The ccpA mutations alsA1 (alsA1 is allelic to ccpA) and ccpA::Tn917 co
mpletely abolished catabolite repression of gluconate kinase and sorbi
tol dehydrogenase synthesis in Bacillus subtilis, whereas they only pa
rtially affected the catabolite repression of inositol dehydrogenase,
histidase and xylose isomerase synthesis. The alsA1 mutation also part
ially affected catabolite repression of sporulation. Analysis of rever
tants from the alsA1 mutant by direct sequencing indicated that this m
utation comprises a base substitution of guanine at nucleotide -14 to
adenine within the Shine-Dalgarno sequence of the ccpA gene (ccpA tran
slation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying th
e ccpA gene was cloned into Escherichia coli plasmid pUC19 and B. subt
ilis plasmid pUB110, resulting in plasmids pCCPA19 and pCCPA110, respe
ctively. The ccpA gene carried in pCCPA110 complemented the alsA1 muta
tion. Western blotting revealed that the level of the CcpA protein in
B. subtilis cells, which seemed to be constitutively synthesized, was
approximately 10 times lower for the alsA1 mutant than for the wild-ty
pe. The CcpA protein synthesized by either E. coli cells bearing pCCPA
19 or B. subtilis cells bearing pCCPA110 was purified to over 90 % hom
ogeneity; the latter cells were grown in the presence of glucose. The
molecular mass of the protein purified from E. coli was 74 kDa, sugges
ting that this protein exists as a dimer because its subunit molecular
mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis i
ndicated that the purified CcpA protein in both cases did not bind to
the cis sequence for catabolite repression of the gnt operon, but it b
ound non-specifically to DNA.