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

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 Δasd::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.Members of the genus Burkholderia, comprising more than 40 different species, are extremely diverse gram-negative, non-spore-forming bacilli. Many Burkholderia species exist as innocuous soil saprophytes or plant pathogens (47), while others cause human and animal diseases. Among these human and animal pathogens are the etiological agents of melioidosis (Burkholderia pseudomallei) and glanders (Burkholderia mallei) (9, 50, 51). Melioidosis is an emerging infectious disease generally considered endemic to Southeast Asia and Northern Australia (12). Positive diagnoses in many tropical countries around the world have expanded the global awareness of melioidosis (3, 15, 24, 25, 28, 35, 39, 42, 52). In contrast to the ubiquitous nature of B. pseudomallei, B. mallei is also a highly infectious agent causing glanders, a predominantly equine disease (34, 50). B. mallei, a clone derived from genomic downsizing of B. pseudomallei, has been used in biowarfare (17). This historical significance, along with the low infectious dose and the route of infection, has contributed to the decision by the Centers for Disease Control and Prevention (CDC) to classify these two microbes as category B select agents (43).Classification of B. pseudomallei as a select agent has stimulated interest and research into the pathogenesis of melioidosis, necessitating the development of appropriate tools for genetic manipulation. In the struggle to elucidate the molecular mechanisms of pathogenesis, selectable markers are indispensable genetic tools (45). Current CDC regulations prohibit the cloning of clinically important antibiotic resistance genes into human, animal, or plant select-agent pathogens if the transfer could compromise the ability to treat or control the disease. The only antibiotic markers currently approved for use in B. pseudomallei are based on resistance to aminoglycosides (gentamicin, kanamycin, and zeocin) (45). However, the efficacy of these markers is limited, due to high levels of aminoglycoside resistance inherent within the Burkholderia genus and high levels of spontaneous aminoglycoside resistance in B. pseudomallei (10, 19, 41). In addition, the use of aminoglycosides (e.g., gentamicin) for selection may require aminoglycoside efflux pump mutants (10, 33). Another potential drawback is that efflux pumps play a major role in bacterial physiology, and mutating them may change the pathogenic traits under investigation (7, 40). A more logical approach employs alternative, non-antibiotic-selectable markers conferring resistance to compounds that are not potentially important in clinical treatment.Very few non-antibiotic resistance markers have been utilized successfully for Burkholderia species. A non-antibiotic-selectable-marker based on tellurite resistance (Tel^r) has been successfully developed and used with Pseudomonas putida, Pseudomonas fluorescens, and Burkholderia thailandensis (2, 27, 44). The engineering of Tel^r-FRT (flip recombination target) cassettes, coupled to FRT sequences, could be used to generate unmarked mutations and allow recycling of the Tel^r selectable-marker (2). In addition, utilization of Flp-FRT resistance cassettes to generate mutants allows downstream modification and manipulation such as fusion integration (29). However, the disadvantage of the Tel^r-cassette is the number of genes required (kilA-telA-telB) and the large size (>3 kb), making it less likely to obtain PCR products for allelic replacement by natural transformation (46). Another potentially useful non-antibiotic-selectable marker is based on the bar gene, encoding resistance to bialaphos or its degradation product, phosphinothricin (PPT) (49). PPT inhibits glutamine synthetase in plants (48), starving the cell for glutamine, and the bar gene has been used successfully as a selection marker in gram-negative bacteria (21). For select-agent Burkholderia species, however, the PPT MIC was found to be greater than 1,024 μg/ml (M. Frazier, K. Choi, A. Kumar, C. Lopez, R. R. Karkhoff-Schweizer, and H. P. Schweizer, presented at the American Society for Microbiology Biodefense and Emerging Diseases Research Meeting, Washington, DC, 2007). We have found the effective concentration of PPT for B. pseudomallei and B. mallei to be ∼2.5% (25,000 μg/ml [data not shown]). The high concentration of PPT required for selection in these species may be costly, considering that purified PPT costs ∼$380 per g. Therefore, further development of non-antibiotic resistance markers, as well as a more economical source of herbicide for use with restricted select-agent species, is needed.Work by Castle et al. (5) generated a highly active glyphosate N-acetyltransferase (GAT) enzyme for plant engineering, making it possible to utilize the gat gene as an effective non-antibiotic resistance marker for bacterial selection with glyphosate (GS). The commonly used herbicide GS inhibits the 5-enolpyruvylshikimate-3-phospate synthase (EPSPS) of plants through competition with phosphoenolpyruvate for overlapping binding sites on EPSPS (14), depriving plants of three aromatic amino acids (Fig. ​(Fig.1).1). Since humans and animals obtain tryptophan and phenylalanine (giving rise to tyrosine) through dietary intake, GS is relatively nontoxic. Like plants, bacteria must make these amino acids, when they are lacking, from basic precursors. GS has been found to be inhibitory to a variety of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, and Bradyrhizobium japonicum (16, 55), while other bacterial strains are able to metabolize low concentrations of GS (26, 31). Although B. pseudomallei has been reported to have two genes (glpA and glpB) for GS degradation and metabolism (38), our searches of all available genomes of Burkholderia species in GenBank yielded no glpA or glpB genes within this genus. GS resistance by bacteria has been documented through EPSPS target mutations or GS detoxification mechanisms (36). However, these mechanisms did not confer resistance to relatively high GS concentrations. More recently, directed evolution of the gat gene, based on various bacterial gat sequences and selection in E. coli, yielded a very active GAT protein sequence with an efficiency increase of nearly 4 orders of magnitude (5), holding promise as an appropriate non-antibiotic resistance marker for select-agent species.Open in a separate windowFIG. 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.Here we engineered and tested a novel non-antibiotic-selectable-marker (gat) for use in the select agent B. pseudomallei. GS is the active ingredient in Roundup, which was used for selection (Fig. ​(Fig.1).1). The effective compound GS is readily available, inexpensive, relatively nontoxic, very soluble, and not clinically important, and it yields tight selection. The engineered gat marker (563 bp) was optimized for Burkholderia codon usage and adapted (with a Burkholderia rpsL promoter) for use in the select agent B. pseudomallei. Effective concentrations of GS for several species of Burkholderia, including the select agents B. pseudomallei and B. mallei, were determined. Using the gat gene, we created deletion mutants of the essential B. pseudomallei asd and B. pseudomallei dapB (asd_Bp and dapB_Bp) genes (encoding aspartate-semialdehyde dehydrogenase and dihydrodipicolinate reductase, respectively) in two wild-type B. pseudomallei strains. The Δasd_Bp mutant of B. pseudomallei showed a phenotypic defect consistent with the lack of diaminopimelate (DAP) for cell wall cross-linking. Complementation of the B. pseudomallei Δasd_Bp mutant with the asd_Bp gene located on a site-specific transposon, mini-Tn7-bar, was successful by using an inexpensive source of PPT for selection.