An improved cloning vector for construction of gene replacements in Listeria monocytogenes

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.