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Applied and Environmental Microbiology, July 2006, p. 5027-5036, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00682-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria{dagger}

Robert M. Q. Shanks, Nicky C. Caiazza,{ddagger} Shannon M. Hinsa,§ Christine M. Toutain, and George A. O'Toole*

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755

Received 24 March 2006/ Accepted 3 May 2006

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1248. Fax: (603) 650-1245. E-mail: georgeo{at}Dartmouth.edu.

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

{ddagger} Present address: Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125.

§ Present address: Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824.

Applied and Environmental Microbiology, July 2006, p. 5027-5036, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00682-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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