ISOLATION AND CHARACTERIZATION OF THE ASPARTOKINASE AND ASPARTATE SEMIALDEHYDE DEHYDROGENASE OPERON FROM MYCOBACTERIA

Diaminopimelic acid (DAP) is a major component of the peptidoglycan la yer of the mycobacterial cell wail. The mycobacterial cell wall has be en implicated as a potential virulence factor and is highly immunogeni c. The pathway for biosynthesis of DAP may serve as a target in the de sign of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorl y understood in mycobacteria. In order to develop a better understandi ng of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehyd rogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guer in (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coil This gene was then used to isolat e the asd genes from other mycobacteria. The asd-comptementing fragmen ts from BCG and M. smegmatis were sequenced. An open reading frame ups tream of the mycobacterial asd gene was identified as the mycobacteria l aspartokinase gene (ask). Primer extension analysis revealed that th e only transcriptional start in this region is found 5' of the ask gen e. This observation indicates that the mycobacterial ask and asd genes are in an operon.