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Appl Environ Microbiol. 2009 Oct;75(19):6062-75. doi: 10.1128/AEM.00820-09. Epub 2009 Jul 31.

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.

Norris MH, Kang Y, Lu D, Wilcox BA, Hoang TT.

Source

Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.

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.

PMID:: 19648360; [PubMed - indexed for MEDLINE]
PMCID:: PMC2753064

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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.

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

Appl Environ Microbiol. 2009 October;75(19):6062-6075.