Improved electrotransformation and decreased antibiotic resistance of the cystic fibrosis pathogen Burkholderia cenocepacia strain J2315

Abstract

The bacterium Burkholderia cenocepacia is pathogenic for sufferers from cystic fibrosis (CF) and certain immunocompromised conditions. The B. cenocepacia strain most frequently isolated from CF patients, and which serves as the reference for CF epidemiology, is J2315. The J2315 genome is split into three chromosomes and one plasmid. The strain was sequenced several years ago, and its annotation has been released recently. This information should allow genetic experimentation with J2315, but two major impediments appear: the poor potential of J2315 to act as a recipient in transformation and conjugation and the high level of resistance it mounts to nearly all antibiotics. Here, we describe modifications to the standard electroporation procedure that allow routine transformation of J2315 by DNA. In addition, we show that deletion of an efflux pump gene and addition of spermine to the medium enhance the sensitivity of J2315 to certain commonly used antibiotics and so allow a wider range of antibiotic resistance genes to be used for selection.

FIG. 1.

Improvement of electrotransformation. (A) Transformants obtained with cells grown with added glycine (gray bars) or without (white bars) and the ratios of TFs with/without glycine (horizontal lines). (B) Transformants obtained with the donor plasmid unmethylated (gray bars) or methylated (white bars) and the ratios of TFs unmethylated/methylated (horizontal lines). (C) Transformants obtained with Ocr added during electrotransformation (gray bars) or without Ocr (white bars) and the TF ratios with/without Ocr (horizontal lines). In panel C, pairs of electrotransformations 1 to 9 are with donor DNA extracted from E. coli dam⁺ dcm⁺, transformants 10 to 16 are from E. coli dam dcm, and transformants 17 to 21 are from B. cenocepacia.

FIG. 2.

Examples of B. cenocepacia genomic recombinants following electrotransformation (A). A single crossover (×) within ctg between the plasmid and c1 integrates pBL2 to give the structure drawn. Integration was confirmed on one Cm^r transformant by PCR amplification across the chromosome-plasmid junctions. Lanes 3 and 4, 1 kb Plus DNA ladder; the relevant sizes are indicated. Lanes 1 and 6, PCRs on the Cm^r transformant primed with P1r-P1f and P8r-P8f, respectively. A specific 2.5-kb amplicon appears (▸). Lanes 2 and 5, control PCRs on the untransformed J2315 strain. (B) A Cm^r Tet^r transformant obtained with pDAG824 was analyzed by PCR with oligonucleotides vrf-A and vrf-B flanking the plasmid-chromosome recombination junction. Lane 1, untransformed control strain; lane 2, Cm^r Tet^r transformant (the expected 2.89 kb amplicon is obtained); lane 3, 2-log DNA ladder. (C) A double crossover between pRF91 and c1, via the regions flanking parA, should exchange parA for Cm^r, so that the PCR primed with F34-F35 should yield an amplicon of 2.12 kb instead of 1.86 kb. Single crossovers integrate the whole plasmid so that the F34-F35-primed PCR should yield both amplicons. Lanes 1 and 7, 1-kb ladder; lanes 2 to 5, PCR on four Cm^r Tet^s transformants, yielding the 2.12-kb amplicon; lane 6, control PCR on the untransformed strain, yielding a wild-type 1.86-kb amplicon; lanes 8 and 9, PCRs on two Cm^r Tet^r clones (plasmid integration), yielding both 2.12 and 1.86-kb amplicons (cat and wild type [wt], respectively).