Development of an alpha-complementation system for mycobacterial promoter analysis.

Abstract

alpha-Complementation is the restoration of beta-galactosidase (beta-Gal) activity to a lacZ mutant via expression of a differentially altered mutant. Here we report the development of an alpha-complementation system for the mycobacteria. Mycobacterium smegmatis alpha-acceptor strains were constructed employing both a novel lacZ alpha-acceptor allele, termed lacZDeltaPvuII, and the widely exploited alpha-acceptor allele lacZDeltaM15. These alpha-acceptor alleles were integrated into M. smegmatis strain mc2-155 using the suicide vector pINT-Delta. The resultant alpha-acceptor strains, TSm-629 (lacZDeltaPvuII) and TSm-630 (lacZDeltaM15), were highly transformable with plasmids, lacked detectable beta-Gal activity, and were not drug resistant. Five potential plasmid-borne alpha-donor fragments were expressed from the Mycobacterium bovis hsp60 promoter in order to determine their abilities to complement either of the two alpha-acceptor strains. beta-Gal activity was restored to both strains TSm-629 and TSm-630 by expression of the lacZ X-90 alpha-donor encoding 1021 amino acids (aa). More importantly, beta-Gal activity was restored to strain TSm-629 by expression of the CyB alpha-donor encoding 85 aa, whereas beta-Gal activity was restored to strain TSm-630 by expression of the AatII a-donor encoding 205 aa. Interestingly, these two alpha-donor/alpha-acceptor pairs were independent and did not complement each other. In addition, expression of the alpha-donor commonly employed in Escherichia coli cloning vectors, such as pUC19, did not restore beta-Gal activity to either of the M. smegmatis alpha-acceptor strains.