Use of Suppression-Subtractive Hybridization To Identify Genes in the
            <i>Burkholderia cepacia</i>
            Complex That Are Unique to
            <i>Burkholderia cenocepacia</i>

Bernier, Steve P.; Sokol, Pamela A.

doi:10.1128/jb.187.15.5278-5291.2005

Cited by 34 publications

(26 citation statements)

References 71 publications

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“…We also observed that BCAM0224 is an exclusive marker of epidemic B. cenocepacia belonging to the ET-12 lineage and we therefore recommend employment of this PCR assay for discrimination of bacteria among Bcc members that belong to this virulent subgroup. These results are compatible with previous studies, which revealed the autotransporter adhesins BCAM0223 and BCAM0225 as unique in the ET-12 lineage (Bernier & Sokol, 2005), but BCAM0223, just like cblA, is present in some B. cenocepacia strains distinct from ET-12 (Turton et al, 2009).…”

Section: Discussionsupporting

confidence: 82%

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

2010

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Members of the Burkholderia cepacia complex (Bcc) are respiratory pathogens in patients with cystic fibrosis (CF). Close repetitive DNA sequences often associate with surface antigens to promote genetic variability in pathogenic bacteria. The genome of Burkholderia cenocepacia J2315, a CF isolate belonging to the epidemic lineage Edinburgh-Toronto (ET-12), was analysed for the presence of close repetitive DNA sequences. Among the 422 DNA close repeats, 45 genes potentially involved in virulence were identified and grouped into 12 classes; of these, 13 genes were included in the antigens class. Two trimeric autotransporter adhesins (TAA) among the 13 putative antigens are absent from the other Burkholderia genomes and are clustered downstream of the cci island that is a marker for transmissible B. cenocepacia strains. This cluster contains four adhesins, one outer-membrane protein, one sensor histidine kinase and two transcriptional regulators. By using PCR, we analysed three genes among 47 Bcc isolates to determine whether the cluster was conserved. These three genes were present in the isolates of the ET-12 lineage but absent in all the other members. Furthermore, the BCAM0224 gene was exclusively detected in this epidemic lineage and may serve as a valuable new addition to the field of Bcc diagnostics. The BCAM0224 gene encodes a putative TAA that demonstrates adhesive properties to the extracellular matrix protein collagen type I. Quantitative real-time PCR analysis indicated that BCAM0224 gene expression occurred preferentially for cells grown under high osmolarity, oxygen-limited conditions and oxidative stress. Inactivation of BCAM0224 in B. cenocepacia attenuates the ability of the mutant to promote cell adherence in vitro and impairs the overall bacterial virulence against Galleria mellonella as a model of infection. Together, our data show that BCAM0224 from B. cenocepacia J2315 represents a new collagen-binding TAA with no bacterial orthologues which has an important role in cellular adhesion and virulence.

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Section: Discussionsupporting

confidence: 82%

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

2010

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“…The loss of hopanoid production and the resulting compromise in the structural integrity of the membrane may affect other structures within or spanning the cell envelope. B. cenocepacia relies on membrane-spanning polar flagella for swimming motility, a form of movement regulated by individual cells perceiving chemical signals, which can be measured using characteristic swim agar plates (3,35). Upon sensing the proper environmental signals, B. cenocepacia modifies its cell morphology to become an elongated, multiflagellated swarm cell (3,24,35).…”

Section: Resultsmentioning

confidence: 99%

“…B. cenocepacia relies on membrane-spanning polar flagella for swimming motility, a form of movement regulated by individual cells perceiving chemical signals, which can be measured using characteristic swim agar plates (3,35). Upon sensing the proper environmental signals, B. cenocepacia modifies its cell morphology to become an elongated, multiflagellated swarm cell (3,24,35). Swarming motility is a coordinated social behavior, allowing the bacteria to act as a multicellular population to rapidly colonize nutrient-rich solid substrates, such as swarm agar plates (21,22,43,63).…”

Section: Resultsmentioning

confidence: 99%

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

2011

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Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the ⌬shc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the ⌬shc mutant produced only very small amounts of the hopanoid peak. The ⌬shc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The ⌬shc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

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“…One approach to identify such virulence factors is to identify genes present in virulent F. tularensis that are absent or have low homology to genes in closely related F. novicida. SSH has been used to successfully identify virulence genes present in pathogens that are absent in closely related species (Bernier & Sokol, 2005;DeShazer et al, 2001;Harakava & Gabriel, 2003;Liu et al, 2003;Newton et al, 2006;Parsons et al, 2003;Winstanley, 2002), and was therefore used to identify genes uniquely expressed by the more virulent type A and B strains. Seventy-six LVS-specific genes with hypothetical or known functions were identified in eight genomic regions in multiple SSH clones (Ahmed & Inzana, 2004).…”

Section: Discussionmentioning

confidence: 99%

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

Ryder

Mandal

et al. 2007

Microbiology

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Francisella tularensis is a zoonotic, Gram-negative coccobacillus that causes tularemia in humans and animals. F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B) are antigenically similar and more virulent than Francisella novicida in humans. The genetic locus that encodes the LPS O antigen was found to be substantially different between the type B live vaccine strain (LVS) and F. novicida. One LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement using a sacB-chloramphenicol expression suicide plasmid, and recombinants were screened for colony morphology on Congo red agar that matched that of F. novicida. Two mutants (WbtI S187Y and WbtI G191V ) were isolated that contained substitutions in conserved motifs in the sugar transamine/perosamine synthetase (WbtI) of the O-antigen locus, and the latter mutant was extensively tested and characterized. WbtI G191V grew at the same rate as the parent strain in Chamberlain's defined medium, completely lacked O antigen, was serum-sensitive but could grow in a mouse macrophage cell line, had increased resistance to sodium deoxycholate, and was highly attenuated in mice. Complementation of WbtI G191V with the wild-type wbtI gene in trans restored normal LPS synthesis, phenotypic properties similar to the parent, and virulence in mice. Immunization with WbtI G191V protected mice against a relatively low-dose intraperitoneal challenge with LVS, but was less protective against a high-dose challenge. These results indicate that complete loss of O antigen alters the surface phenotype and abrogates virulence in F. tularensis, but also compromises the induction of full protective immunity against F. tularensis infection in mice.

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Use of Suppression-Subtractive Hybridization To Identify Genes in the Burkholderia cepacia Complex That Are Unique to Burkholderia cenocepacia

Cited by 34 publications

References 71 publications

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

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