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Infection and Immunity, July 2004, p. 4010-4022, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4010-4022.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Burkholderia cenocepacia Genes Required for Bacterial Survival In Vivo

Tracey A. Hunt,1,{dagger} Cora Kooi,2 Pamela A. Sokol,2 and Miguel A. Valvano1,3*

Departments of Microbiology and Immunology,1 Medicine, University of Western Ontario, London, Ontario N6A 5C1,3 Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta T2N 4N1, Canada2

Received 11 February 2004/ Returned for modification 10 March 2004/ Accepted 18 March 2004

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.

* Corresponding author. Present address: Department of Microbiology and Immunology, Dental Sciences Building, Rm. 3014, University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone: (519) 661-3996. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca.

Editor: J. N. Weiser

{dagger} Present address: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Infection and Immunity, July 2004, p. 4010-4022, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4010-4022.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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