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Applied and Environmental Microbiology, July 2005, p. 3917-3927, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3917-3927.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a recA Gene-Based Identification Approach for the Entire Burkholderia Genus

George W. Payne,1 Peter Vandamme,2 Sara H. Morgan,1 John J. LiPuma,3 Tom Coenye,2 Andrew J. Weightman,1 T. Hefin Jones,1 and Eshwar Mahenthiralingam1*

Cardiff School of Biosciences, Cardiff University, Cardiff, Wales CF10 3TL, United Kingdom,1 Laboratorium voor Microbiologie, Faculteit Wetenschappen Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, B-9000 Ghent, Belgium,2 Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109-06463

Received 15 November 2004/ Accepted 4 February 2005

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.

* Corresponding author. Mailing address: Cardiff School of Biosciences, Main Building, Museum Avenue, PO Box 915, Cardiff University, Cardiff, Wales CF10 3TL, United Kingdom. Phone: 44(0)29 20875875. Fax: 44 (0)29 20874305. E-mail: MahenthiralingamE{at}cardiff.ac.uk.

Applied and Environmental Microbiology, July 2005, p. 3917-3927, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3917-3927.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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