Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host.
We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotics resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltransferase (cat) and lacZY genes. A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with intraperitoneal injections of chloramphenicol. Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium. These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids. The pIVET8-based selection system was also used to identify S. typhimurium genes that are induced in cultured macrophages. The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
M J
MJ
Mahan
J W
JW
Tobias
J M
JM
Slauch
P C
PC
Hanna
R J
RJ
Collier
J J
JJ
Mekalanos
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002702:Chloramphenicol Resistance; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012481:Salmonella Infections, Animal / Q000382:microbiology; D012486:Salmonella typhimurium / Q000235:genetics / Q000472:pathogenicity*; D014169:Transformation, Bacterial; D014774:Virulence / Q000235:genetics
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase