Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1.
(25/244)
The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism. (+info)
Variation in Bordetella bronchiseptica flaA does not correlate with typing by macro-restriction analysis by pulsed-field gel electrophoresis.
(26/244)
A genotyping method based on PCR-RFLP analysis of the flagellin gene (flaA) was applied to 30 mainly feline isolates of Bordetella bronchiseptica. These isolates were separated into three PCR-RFLP groups with the restriction endonucleases HaeIII, MspI, MboI and RsaI. flaA nucleotide sequences representing each of the three groups differed from each other by 11-13%. One of the groups exhibited far greater flaA sequence identity with the cryptic flagellin gene sequence of B. pertussis (>97%) than with flaA sequences from representatives of the other B. bronchiseptica PCR-RFLP groups. Amongst the 30 isolates were at least 10 representing each of the two major genotypes (A and B) identified in a previous study by macro-restriction analysis and pulsed-field gel electrophoresis (PFGE), as well as representatives of other less common genotypes. Each of the major PFGE genotypes contained strains representing more than one flagellin genotype. Indeed, there was no correlation between the two molecular typing methods. PFGE analysis may identify differences due to genomic re-arrangements rather than genuine variations in gene content. If so, relationships inferred on the basis of PFGE or other molecular methods for whole genome comparison should be treated with caution. (+info)
Reduced virulence of a Bordetella bronchiseptica siderophore mutant in neonatal swine.
(27/244)
One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs. (+info)
Expression of the putative siderophore receptor gene bfrZ is controlled by the extracytoplasmic-function sigma factor BupI in Bordetella bronchiseptica.
(28/244)
A new gene from Bordetella bronchiseptica, bfrZ encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay. A bfrZ null mutant was constructed by allelic exchange. The protein profile of this mutant is similar to that of the wild-type parent strain. The BfrZ(-)-BfrZ(+) isogenic pair was tested for utilization of 132 different siderophores as iron sources. None of these iron sources acted as a ligand for BfrZ. Translational bfrZ::phoA and transcriptional bfrZ::lacZ fusions were introduced into the B. bronchiseptica bfrZ locus. No alkaline phosphatase or beta-galactosidase activity was detected. Sequence analysis of the bfrZ upstream region revealed the presence of two tightly linked genes, bupI and bupR. Both of these genes are located downstream from a Fur-binding sequence. BupI is homologous to Escherichia coli FecI and Pseudomonas putida PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions. BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane. Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension. We found that bfrZ is not transcribed when bupI and bupR are expressed at the same level. However, overexpression of bupI from a multicopy plasmid triggers bfrZ transcription, and under these conditions BfrZ was detected in membrane fractions. By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that bfrZ expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope. (+info)
Infected branchial cleft cyst due to Bordetella bronchiseptica in an immunocompetent patient.
(29/244)
A healthy 23-year-old man with fever and a tender mass in his right anterior neck was found to have a branchial cleft cyst infected with Bordetella bronchiseptica. Initial testing suggested a Brucella species, but further laboratory testing identified the organism definitively. B. bronchiseptica infection in healthy adults is an unusual event. (+info)
Role of the dermonecrotic toxin of Bordetella bronchiseptica in the pathogenesis of respiratory disease in swine.
(30/244)
Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT(+) parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism. (+info)
In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure.
(31/244)
Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic infections, the possibility that this phenotype arises as a consequence of selection pressure within the host at a late stage of the infection process is discussed. (+info)
Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage.
(32/244)
Host-pathogen interactions are often driven by mechanisms that promote genetic variability. We have identified a group of temperate bacteriophages that generate diversity in a gene, designated mtd (major tropism determinant), which specifies tropism for receptor molecules on host Bordetella species. Tropism switching is the result of a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within mtd. This cassette-based mechanism is capable of providing a vast repertoire of potential ligand-receptor interactions. (+info)