ISOLATION OF A FRANCISELLA-TULARENSIS MUTANT THAT IS SENSITIVE TO SERUM AND OXIDATIVE KILLING AND IS AVIRULENT IN MICE - CORRELATION WITH THE LOSS OF MIND HOMOLOG EXPRESSION

We constructed mutant strains of Francisella tularensis biotype novici da by insertional mutagenesis with a kanamycin resistance (Km(R)) cass ette. One mutant, KEM7, was defective for survival in macrophages in c omparison with the wild-type (WT) strain and a random insertion strain , KEM21. While all three strains exhibited intracellular growth, the n umber of viable KEM7 present after 24-48 h of infection was approximat ely 10 times less than that of WT or KEM21. This observation was appar ently due to a reduced number of viable KEM7 associated with the macro phages one hour after phagocytosis. KEM7 was approximately 3 times mor e susceptible than WT or KEM21 to killing by the products of the xanth ine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also f ound to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 10 00- to 10000-times less than that of either KEM21 or WT. DNA sequence analysis at the Km(R) insertion site suggested that the F. tularensis homologue of min D had been interrupted. Western immunoblot analysis c onfirmed the presence of a MinD homologue in F. tularensis WT and KEM2 1, and demonstrated its absence in KEM7.