Construction of a reporter plasmid for screening in vivo promoter activityin Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that surviv es and multiplies inside macrophages. Here we constructed a new promoter pr obe plasmid denoted pKK214 by introduction of a promoter-less chloramphenic ol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F tularensis strain LVS by cloning random chromosoma l DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also u sed to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F tularens is genes that are expressed during infection. (C) 2001 Federation of Europe an Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.