Identification of in vivo induced genes in Actinobacillus pleuropneumoniae

We have developed an in vivo expression technology (IVET) system to identif y Actinobacillus pleuropneumoniae gene promoters that are specifically indu ced in vivo during infection. This system is based upon an avirulent ribofl avin-requiring A. pleuropneumoniae mutant and a promoter-trap vector (pTF86 ) that contains, in sequence, the T4 terminator, a unique BamHI site, a pro moterless copy of the V. harveyi luxAB genes, and a promoterless copy of th e B. subtilis ribBAH genes in the E. coli-A. pleuropneumoniae shuttle vecto r pGZRS19. Sau3A fragments of A. pleuropneumoniae genomic DNA were cloned i nto the BamHI site in pTF86 and transformed into the A. pleuropneumoniae Ri b- mutant. Pigs were infected with pools of 300-600 transformants by endobr onchial inoculation and surviving bacteria were isolated from the pigs' lun gs at 12-16 h post-infection. Infection strongly selected for transformants containing cloned promoters which drove expression of the vector ribBAH ge nes and allowed survival of the Rib- mutant in vivo. Strains that survived in vivo, but which minimally expressed luciferase activity in vitro, should contain cloned promoters that are specifically induced in vivo. Ten clones , designated iviA-J, were isolated which contain promoters that are induced in vivo during infection. These ivi clones were shown to be induced in the animal by luminescence of infected tissue and by direct assay of bacteria recovered from bronchoalveolar lavage. Four of these clones were putatively identified by amino acid sequence similarity as ilvl, the ilvDA operon, th e secE-nusG operon, and the mrp gene. This is the first report of an IVET s ystem for use in the family Pasteurellaceae, as well as the first report of an IVET system utilizing an infection model of pneumonia in the natural ho st. (C) 1999 Academic Press.