Open AccessAnalysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium
Author(s) -
Batoni Giovanna,
Maisetta Giuseppantonio,
Florio Walter,
Freer Giulia,
Campa Mario,
Senesi Sonia
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb13307.x
Subject(s) - mycobacterium bovis , microbiology and biotechnology , escherichia coli , biology , intracellular , phagocytosis , mycobacterium , lac operon , bacteria , exponential growth , recombinant dna , incubation , gene , biochemistry , mycobacterium tuberculosis , medicine , tuberculosis , genetics , pathology , mathematical analysis , mathematics
A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli β‐galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette‐Guérin (BCG) hsp60 promoter. β‐Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain ( M. avium::lacZ ) and used for quantification of the hsp60 promoter strength in different conditions of extra‐ and intracellular growth. Very low levels of β‐galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases. A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45°C, but not from 37 to 42°C nor from 30 to 42°C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures. Finally, β‐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 of bacterial β‐galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.