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Appl. Environ. Microbiol. doi:10.1128/AEM.00415-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A novel bacterial artificial chromosome vector pUvBBAC for functional genomics in Listeria spp

Institute for Medical Microbiology, Justus-Liebig-University, D-35392 Giessen, Germany

^* To whom correspondence should be addressed. Email: trinad.chakraborty{at}mikrobio.med.uni-giessen.de.

	Abstract

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector pUvBBAC for replication in both Gram-negative and Gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a non-pathogenic Listeria innocua strain. Recombinant L. innocua strains harboring the pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin-assembly protein ActA, and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad host range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of Gram-positive bacteria as well as its use in explorative functional metagenomics.

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