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-agentcompliant 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 ⌬asd::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.

The Burkholderia pseudomallei Δ asd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice

“…All manipulations of B. pseudomallei were conducted in CDC/USDA-approved and -registered biosafety level 3 (BSL3) facilities at the University of Hawaii at Manoa and Colorado State University, and experiments with select agents were performed in accordance with the recommended BSL3 practices (32). Derivatives of Escherichia coli strains EPMax10B (Bio-Rad), E1345, E1354, E1869, and E1889 (Table 1) were routinely used for cloning or plasmid mobilization into B. pseudomallei as described previously (24,28). Luria-Bertani (LB) medium (Difco) was used to culture E. coli strains.…”

“…Selections for bar and gat genes in E. coli and B. pseudomallei strains were performed as previously described (28). B. pseudomallei ⌬asd::gat strains were grown on LB containing 200 g/ml DAP or on MG containing 1 mM Lys, 1 mM Met, 1 mM Thr, and 200 g/ml meso-DAP, as described previously (28).…”

“…Molecular methods, PCR conditions, and conjugation into select agents were conducted as described previously (2,28,35).…”

“…In a recent publication, we described an engineered B. pseudomallei strain with a deletional mutation in the aspartate-␤-semialdehyde dehydrogenase (asd) gene, which is auxotrophic for diaminopimelate (DAP) in rich medium and auxotrophic for DAP, lysine, methionine, and threonine in minimal medium (28); this is consistent with similar mutations in many other bacterial species (13,(18)(19)(20). DAP is a diamino acid that cross-links to D-alanine in neighboring peptidoglycan strands, and the ⌬asd mutant exhibits the "pop-and-die" phenotype associated with an inability to synthesize DAP for cell wall biosynthesis.…”

Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria

“…An RFP-labeled version of B1qs70 was constructed by performing a triparental mating procedure using the E. coli helper strain E1354 (pTNS3-asd Ec ) and the E. coli donor strain E2072 with pmini-Tn7-gat-P1-rfp as previously described (61,62). RFP-positive Burkholderia conjugants were confirmed using Burkholderia-specific PCR.…”