Novel bacterial artificial chromosome vector pUvBBAC for use in studies of the functional genomics of Listeria spp.
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.
Hain
Torsten
T
Institute for Medical Microbiology, Justus Liebig University, Frankfurter Strasse 107, 35392 Giessen, Germany.
Otten
Sonja
S
von Both
Ulrich
U
Chatterjee
Som S
SS
Technow
Ulrike
U
Billion
André
A
Ghai
Rohit
R
Mohamed
Walid
W
Domann
Eugen
E
Chakraborty
Trinad
T
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics*; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D015151:Immunoblotting; D008087:Listeria / Q000235:genetics* / Q000254:growth & development; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic