POSSIBLE FUNCTION AND SOME PROPERTIES OF THE CCPA PROTEIN OF BACILLUS-SUBTILIS

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.