Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium.
A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) hsp60 promoter. beta-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 beta-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 degrees C, but not 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 of bacterial beta-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.
Batoni
G
G
Dipartimento di Patologia Sperimentale Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Italy.batoni@biomed.unipi.it
Maisetta
G
G
Florio
W
W
Freer
G
G
Campa
M
M
Senesi
S
S
D000818:Animals; D018834:Chaperonin 60 / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter; D018869:Heat-Shock Response; D007763:Lac Operon; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009162:Mycobacterium avium / Q000235:genetics* / Q000254:growth & development; D009163:Mycobacterium bovis / Q000235:genetics*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D013154:Spleen / Q000166:cytology / Q000382:microbiology
D018834:Chaperonin 60