Development of an alpha-complementation system for mycobacterial promoter analysis.
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.
Knipfer
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Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA.
Nooruddin
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Shrader
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TE
D000483:Alleles; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test / Q000379:methods*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018834:Chaperonin 60; D009838:Oligodeoxyribonucleotides; D001616:beta-Galactosidase