IN-VIVO GROWTH-CHARACTERISTICS OF LEUCINE AND METHIONINE AUXOTROPHIC MUTANTS OF MYCOBACTERIUM-BOVIS BCG GENERATED BY TRANSPOSON MUTAGENESIS

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the sl ow-growing M tuberculosis complex, was accomplished with transposons e ngineered from the Mycobacterium smegmatis insertion element IS1096. T ransposons were created by placing a kanamycin resistance gene in seve ral different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses dem onstrated that only one of the two open reading frames was necessary f or transposition. A library of insertions was generated. Southern anal ysis of 23 kanamycin-resistant clones revealed that the transposons ha d inserted directly, with no evidence of cointegrate formation, into d ifferent restriction fragments in each clone. Sequence analysis of nin e of the clones revealed junctional direct 8-bp repeats with only a sl ight similarity in target sites. These results suggest that IS1096-der ived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, we re isolated from the library of transposon insertions in BCG. They wer e characterized by sequencing and found to be homologous to the leuD g ene of Escherichia coli and a sulfate-binding protein of cyanobacteria , respectively. When inoculated intravenously into C57BL/6 mice, the l eucine auxotrophs, in contrast to the parent BCG strain or the methion ine auxotroph, showed an inability to grow in vivo and were cleared wi thin 7 weeks from the lungs and spleen.