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The two-component response regulator RisA, encoded by open reading frame BP3554 in the Bordetella pertussis Tohama I genomic sequence, is a known activator of vrg genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the bvgAS virulence regulon. Here we demonstrate that RisA is phosphorylated in vivo and that RisA phosphorylation is required for activation of vrg genes. An adjacent histidine kinase gene, risS, is truncated by frameshift mutation in B. pertussis but not in Bordetella bronchiseptica or Bordetella parapertussis. Neither deletion of risS' or bvgAS nor phenotypic modulation with MgSO4 affected levels of phosphorylated RisA (RisA~P) in B. pertussis. However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the vrg genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisAD60E mutant, indicating that an active conformation of RisA, but not phosphorylation per se, is crucial for vrg activation. Interestingly, expression of vrg genes is still modulated by MgSO4 in cells harboring the RisAD60E mutation, suggesting that the activated RisA senses additional signals to control vrg expression in response to environmental stimuli. [ABSTRACT FROM AUTHOR]

ordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg+ mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dit operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of D-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens. [ABSTRACT FROM AUTHOR]

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes. [ABSTRACT FROM AUTHOR]

The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussis adenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. [ABSTRACT FROM AUTHOR]

Pertussis toxin (PT) is secreted from Bordetella pertussis by a type IV secretion system, known as the Ptl transporter, that comprises nine different proteins, PtlA to PtlI. In this study, we found that PtlD is required for the stability of three Ptl proteins, PtlE, PtlF, and PtlH. A region limited to the C-terminal 72 amino acids of PtlD (amino acids 392 to 463) was sufficient for maintaining the stability of PtlE, PtlF, and PtlH, although this region was not sufficient to support secretion of the toxin. Further analysis demonstrated that a stretch of 10 amino acids at the C-terminal end of PtlD (amino acids 425 to 434) contributes to transporter stability. [ABSTRACT FROM AUTHOR]

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochcome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed. [ABSTRACT FROM AUTHOR]

Bordetella pertussis and Bordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efllux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study, alcS null mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export, trans complementation using cloned alcS genes of B. pertussis or B. bronchiseptica restored the wild-type phenotype to the alcS mutants. Although the levels of extracellular alcaligin measured in alcS strain culture fluids were severely reduced compared with the wild-type levels, alcS mutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a Δa/cA mutation that eliminated alcaligin production suppressed the growth defects of alcS mutants. This suppression and the alcaligin production defect were reversed by trans complementation of the ΔalcA mutation in the double-mutant strain, confirming that the growth-defective phenotype of alcS mutants is associated with alcaligin production. In an alcA::mini-Tn5 lacZ1 operon fusion strain background, an alcS null mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer;, conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression. [ABSTRACT FROM AUTHOR]

The BvgAS two-component system positively regulates the expression of the virulence genes of Bordetella pertussis and negatively regulates a second set of genes whose function is unknown. The BvgAS-mediated regulation of the bvg-repressed genes is accomplished through the activation of expression of the negative regulator, BvgR. A second two-component regulatory system, RisAS, is required for expression of the bvg- repressed surface antigens VraA and VraB. We examined the roles of BvgR and RisA in the regulation of four bvg-repressed genes in B. pertussis. Our analyses demonstrated that all four genes are repressed by the product of the bvgR locus and are activated by the product of the nsA locus. Deletion analysis of the vrg6 promoter identified the upstream and downstream boundaries of the promoter and, in contrast to previously published results, demonstrated that sequences downstream of the start of transcription are not required for the regulation of expression of vrg6. Gel mobility-shift experiments demonstrated sequence-specific binding of RisA to the vrg6 and vrg18 promoters, and led to the identification of two putative RisA binding sites. Finally, transcriptional analysis and Western blot analysis demonstrated that BvgR regulates neither the expression nor the stability of RisA. [ABSTRACT FROM AUTHOR]

Utilization of the enterobactin siderophore by the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica is dependent on the BfeA outer membrane receptor. This study determined that production of BfeA was increased significantly in iron-starved bacteria upon supplementation of cultures with enterobactin. A 1.01-kb open reading frame, designated bfeR, encoding a predicted positive transcriptional regulator of the AraC family was identified upstream and divergently oriented from bfeA. In iron-depleted cultures containing enterobactin, a Bordetella bfeR mutant exhibited markedly decreased BfeA receptor production compared to that of the wild-type strain. Additionally, B. pertussis and B. bronchiseptica bfeR mutants exhibited impaired growth with ferric enterobactin as the sole source of iron, demonstrating that effective enterobactin utilization is bfeR dependent. Transcriptional analysis using bfeA-lacZ reporter fusions in wild-type strains demonstrated that bfeA transcription was stimulated in iron-depleted conditions in the presence of enterobactin, compared to modest expression levels in cultures lacking enterobactin. In contrast, bfeA transcription in B. pertussis and B. bronchiseptica bfeR mutants was completely unresponsive to the enterobactin inducer. bfeA transcriptional analyses of a bfeA mutant demonstrated that induction by enterobactin did not require BfeA receptor-mediated uptake of the siderophore. These studies establish that bfeR encodes an enterobactin-dependent positive regulator of bfeA transcription in these Bordetella species. [ABSTRACT FROM AUTHOR]

Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected. [ABSTRACT FROM AUTHOR]

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type HI secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells. [ABSTRACT FROM AUTHOR]

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers. [ABSTRACT FROM AUTHOR]

The Bordetella pertussis heme utilization gene cluster hurIR bhuRSTUV encodes regulatory and transport functions required for assimilation of iron from heme and hemoproteins. Expression of the bhu genes is iron regulated and heme inducible. The putative extracytoplasmic function (ECF) σ factor, HurI, is required for heme-responsive bhu gene expression. In this study, transcriptional activation of B. pertussis bhu genes in response to heme compounds was shown to be dose dependent and specific for heme; protoporphyrin IX and other heme structural analogs did not activate bhu gene expression. Two promoters controlling expression of the heme utilization genes were mapped by primer extension analysis. The hurI promoter showed similarity to σ70-like promoters, and its transcriptional activity was iron regulated and heme independent. A second promoter identified upstream of bhuR exhibited little similarity to previously characterized ECF σ factordependent promoters. Expression of bhuR was iron regulated, heme responsive, and hurI dependent in B. pertussis, as shown in a previous study with Bordetella bronchiseptica. Further analyses showed that transcription originating at a distal upstream site and reading through the hurR-bhuR intergenic region contributes to bhuR expression under iron starvation conditions in the absence of heme inducer. The pattern of regulation of the readthrough transcript was consistent with transcription from the hurI promoter. The positions and regulation of the two promoters within the hur-bhu gene cluster influence the production of heme transport machinery so that maximal expression of the bhu genes occurs under iron starvation conditions only in the presence of heme iron sources. [ABSTRACT FROM AUTHOR]

Pertussis toxin is an AB5 toxin comprised of protein subunits SI through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Pti 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/rain/cell from 2 to 18 h. More of toxin subunits SI, 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 Pti complex assembly, is rate limiting. [ABSTRACT FROM AUTHOR]

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 are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein. It is hypothesized 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 products of the bvg locus. The repression of these genes is dependent upon the third gene, bvgR. Expression of bvgR is dependent upon the function of BvgA and BvgS. This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR. We undertook an analysis of the transcriptional activation of bvgR expression. We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension. We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA. Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses. A model of BvgA binding to the bvgR promoter is presented. [ABSTRACT FROM AUTHOR]

A family of genes that are likely to encode extracytoplasmic solute receptors is strongly overrepresented in several β-proteobacteria, including Bordetella pertussis. This gene family, of which members have been called bug genes, contains some examples that are contained within polycistronic operons coding for tripartite uptake transporters of the TTT family, while the vast majority are "orphan" genes. Proteomic and functional analyses demonstrated that several of these genes are expressed in B. pertussis, and one is involved in citrate uptake. The bug genes probably form an ancient family that has been subjected to a large expansion in a restricted phylogenic group. [ABSTRACT FROM AUTHOR]

Bordetella pertussis and Bordetella bronchiseptica, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the bhuRSTUV genes. Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions. The hurIR (heme uptake regulator) genes were previously identified upstream of the bhuRSTUV gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators. In this study, B. pertussis and B. bronchiseptica ΔhurI mutants, predicted to lack an ECF σ factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin. Genetic complementation of ΔhurI strains with plasmid-borne hurI restored wild-type levels of heme utilization. B. bronchiseptica ΔhurI mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor; hurI in trans restored heine-inducible BhuR expression to the mutant and resulted in BhuR overproduction. Transcriptional analyses with bhuR-lacZ fusion plasmids confirmed that bhuR transcription was activated in iron-starved cells in response to heme compounds. Heme-responsive bhuR transcription was not observed in mutant BRM23, indicating that hurI is required for positive regulation of bhu gene expression. Furthermore, bhuR was required for heme-inducible bhu gene activation, supporting the hypothesis that positive regulation of bhuRSTUV occurs by a surface signaling mechanism involving the heme-iron receptor BhuR. [ABSTRACT FROM AUTHOR]

The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "α-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "β-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal α-domain and a 30-kDa C-terminal β-domain. We have previously shown that a recombinant form of the β-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31- to 39-amino-acid region found immediately upstream of the β-domain that was essential for surface expression. This 31- to 39-amino-acid linker region, together with the β-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface. [ABSTRACT FROM AUTHOR]

Examines the peptidoglycanase activity of the Pt1E protein of Bordetella pertussis. Components of pertussis toxin; Role of peptidoglycan in the periplasm of large folded molecules; Influence of the amino acid substitutions at the glycohydrolase active site on the peptidoglycanase activity.

Examines adenylate cyclase (AC) toxin essential for virulence factor produced by bordetella pertussis. Details on the pathophysiologic function of AC toxin; Importance of deletion mutants; Prevention of delivery of catalytic domain.

Identifies a cluster of genes coding a putative bacterial heme iron acquisition system in a Bordetella pertussis genomic sequence database. Construction of B. pertussis bhuR mutant PM5; Characterization of DNA sequences upstream of bhuRSTUV gene; Identification of open reading frames encoding FecI and FecR protein homologs; Analysis on heme-responsive protein expression; Production of BhuR protein by iron-starved B. bronchiseptica.

Presents a genetic and biochemical analyses of the cognate response regulator BvgA interaction with the secondary binding region of the fha promoter of Bordetella pertussis. Positive control of bacterial transcription; Intermolecular interactions involved in fha promoter activation.

Details the identification of virulence-activated and -repressed genes in Bordetella pertussis. Use of systematic gene inactivation and generation of transcriptional fusions in the study; Regulation of genes encoding adhesins and an autotransporter protein; Repression of gene encoding a putative intimin-like protein under virulent conditions.

Focuses on the characterization of a Bordetella pertussis diaminopimelate (DAP) biosynthesis locus that identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase. Cloning and characterization of the dap locus of Bordetella pertussis; Search for sequence similarities of the putative open reading frames.

The two-component system BvgAS controls the virulence regulon in Bordetella pertussis. BvgS is the prototype of a family of sensor histidine kinases harboring periplasmic Venus flytrap (VFT) domains. The VFT domains are connected to the cytoplasmic kinase moiety by helical linkers separated by a Per-ARNT-Sim (PAS) domain. Antagonism between the two linkers, as one forms a coiled coil when the other is dynamic and vice versa, regulates BvgS activity. Here, we solved the structure of the intervening PAS domain by X-ray crystallography. Two forms were obtained that notably differ by the connections between the PAS core domain and the flanking helical linkers. Structure-guided mutagenesis indicated that those connections participate in the regulation of BvgS activity. Thus, the PAS domain appears to function as a switch facilitator module whose conformation determines the output of the system. As many BvgS homologs have similar architectures, the mechanisms unveiled here are likely to generally apply to the regulation of sensor histidine kinases of that family. [ABSTRACT FROM AUTHOR]

Investigates the putative pore-forming ability of the C-terminal domain of the Bordetella pertussis autotransporter BrkA through lipid bilayer analysis. Purification of the BrkA C-terminal domain; Performance of black lipid bilayer experiments with purified recombinant proteins; Translocation of the N-terminal domain of the protein across the outer membrane.

Determines the genomic organization of 14 clinical strains of Bordetella pertussis isolated over an 18-month period in Alberta, Canada. Bacterial strain mass; General similarity of the gene order; Genomic rearrangements in the form of large chromosomal inversions.

Analyzes the transcription activator protein BvgA activation of the pertactin gene promoter in the whooping cough agent Bordetella pertussis. Two-component signal transduction system encoding by the bvg regulatory locus; Virulence factors; Fha transcription; DNA manipulations and allelic exchange; DNA sequence analysis; 5' RACE analysis.

Compares the 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) content of endotoxins of Bordetella pertusis and Escherichia coli. Role of the waaA gene in encoding the Kdo; Structural analysis of the lipopolysaccharides in both pathogens.

Cites a study which detected the insertion sequences IS481 and IS102, in the bacterium Bordetella pertussis. Determination of the DNA sequence of the insertions sequences; Characterization of the insertion sequences; Results of the study.

Provides information on a study examining the effects of short6 deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system in Bordetella pertussis. Methodology and materials used to conduct the study; Results of the study.

Presents a study which gives a characterization of the bvgR locus of Bordetella pertusis. How gram-negative bacterium Bordetella pertussis relates to the whooping cough disease; Description of a class of genes which is repressed by the bvg locus; Demonstration of an activation of expression of the bvgR gene by BvgA.

Identifies the target genes of the global iron regulator, which is the first step to elucidate the iron regulatory network in Bordetella pertussis (B. pertussis). Characterization of B. pertussis and B. bronchiseptica alcR mutants; Colonization assay in the murine model; Information on the presence of the Fur-repressed gene in other Bordetella genomes; Results and discussion of the study.

Examines the DNA locus for Bordetella pertussis in humans in relation to the whooping cough. Background information on the DNA gene in relation to proteins; Functions of this gene; Information on the structure of the Bordetella pertussis; Methodology used in this experiment.

Examines the activities of the host species of Bordetella pertussis while relating to the structural subunit gene fimA. Characterization of genetic basis for fimbrial expression in Bordetella pertussis; Location of fimbrial operon; Observation of fimbrial phase variation; Contents of gene; Reflection of differences in host species specificities of three bordetella species.

Analyzes the role of the consensus nucleotide-binding site of PtlH in secretion of pertussis toxin (PT) from Bordetella pertussis. Effect of mutation of ptlH on secretion of PT from Bordetella pertussis; How the secretion of the SI subunit represents the secretion of the holotoxin molecule; What results indicate.

Provides information on a study of a method for examining the genomic organization of Bordetella pertussis strains. Materials and methodology used to conduct the study; Results of the study; Discussion on the results of the study.

Investigates the cloning of a manganese-containing superoxide dismutase (SodA) of Bordetella pertussis. Identification of an open reading frame coding for a potential superoxide dismutase (SOD); Regulation of the expression of the sodA gene; Characterization of the enzyme activity of SOD in bacterial lysates.

Investigates the nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the filamentous hemagglutinin promoter. Correlation of the BvgA phosphate binding with in vitro transcriptional activity; Use of mutant Escherichia coli proteins; Performance of in vitro transcription analyses.

Presents a study on the lack of functional complementation between Bordetella pertussis filementous hemagglutinin (FHA) and Proteus mirabilis HpmA hemolysin secretion machineries. Simalarities and differences between the secretion of FHA and the hemolysins; Effect of alterations of the conserved asparagine residues on Fha44 secretion; Results of the study.

Studies the identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes. Restoration of alcaligin production; Phenotypic complementation analysis; Demonstration of iron regulation at the transcriptional level.

Examines the overexpression of RNA polymerase alpha subunit in Bordetella pertussis resulting from differential effects on virulence genes' expression of a novel type of regulatory mutation. Interaction between BvgA and the RNA alpha polymerase subunit during transcription initiation process; Significance of identified proteins in the transcription machinery.

Reports on the identification of a second protein of Bordetella pertussis with homoloy to eukaryotic histone H1 protein named BpH2. Bacterial strains and growth conditions; Plasmids; Recombinant DNA methods; Southwestern blot; Chromosomal integration; Assay of enzymatic activities.

Assesses the functional role of the C-terminal and N-terminal regions of the 220-kilodalton Bordetella pertussis filamentous hemagglutinin (FHA). Role of C-terminal domain in the biogenesis of FHA and truncated analogs; Fha44 biogenesis and fhaC expression; Functional roles of the N-terminal domain of FHA encoded by pBG4.

Demonstrates the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the pertussis toxin (ptx) promoter of Bordetella pertussis. Gel shift analyses of the ptx promoter; Partial purification of recombinant BvgA.

Examines the role of BvgAS, member of the signal transductional family, in the activation of the Bordetella pertussis ptx locus in Escherichia coli. Effects of growth of media; Expression of a ptx-A-lacZYA transcriptional fusion in Escherichia coli.

Investigates the identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli. Positive regulation of the transcription of pertussis toxin operon by bvgAS locus; Ability of a gene encoding a Byg accessory factor to activate an Escherichia coli ptx-lacZ fusion in the presence of bvgAS.

Identifies accessory proteins involved in the regulation of bvg-repressed genes in Bordetella pertussis. Isolation of transposon insertion mutants; Mapping of transposon insertion sites; Construction and characterization of an in-frame insertion within the putative bvg-regulated repressor; Complementation analysis; Sequence analysis.

Discusses the reproduction of bvg-regulated porin-like proteins OmpQ by Bordetella pertussis. Effect of environmental signals on gene expression; Plasmid and bacteria cloning; Nucleotide sequence analysis; Amino acid sequence analysis; Presence of vag-49 in other Bordetella species.