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Journal of Bacteriology, June 2006, p. 4037-4050, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.02000-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa

Bridget R. Kulasekara,^1, Hemantha D. Kulasekara,^1, Matthew C. Wolfgang,^1, Lisa Stevens,¹ Dara W. Frank,² and Stephen Lory^1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115,¹ Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, Wisconsin 53226²

Received 31 December 2005/ Accepted 22 March 2006

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA^Lys gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of ß- and -Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA^Lys-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA^Lys gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

Present address: Seattle Biomedical Research Institute, 307 Westlake Avenue N, Seattle, WA 98109.

Present address: Department of Genome Sciences, University of Washington, 1705 NE Pacific, Seattle, WA 98195.

Present address: Department of Microbiology and Immunology and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599.

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