Screening of genes involved in carbon source utilization (correspondence, Yasutaro Fujita)

      The genes involved in utilization of glucose, fructose, glycerol, myo-inositol, ribose or malate as sole carbon source were screened among genes inactivated by pMUTIN integration. The integrants were examined for their colony-forming abilities on minimal medium plates containing each of these as sole carbon source. As 5-bromo-4-chloro-3-indolyl β-galactoside (X-gal) was added to the plates, the genes, the expression of which was induced by these carbon sources, are also able to be found.



     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 formed colonies of the integrants were stung with toothpicks, and the cells adsorbed to one toothpick were inoculated into 1 ml of MM medium containing tryptophan (50 μg/ml), a glucose minimal medium adopted in the project, and incubated at 37˚C overnight.

     The cultures were appropriately diluted 10- to 104-fold with PM solution (100 mM K-phosphate, pH 6.5 and 1 mM MgSO4).  The diluted cell suspensions (2μl each) were spotted on N medium (see J. Bacteriol. 154:864-869) plates containing 1.5% Bacto-Agar (Difco), tryptophan (50μg/ml), X-gal (120μg/ml) and erythromycin (0.3 μg/ml), and one of carbon sources, and incubated at 37˚C until the appropriate size of the colonies was formed. The concentrations of carbon sources (glucose, fructose, glycerol, myo-inositol, ribose and malate) were 25, 25, 50, 25, 30 and 38 mM, respectively.

     The sizes of the formed colonies and their blue intensities were classified into 5 levels (-2 to +2) and 7 levels (0 to 6), respectively, in comparison with those of YXKCd and YXNBd strains used as references. These two strain gave the standard colony size (level 0), but the blue intensities of colonies of YXKCd and YXNBd strains gave the levels 3 and 6 on the plates using any of these carbon sources, respectively.