Genetic tools for allelic replacement in Burkholderia species

Abstract

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

FIG. 1.

Killing of four different Burkholderia species by a pheS plasmid (pBBR1MCS-Km-Tp-pheS) in the presence of 0.1% cPhe. The control plasmid used was pBBR1MCS-Km-Tp. Tp was used at 200 μg/ml for B. cenocepacia K56-2 (A), 500 μg/ml for B. dolosa (B), and 700 μg/ml for B. cenocepacia J2315 (C); kanamycin (Km) was used at 500 μg/ml for B. thailandensis (D). The same numbers of CFU were plated for each strain on three different media, 1× M9-glucose medium (MG), 1× M9-glucose medium containing Tp (MG+Tp), and 1× M9-glucose medium containing Tp and cPhe (MG+Tp+cPhe); more bacteria were plated in the bottom picture for each species to show effective killing at a high number of CFU. The four Burkholderia species containing the mutant pheS died in the presence of cPhe. No synergistic effect of Tp/Km and cPhe was observed. There was no effect on the number of CFU when each control strain was grown on cPhe-Tp or cPhe-Km.

FIG. 2.

Allelic replacement vectors based on the mutant pheS gene. Each vector contains a different selectable marker for resistance to Ap (A), Gm (B), Km (C), Tet (D), and Tp (E). (F) The asd_Pa gene as a non-antibiotic-selectable marker. All plasmids can be maintained in regular laboratory E. coli strains, with the exception of pBAKA, which was maintained in E. coli strain E1345 or E1354 (Table 1). aacC1, Gm acetyltransferase-encoding gene; bla, β-lactamase-encoding gene; lacZα, β-galactosidase α-peptide; ori, ColE1 origin of replication; oriT, conjugal origin of transfer; P_lac, lac promoter; P_S12, the B. pseudomallei rpsL gene promoter; asd_Pa, P. aeruginosa aspartate semialdehyde dehydrogenase gene; pheS, mutant gene for the α-subunit of phenylalanyl tRNA synthase; and T1T2, transcriptional terminators.

FIG. 3.

Maps of FRT and Flp plasmids. Tel^r and Tp^r FRT cassettes can be removed by restriction digestion from pwFRT-Tel^r (A) and pwFRT-Tp^r (B), respectively. Not shown are four other unique FRT cassettes for each resistance determinant, which yield eight other plasmids (pmFRT-Tel^r, pmFRT-Tp^r, pFRT1-Tel^r, pFRT1-Tp^r, pFRT2-Tel^r, pFRT2-Tp^r, pFRT3-Tel^r, and pFRT3-Tp^r), where each selectable marker is flanked by identical FRTs with unique spacer sequences. The DNA sequences and restriction sites for all five Tel^r-FRT plasmids are identical, with the exception of the FRT spacer sequences on both sides of the resistant marker; similarly, all Tp^r-FRT plasmids have identical DNA sequences, with the exception of the spacers. Two Flp-containing replicative plasmids, pFLP-AB4 (C) and pFLP-AB5 (D), were engineered to excise chromosomal markers based on Tel^r and Tp^r, respectively. cI₈₅₇, temperature-sensitive λ cI repressor; flp, gene encoding flippase (Flp); Ω, tonB transcriptional terminator; ori₁₆₀₀-rep, broad-host-range replicon; P_S12, promoter of the B. pseudomallei rpsL gene; PC_S12, promoter of the B. cenocepacia rpsL gene.

FIG. 4.

(A) Gene replacement scheme using a Tel^r-FRT or Tp^r-FRT cassette to inactivate the B. thailandensis betBA operon. We utilized the Tel^r-FRT cassette. The betBA operon was amplified and cloned into pBAKA with oligonucleotides 861 and 862, and the inactivation and selection procedure performed was described in Materials and Methods. After the Flp excision, the resulting ΔbetBA::FRT mutant has one remaining FRT sequence (∼100 bp) that inactivates the betBA operon. (B) PCR confirmation of the ΔbetBA mutant with outside oligonucleotides 867 and 868. Numbers in circles from 1 to 3 corresponds to lanes 1 to 3. Lane 1, wild-type betBA operon; lane 2, ΔbetBA::Tel^r-FRT mutant before Flp excision; lane 3, ΔbetBA::FRT mutant after Flp excision; M, 1-kb ladder.

FIG. 5.

Growth defect of the ΔbetBA::Tel^r-FRT and ΔbetBA::FRT mutants in choline, succinate, and glucose media. (A) The ΔbetBA mutation abolished the growth of B. thailandensis in 1× M9 minimal medium-choline, while the wild type grew well on choline as a sole carbon source. The ΔbetBA::FRT nonpolar mutation does not affect growth on the other carbon sources, succinate (B) and glucose (C), compared to that of wild-type B. thailandensis. However, quicker death was observed for the ΔbetBA::Tel^r-FRT polar mutant (B and C).