f Possible function and some properties of the CcpA protein of Bacillus subtilis
- Authors: Yasuhiko Miwa, Mitsuyuki Saikawa, Yasutaro Fujita*
- *Author for correspondence: Y. Fuiita. Tel: +81 849 36 2111. Fax: +81 849 36 2023.
- Microbiology, October 1994 140: 2567-2575, doi: 10.1099/00221287-140-10-2567
- Subject: Genetics And Molecular Biology
- Published Online:
The ccpA mutations alsA1 (alsA1 is allelic to ccpA) and ccpA::Tn917 completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis. The alsA1 mutation also partially affected catabolite repression of sporulation. Analysis of revertants from the alsA1 mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide −14 to adenine within the Shine-Dalgarno sequence of the ccpA gene (ccpA translation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying the ccpA gene was cloned into Escherichia coli plasmid pUC19 and B. subtilis plasmid pUB110, resulting it plasmids pCCPA19 and pCCPA110, respectively. The ccpA gene carried in pCCPA110 complemented the alsA1 mutation. 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-type. The CcpA protein synthesized by either E. coli cells bearing pCCPA19 or B. subtilis cells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose The molecular mass of the protein purified from E. coli was 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cis sequence for catabolite repression of the gnt operon, but it bound non-specifically to DNA.
© Society for General Microbiology 1994 | Published by the Microbiology Society
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