Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of Burkholderia spp. and Characterization of the Burkholderia thailandensis betBA Operon▿ †

There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei , necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel r ) and Escherichia coli aspartate-semialdehyde dehydrogenase ( asd Ec ), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn 7 -derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ , gfp , and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Δ bla -Tel r - FRT efficiently transposed within Burkholderia cenocepacia , Burkholderia thailandensis , B. pseudomallei , and B. mallei . We then utilized the mini-Tn 7 -Tel r -based transposon vector (mini-Tn7-Tel r - betBA ) and a transposase-containing helper plasmid (pTNS3- asd Ec ) to complement the B. thailandensis Δ betBA mutation. Next, one of the FRT - lacZ fusion vectors (pFRT1- lacZ -Tel r ) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp- oriT - asd Ec ) to construct the B. thailandensis Δ betBA :: FRT - lacZ -Tel r reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the B. thailandensis Δ betBA :: FRT - lacZ -Tel r fusion strain. Finally, we engineered B. thailandensis Δ betBA :: FRT - gfp -Tel r and Δ betBA :: FRT - lux -Tel r fusion strains by utilizing fusion vectors pFRT1- gfp -Tel r and pFRT1- lux -Tel r , respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.