Burkholderia pseudomallei natural competency and DNA catabolism: Identification and characterization of relevant genes from a constructed fosmid library

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.

The heritable natural competency trait of Burkholderia pseudomallei in other Burkholderia species through comE and crp

“…An unknown nuclease generates a single-stranded DNA molecule that is transported into the cytoplasm through the ComA channel in the inner membrane 3 . The single-stranded DNA molecule is further broken down into nucleotide components or recombined into the chromosome of the recipient organism 2 , 5 . The ability of bacteria to be naturally transformable impart an evolutionary advantage and has driven diversification of species over time 2 , 6 .…”

“…Thus, the ability to make these Burkholderia species uptake DNA would be very significant in manipulating their genomes. Some Bp strains are naturally transformable (i.e., ~50% of Bp strains) 5 , 24 . Bp strain 1026b is able to naturally uptake extracellular DNA allowing for easy genetic manipulation 23 – 25 .…”

“…cenocepacia ( Bc ) K56-2 are commonly used in the field but are non-naturally competent making genome manipulation tedious and requiring many steps. To further investigate the natural transformation mechanisms of some Bp strains and increase the utility of natural transformation-based genetic manipulation techniques, our lab has sought to identify specific genetic properties that confer this phenotype 5 .…”

“…Four fosmids containing genomic regions from naturally competent Bp 1026b were previously isolated and able to confer natural transformation to non-naturally competent Burkholderia species 5 . The fosmids isolated encode ~30–40 genes each, which allowed the non-naturally competent Bp K96243, Bm ATCC23344 and Bc K56-2 to uptake gfp DNA and grow on DNA as a sole carbon source 5 . A bioinformatics analysis of each genomic region revealed several candidate genes for natural competency 5 .…”

“…The fosmids isolated encode ~30–40 genes each, which allowed the non-naturally competent Bp K96243, Bm ATCC23344 and Bc K56-2 to uptake gfp DNA and grow on DNA as a sole carbon source 5 . A bioinformatics analysis of each genomic region revealed several candidate genes for natural competency 5 . Mutation in five of these genes, in the naturally competent Bp 1026b background, led to a reduction in growth on DNA and gfp DNA uptake indicating their involvement in natural transformation 5 .…”