DEVELOPMENT OF AN ALPHA-COMPLEMENTATION SYSTEM FOR MYCOBACTERIAL PROMOTER ANALYSIS

alpha-Complementation is the restoration of beta-galactosidase (beta-G al) activity to a lacZ mutant via expression of a differentially alter ed mutant. Here we report the development of an alpha-complementation system for the mycobacteria. Mycobacterium smegmatis alpha-acceptor st rains were constructed employing both a novel lacZ a-acceptor allele, termed IncZ Delta PvuII, and the widely exploited alpha-acceptor allel e lacZ Delta M15. These alpha-acceptor alleles were integrated into M. smegmatis strain mc(2)-155 using the suicide vector pINT-Delta. The r esultant alpha-acceptor strains, TSm-629 (lacZ Delta PvuII) and TSm-63 0 (lacZ Delta M15), were highly transformable with plasmids, lacked de tectable beta-Gal activity, and were not drug resistant. Five potentia l plasmid-borne alpha-donor fragments were expressed from the Mycobact erium bovis hsp60 promoter in order to determine their abilities to co mplement either of the two alpha-acceptor strains. beta-Gal activity w as restored to both strains TSm-629 and TSm-630 by expression of the l acZ 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 restore d to strain TSm-630 by expression of the AatII alpha-donor encoding 20 5 aa. Interestingly, these two alpha-donor/alpha-acceptor pairs were i ndependent and did not complement each other. In addition, expression of the alpha-donor commonly employed in Escherichia coli cloning vecto rs, such as pUC19, did not restore beta-Gal activity to either of the M. smegmatis alpha-acceptor strains. (C) 1998 Elsevier Science B.V. Al l rights reserved.