Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen
            <i>Burkholderia cenocepacia</i>
            Strain J2315

Dubarry, Nelly; Du, Weian; Lane, David; Pasta, Franck

doi:10.1128/aem.02123-09

Cited by 29 publications

(29 citation statements)

References 42 publications

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“…PCR fragments were cut with ApaI and HindIII or ApaI and HpaI and cloned in pMMB206 similarly cut. Recombinant plasmids were introduced into the J2315 derivative strain Mex1 by electrotransformation as described previously (14). Cells were spread on LB agar with chloramphenicol (40 g/ml) and incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

et al. 2012

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How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

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Section: Methodsmentioning

confidence: 99%

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

et al. 2012

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“…A 1.6-kb XbaI/HindIII fragment from pRM306 was subcloned into pUCP26 (47), yielding pRM308. The pUCP28T-based vectors were conjugated into the parent and the RMI19 mutant by triparental mating using pRK2013 (14) as the mobilization strain, and the pUCP26-based vectors were transferred into the parent and the 26D7 mutant by electroporation (12).…”

Section: Methodsmentioning

confidence: 99%

Identification of Hopanoid Biosynthesis Genes Involved in Polymyxin Resistance in Burkholderia multivorans

Malott

Steen-Kinnaird

Lee

et al. 2012

Antimicrob Agents Chemother

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A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8-and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.

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“…E. coli cells were transformed by heat shock using frozen competent cells prepared by using the calcium chloride method described in Molecular Cloning (24). Burkholderia strains were transformed by the electrotransformation method (7). Plasmid construction and plasmid DNA extraction were carried out according to standard molecular biology techniques (25).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Poly-β-1,6- N -Acetylglucosamine Polysaccharide Component of Burkholderia Biofilms

Yakandawala

Gawande

LoVetri

et al. 2011

Appl Environ Microbiol

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We demonstrated the production of poly-␤-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ⌬pgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMG⌬C (⌬pgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

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Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen Burkholderia cenocepacia Strain J2315

Cited by 29 publications

References 42 publications

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

Identification of Hopanoid Biosynthesis Genes Involved in Polymyxin Resistance in Burkholderia multivorans

Characterization of the Poly-β-1,6- N -Acetylglucosamine Polysaccharide Component of Burkholderia Biofilms

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