Screening of catabolite repressive genes (correspondence, Yasutaro Fujita)

     When the genes are inactivated by pMUTIN-integration, their expression is able to be monitored by examining the synthesis of β-galactosidase in the integrants as a reporter. To examine whether or not the inactivated genes are under catabolite repression, we compared the blue intensities of the colonies grown on nutrient sporulation medium plates containing 5-bromo-4-chloro-3-indolyl β-galactoside (X-gal) with and without glucose.

METHODS

     The integrant cells were grown on Tryptose Blood Agar Base (Difco) plates containing 10 mM glucose and 0.3μg/ml erythromycin at 30˚C overnight. The integrants, GALEd and IOLHd, were used as catabolite repression-negative and -positive controls, respectively

     The formed cell colonies were stung with toothpicks and transferred onto DSM medium plates, a nutrient sporulation medium adopted in the project, containing 1.5% Bacto-Agar (Difco), 0.3 mg/ml erythromycin, 100 mM K-MOPS (pH 7) and 120 μg/ml X-gal, and incubated at 37˚C overnight. The blue intensities of the formed colonies were classified into 7 levels (0 to 6) in comparison with those of the GALEd and IOLHd colonies as references. 

   The blue intensities of the GALEd colonies were considered as the level 3 either with or without glucose, while those of the IOLHd colonies were as the levels 2 and 6 with and without glucose, respectively.