Fig 1.
(A) Model of DNA uptake in Gram-negative bacteria (see text for detail). (B) Utilization of DNA as a sole carbon and energy source in selected Burkholderia species. Bp 1026b and B. thiailandensis E264 strains exhibited heavy growth; Bp K96243 showed intermediate growth, while Bc K56-2 and B. mallei ATCC23344 were unable to grow on DNA.
Fig 2.
Library production and counter-selection scheme.
(A) Plasmid map of an in-lab created pBBR-FOSGAT and strategy for creating the fosmid library. Bp 1026b chromosomal DNA (1) was sheared (2), and approximately 50-Kbp size fragments were ligated to pBBR-FOSGAT fosmid vector DNA (3 and 4; fosmid vector indicated in red). The ligated DNA was then packaged into λ-phage particles (5) and the particles are used to infect E. coli host cells (6). (B) Counter-selection scheme to screen for fosmid clones enabling DNA uptake or DNA catabolism. (1) pheS and sacB counter-selectable markers were inserted into Bc K56-2 and Bp K96243 chromosomes through minTn7 integration. (2) The E. coli library containing fosmid pool from (A) was conjugated with Bc K56-2 and Bp K96243 containing pheS and sacB counter-selectable markers. (3) Colonies from these two Burkholderia strains were individually pooled and incubated with flp-DNA, allowing transiently expressed Flp to excise the chromosomally integrated counter-selection markers and counter-selected on media containing cPhe and sucrose. (4) Alternatively, the Bc K56-2 pool was selected on minimal medium containing purified salmon sperm DNA as a sole carbon source.
Fig 3.
Gene content of four fosmid clones conferring natural competency and/or DNA catabolism.
(A) The fosmid backbone is indicated in black and the red region depicts the extent of Bp 1026b genomic DNA inserted in each fosmid. Gene products with known predicted functions are indicated by gene names and those with unknown functions are labeled with gene identifications. (B) Synteny map comparing fosmid regions of Bp K96243 and 1026b. Red regions indicate the high level of similarity between fosmid regions in strain K96243 (non-competent) versus 1026b (competent) aligned using Artemis webACT. Identical regions are indicated in red, non-identical regions in white, and blue indicates a region that is inverted in the two strains.
Table 1.
Characterization of selected genes from fosmids.
Fig 4.
Confirmation of the ability of four fosmid clones to individually confer natural competency or growth on DNA.
(A) gfp-DNA uptake assay to assess natural competency. Individual fluorescent bacteria are visible in the representative images (green fluorescence and DIC overlay). The percentages of cells that fluoresce following gfp-DNA uptake are shown in (B). Numbers are from 3 representative images from duplicate experiments. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher %GFP positive comparing to the corresponding empty vector control in three fields (**, P<0.005 based on unpaired t-test). (C) Growth of various fosmid-containing strains in 1x M9 with 0.25% DNA done in triplicate. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher growth in DNA comparing to the corresponding empty vector control (*, P<0.05 based on unpaired t-test; **, P<0.005 based on unpaired t-test).
Fig 5.
Allelic-replacement in B. mallei strain ATCC23344 and B. pseudomallei K96243 containing fosmids by natural transformation.
(A) λ-Red plasmid pKaKa2 and individual fosmid clones were co-transformed into B. mallei ATCC23344 and B. pseudomallei K96243. (B) PCR products were generated containing the tellurite resistance cassette (kilA-telA-telB) flanked with 45-bp homologies to the vacJ gene. (C) Various numbers of distinct colonies were obtained. PCR was performed for the kilA-vacJ junction (D) or the telA gene (E) and all were positive. Two gels were shown here as representative results; “-” denotes negative control using wildtype B. mallei chromosomal DNA as template for kilA-vacJ junction PCR, and “+” denotes positive control using pwFRT-tel as template for telA PCR.