Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Abstract

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

FIG. 1.

pMLBAD and pMLS7 expression vector maps. (A) Construction of pMLBAD and pMLS7 vectors. Sources of DNA fragments described in Materials and Methods are indicated by arrows above each construct. Primers used in plasmid constructions are indicated by the numbers above or below the small arrows: 1, DHFRNT; 2, DHFRCT; 3, PBADNT; 4, PBADCT; 5, S7NT; 6, S7CT; 7, P6000NT; and 8, P6000CT. Blunt, end of the fragment cloned directly from the PCR product; dhfr, dihydrofolate reductase gene encoding trimethoprim resistance; P_BAD, arabinose-inducible promoter; araC, transcriptional regulator gene; rrnB, transcriptional terminator; MCS, multiple cloning site; P_S7, region carrying the S7 ribosomal protein promoter from Burkholderia sp. strain LB400; p6000, backbone sequence derived from broad-host-range vector pME6000; mob, gene required for conjugal transfer of plasmid; rep, replication protein gene; ori, origin of replication. (B) Map of pMLBAD and pMLS7. Unique restriction endonuclease sites in the MCS of both plasmids are indicated. Plasmids are not drawn to scale.

FIG. 2.

Analysis of pMLS7 stability in the absence of antibiotic selection. Cultures of Burkholderia strains C5424 (□) and CEP040 (⋄) and E. coli DH5α (trio), all containing pMLS7, were grown in LB without trimethoprim for 20 generations. Aliquots were removed after every four generations and plated on LB agar with or without trimethoprim. The percent stability of pMLS7 in each strain was determined by dividing the number of colonies obtained on LB-trimethoprim plates by the number of colonies obtained on LB plates with no trimethoprim. The data points represent the means and standard deviations from the experiments completed in triplicate.

FIG. 3.

Western blot analysis of arabinose-induced eGFP expression from CEP040(pMLBAD-eGFP) cultures. Cultures were grown in LB alone or with the addition of various concentrations of arabinose and glucose. Samples were processed as described in Materials and Methods. Lanes: 1, LB alone; 2, 0.2% glucose; 3, 1% arabinose; 4, 2% arabinose.

FIG. 4.

eGFP expression in B. cepacia cells in culture and following the infection of A. polyphaga. (A and B) Images representing the same field of view of CEP040(pMLBAD-eGFP) cells grown in the presence of 2% arabinose visualized by differential interference phase contrast and fluorescence microscopy, respectively. (C and D) Images representing the same field of view of CEP040(pMLBAD-eGFP) cells grown in the presence of 1% glucose visualized by differential interference phase contrast and fluorescence microscopy, respectively. (E) Image of an A. polyphaga cell following 12 h of infection with C5424(pMLBAD-eGFP) visualized by fluorescence microscopy. (F) Overlay of a phase contrast image with the fluorescent image in panel E. All images were obtained at magnification ×1,000.