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Applied and Environmental Microbiology, December 2002, p. 5956-5964, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5956-5964.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction and Evaluation of Plasmid Vectors Optimized for Constitutive and Regulated Gene Expression in Burkholderia cepacia Complex Isolates

Matthew D. Lefebre1 and Miguel A. Valvano1,2*

Departments of Microbiology and Immunology,1 Medicine, The University of Western Ontario, London, Ontario, Canada N6A 5C12

Received 17 May 2002/ Accepted 29 August 2002

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 PBAD 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dental Sciences Building, Room 3000, The University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone: (519) 661-3996. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca.

Applied and Environmental Microbiology, December 2002, p. 5956-5964, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5956-5964.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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