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Applied and Environmental Microbiology, August 2009, p. 4936-4949, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.02564-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus ^,

Paul A. Gulig,^* Matthew S. Tucker, Patrick C. Thiaville, Jennifer L. Joseph, and Roslyn N. Brown

Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, P.O. Box 100266, Gainesville, Florida 32610-0266

Received 10 November 2008/ Accepted 29 May 2009

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.

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* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, P.O. Box 100266, Gainesville, FL 32610-0266. Phone: (352) 392-0050. Fax: (352) 273-8905. E-mail: gulig{at}ufl.edu

Published ahead of print on 5 June 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, August 2009, p. 4936-4949, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.02564-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Brown, R. N., Gulig, P. A. (2009). Roles of RseB, {sigma}E, and DegP in Virulence and Phase Variation of Colony Morphotype of Vibrio vulnificus. Infect. Immun. 77: 3768-3781 [Abstract] [Full Text]