Glyphosate resistance as a novel select-agent-compliant, non-antibiotic-selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei

Abstract

Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Deltaasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

FIG. 1.

(A) A 946-ml bottle of the “superconcentrated” herbicide Roundup used in this study, available for ∼$50 from most local hardware stores and garden or farm supply centers. The active ingredient, 50% GS, is indicated on the label, and the chemical structure of GS is shown. GAT, encoded by the gat gene, catalyzes the inactivation of GS via N acetylation. (B) Pathways of aromatic amino acid biosynthesis. GS inhibits the enzyme EPSPS, which is required for the biosynthesis of aromatic amino acids, thus starving bacteria for tyrosine, phenylalanine, and tryptophan. PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

FIG. 2.

Bacterial survival after incubation with different concentrations of GS for 24 h. B. mallei was more sensitive to GS than both B. pseudomallei strains, and killing of B. mallei by GS was observed at 0.25% GS. B. pseudomallei strain 1026b is significantly more resistant to GS than strain K96243. Minimal replication of both B. pseudomallei strains (less than doubling) after 24 h was observed at 0.25% GS. Killing was observed at 2% GS for strain K96243 and at 3% GS for strain 1026b.

FIG. 3.

Schematic diagram of the engineered 563-bp gat gene on pwFRT-PC_S12-gat. The B. cenocepacia rpsL promoter (PC_S12) and ribosomal binding site (rbs) are shown in relation to the gat gene. Below the schematic are the corresponding nucleotide and protein sequences. Codons were optimized according to the codon preference within the B. pseudomallei K96243 asd gene. Also indicated are the −35 and −10 regions of the PC_S12 promoter. Restriction sites (in boldface) were positioned strategically for subsequent cloning and manipulation.

FIG. 4.

Maps of pwFRT-PC_S12-gat (A), pwFRT-PC_S12-bar (B), mini-Tn7-gat (C), and mini-Tn7-bar (D). (A) pwFRT-PC_S12-gat is flanked with symmetrical restriction-sites (HindIII to SacI) that will cut to remove the gat cassette flanked with identical wild-type FRT sequences. Not shown are four other FRT-gat cassettes with unique flanking FRT-sequences (pmFRT-gat, pFRT1-gat, pFRT2-gat, and pFRT3-gat), where the gat marker is flanked by identical FRTs with unique spacer sequences. The DNA sequences and restriction sites for all five gat-FRT cassettes are identical with the exception of the spacers. (B) pwFRT-PC_S12-bar, with bar flanked by wild-type FRT sequences and symmetrical restriction enzyme sites. (C and D) mini-Tn7-gat (C) and mini-Tn7-bar (D) were engineered to allow site-specific integration of the cloned gene(s), using the non-antibiotic resistance bar or gat selectable marker, with the assistance of a helper plasmid (pTNS3-asd_Ec). bla, β-lactamase-encoding gene; ori, ColE1 origin of replication; oriT, conjugal origin of transfer; R6Kγori, π protein-dependent R6K origin of replication; Tn7L and Tn7R, left and right transposase recognition sequences; T₀T₁, transcriptional terminator.

FIG. 5.

(A) Gene replacement strategy using a gat-FRT cassette to inactivate the B. pseudomallei strain K96243 and 1026b asd_Bp genes. Oligonucleotides 892 and 893 were used in the initial cloning of the asd_Bp gene into the allelic-replacement vector pBAKA, and the asd_Bp gene was inactivated with the gat-FRT cassette. Deletion of the chromosomal asd_Bp gene with pBAKA-Δasd_Bp::gat was performed as shown. PCR verification of the Δasd_Bp mutant was done using outside oligonucleotides 1062 and 1063. The asd_Bp genes of both the K96243 and the 1026b strain were inactivated using pBAKA and pheS for counterselection. Similarly, the dapB_Bp gene of strain K96243 was inactivated using pBAKA and pheS for counterselection (not shown) (see Materials and Methods). Oligonucleotides 1049 and 1051 were used to amplify the ΔdapB_Bp::gat cassette from plasmid pBAKA-ΔdapB_Bp::gat in order to inactivate the dapB_Bp gene from strain 1026b using the DNA incubation method (46) (see Materials and Methods). (B) Bacterial amino acid biosynthetic pathway of the aspartate family, where aspartate is used to synthesize DAP, Lys, Met, Thr, and Ile. The indicated reactions catalyzed by Asd and DapB are central to this pathway, and mutants of these genes cannot cross-link their cell walls due to the lack of DAP. (C) PCR verification of the Δasd_Bp and ΔdapB_Bp mutants. In each case, as expected, the PCR products indicated that the chromosomal fragment of the mutant is larger than that of the wild type (wt), and the no-template negative control (nc) showed no PCR product. asd_Bp, B. pseudomallei asd gene encoding aspartate-semialdehyde dehydrogenase; asd_Pa, P. aeruginosa asd gene; dapB_Bp, B. pseudomallei gene encoding dihydrodipicolinate reductase; M, 1-kb ladder (New England Biolabs); Plac, lac promoter; pheS, mutant B. pseudomallei gene encoding the α subunit of phenylalanyl tRNA synthase.

FIG. 6.

Phenotypic characterization of B. pseudomallei K96243 Δasd_Bp and ΔdapB_Bp mutants. Wild-type K96243 was rod shaped when grown in the absence or presence of DAP (left). The Δasd_Bp (center) and ΔdapB_Bp (right) mutant strains grow, but “pop and die” without the ability to cross-link their cell walls in the absence of DAP. The majority of the bacteria are in the process of forming protoplasts. Some protoplasts could be observed (black arrows), as well as cell debris (white arrows) due to bacterial lysis; these were absent when mutants were grown in the presence of DAP (bottom).

FIG. 7.

Growth characteristics of the B. pseudomallei K96243 Δasd_Bp mutant and five complemented isolates relative to that of the wild type (wt) on medium lacking amino acids (aa) of the aspartate family. (A) On 1× MG medium, the Δasd_Bp mutant did not grow compared to the wt, whereas five strains (numbered 1 to 5) complemented using the mini-Tn7-bar-asd_Bp transposon all grew as well as the wt. Spots 1 and 2 are Tn7-bar-asd_Bp-complemented isolates transposed at the glmS1 site, while spots 3 to 5 are complemented isolates transposed at the glmS2 site. (B) The Δasd_Bp mutant grew similarly to the wt on MG medium when provided with all five aa of the aspartate family (DAP, Lys, Met, Thr, and Ile). (C through F) The Δasd_Bp mutant could not grow when four of the five aa were present in the MG medium and only Met (C), Thr (D), or DAP (F) was omitted, whereas the wt and all complemented strains grew well on these media. The Δasd_Bp mutant still grew when Ile was omitted from MG medium containing the other four aa (E), because Thr in the medium could be converted to Ile in this pathway. (G) Surprisingly, no growth was observed when Lys was omitted from the MG medium supplemented with four aa (Met, Thr, DAP, and Ile). We suspect that the amount of DAP provided was shuffled for use in cell wall biosynthesis and that very little was converted to Lys for growth. The Δasd_Bp mutant grew slowly on this medium. (H) When the plate in panel G was incubated for another 6 days, growth was observed for the Δasd_Bp mutant, indicating that some DAP did get converted to Lys. All other plates on which the Δasd_Bp mutant did not grow after 1 day also did not show growth of this mutant after 7 days (data not shown).

FIG. 8.

Single-copy complementation of the B. pseudomallei 1026b Δasd_Bp mutant using mini-Tn7-bar-asd_Bp. (A) The suicidal plasmid mini-Tn7-bar-asd_Bp and its suicidal helper plasmid, pTNS3-asd_Ec, were introduced into the B. pseudomallei 1026b Δasd_Bp mutant by conjugation. Tn7 has three possible integration sites on different chromosomes (indicated by red triangles), as previously described (10), which can result in complementation of the Δasd_Bp mutation from three different chromosomal loci, as depicted according to the annotation of B. pseudomallei strain K96243. Ten random complemented isolates were screened using oligonucleotide Tn7L (876) and an oligonucleotide specific for each potential integration site (oligonucleotide 1079, 1080, or 1081), as indicated by arrows. (B) For each isolate, PCR verification of 10 random complemented isolates was performed for all three glmS sites (lanes 1, 2, and 3). Insertion downstream of glmS1 would result in a 218-bp PCR product; insertion downstream of glmS2 would result in a 263-bp fragment; and insertion downstream of glmS3 would result in a 309-bp PCR product. Isolates 1, 2, 3, 4, 5, 6, 8, and 10 had Tn7 inserted downstream of glmS2. Isolates 7 and 9 showed PCR products near 200 bp, indicating Tn7 integration downstream of glmS1. P1, P1 integron promoter; glmS1, glmS2, and glmS3 encode three different B. pseudomallei glucosamine 6-phosphate synthetases; M, 100-bp ladder (New England Biolabs); tnsABCD, Tn7 transposase-encoding genes.