Recombinant Fusion Proteins/biosynthesis , Restriction Mapping , Virulence , Virulence Factors, Bordetella/analysis , Virulence Factors, Bordetella/genetics
19.
J Bacteriol ; 174(23): 7770-7, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1447143

ABSTRACT

We have used pulsed-field gel electrophoresis to derive a restriction map of the chromosome of Bordetella pertussis for the enzymes XbaI, SpeI, PacI, and PmeI, which cleave 25, 16, 2, and 1 times, respectively. The apparent size of the genome is 3,750 kb. The positions of genes for major virulence determinants in the vir regulon and of some housekeeping genes were determined. Apart from the previously known linkage of the vir and fha loci, no significant linkage of virulence genes was demonstrated.


Subject(s)
Bordetella pertussis/genetics , Chromosome Mapping , Chromosomes, Bacterial , Base Sequence , DNA, Bacterial/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Electrophoresis, Gel, Pulsed-Field , Genes, Bacterial/genetics , Molecular Sequence Data
20.
J Bacteriol ; 173(14): 4288-96, 1991 Jul.
Article in English | MEDLINE | ID: mdl-2066330

ABSTRACT

The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded. Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane. We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins. PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting. Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems. We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS.


Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/genetics , Escherichia coli Proteins , Genes, Bacterial , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Bordetella pertussis/isolation & purification , Bordetella pertussis/pathogenicity , DNA, Bacterial/genetics , Humans , Molecular Sequence Data , Oligonucleotide Probes , Plasmids , Restriction Mapping , Transcription Factors/analysis , Virulence/genetics
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