ANALYSIS OF THE MYCOBACTERIUM-BOVIS HSP60 PROMOTER ACTIVITY IN RECOMBINANT MYCOBACTERIUM-AVIUM

A clinical isolate of Mycobacterium avium was transformed with a new s huttle plasmid containing the Escherichia coli beta-galactosidase repo rter gene under the control of the Mycobacterium bovis bacillus Calmet te-Guerin (BCG) hsp60 promoter. beta-Galactosidase activity was assaye d spectrophotometrically in bacterial homogenates of the recombinant s train (M. avium.. lacZ) and used for quantification of the hsp60 promo ter strength in different conditions of extra- and intracellular growt h. Very low levels of beta-galactosidase were recorded during the expo nential phase of in vitro growth, while they increased progressively d uring the late exponential and stationary phases. A significant increa se in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but no t from 37 to 42 degrees C nor from 30 to 42 degrees C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures . Finally, beta-galactosidase levels were quantified during growth of M. avium::lacZ in murine macrophages, Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels o f bacterial beta-galactosidase were observed indicating an increment i n transcriptional activity of hsp60 promoter both at early phases of i nfection and during the course of intracellular growth. (C) 1998 Feder ation of European Microbiological Societies. Published by Elsevier Sci ence B.V. All rights reserved.