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
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.
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.
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.