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Applied and Environmental Microbiology, May 2003, p. 3020-3023, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.3020-3023.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

An Improved Cloning Vector for Construction of Gene Replacements in Listeria monocytogenes

Guojie Li1,{dagger} and S. Kathariou2*

Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822,1 Department of Food Science and Graduate Program in Genomic Sciences, North Carolina State University, Raleigh, North Carolina 27695-76242

Received 4 October 2002/ Accepted 10 February 2003

Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.

* Corresponding author. Mailing address: North Carolina State University, Department of Food Science, Raleigh NC 27695-7624. Phone: (919) 513-2075. Fax: (919) 515-7124. E-mail: skathar{at}unity.ncsu.edu.

{dagger} Present address: Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710.

Applied and Environmental Microbiology, May 2003, p. 3020-3023, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.3020-3023.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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