THE URAA LOCUS AND HOMOLOGOUS RECOMBINATION IN MYCOBACTERIUM-BOVIS BCG

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic mark ers and as sequences used to target homologous integration of recombin ant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by compl ementing an Escherichia coli mutant defective in this activity. The BC G OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predi cted BCG OMP-DCase protein sequence is closely related to the Myxococc us xanthus OMP-DCase and more distantly related to the other known pro karyotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transf ormation of BCG was developed and a linear fragment of mycobacterial D NA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resis tant BCG transformants all contained vector DNA integrated into the ge nome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implication s for understanding the role of mycobacterial genes in disease pathoge nesis and for the genetic engineering of improved mycobacterial vaccin es.