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Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp. ^,

Torsten Hain, Sonja Otten, Ulrich von Both, Som S. Chatterjee, Ulrike Technow, André Billion, Rohit Ghai, Walid Mohamed, Eugen Domann, and Trinad Chakraborty^*

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

Received 22 February 2007/ Accepted 18 November 2007

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 nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring 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.

Published ahead of print on 25 January 2008.

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

These authors contributed equally to this work.

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• Monk, I. R., Gahan, C. G. M., Hill, C. (2008). Tools for Functional Postgenomic Analysis of Listeria monocytogenes. Appl. Environ. Microbiol. 74: 3921-3934 [Abstract] [Full Text]