Abstract
Burkholderia spp. are genetically and physiologically diverse. Some strains are naturally transformable and capable of DNA catabolism. Burkholderia pseudomallei (Bp) strains 1026b and K96243 and B. thailandensis strain E264 are able to utilize DNA as a sole carbon source for growth, while only strains 1026b and E264 are naturally transformable. In this study, we constructed low-copy broad-host-range fosmid library, containing Bp strain 1026b chromosomal DNA fragments, and employed a novel positive selection approach to identify genes responsible for DNA uptake and DNA catabolism. The library was transferred to non-competent Bp K96243 and B. cenocepacia (Bc) K56-2, harboring chromosomally-inserted FRT-flanked sacB and pheS counter-selection markers. The library was incubated with DNA encoding Flp recombinase, followed by counter-selection on sucrose and chlorinated phenylalanine, to select for clones that took up flp-DNA, transiently expressed Flp, and excised the sacB-pheS cassette. Putative clones that survived the counter-selection were subsequently incubated with gfp-DNA and bacteria were visualized via fluorescent microscopy to confirm natural competency. Fosmid sequencing identified several 1026b genes implicated in DNA uptake, which were validated using chromosomal mutants. One of the naturally competent clones selected in Bc K56-2 enabled Bc, Bp and B. mallei to utilize DNA as a sole carbon source, and all fosmids were used to successfully create mutations in non-naturally-competent B. mallei and Bp strains.
Highlights
Several Gram-negative bacteria are naturally competent for DNA uptake
20,000 Escherichia coli clones were initially created, representing more than 20-fold coverage of the Burkholderia species genomes. This library was mobilized into Burkholderia pseudomallei (Bp) strain K96243 and B. cenocepacia (Bc) strain K56-2
Investigators have harnessed the bacterial ability of natural transformation to create genetic tools and methodologies that allow manipulation of the Burkholderia genome [12, 16]
Summary
Several Gram-negative bacteria are naturally competent for DNA uptake. The natural transformation system of these bacteria shares protein components common with type IV pili (T4P) and type II secretion systems (Fig 1A) [1,2,3], with the exception of Helicobacter pylori which. Burkholderia pseudomallei natural competency and DNA catabolism design, data collection and analysis, decision to publish, or preparation of the manuscript
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