NUCLEOTIDE SEQUENCE AND CONTROL OF THE GDH OPERON

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During the developmental process leading to spore production in Bacillus subtilis, numerous proteins are synthesized. We have been studying one such protein, glucose dehydrogenase, which is made exclusively during sporulation and only in the forespore compartment. The gene encoding glucose dehydrogenase (gdh) is part of a bicistronic unit. From S1 nuclease mapping experiments, the location of the transcriptional initiation site was determined to be 12 base pairs from the ribosome binding site preceding the first gene of the operon. The region containing the nucleotide sequences needed for gdh expression was determined. Bal31 nuclease digestions of DNA fragments with the promoter region were fused to a promoterless lacZ gene and the gene fusions were inserted into the B. subtilis chromosome. Also, by oligonucleotide site directed mutagenesis, alterations in the base sequence at the -35 and -22 region were made. Changes in the - 35 region resulted in the loss of lacZ expression whereas no effect on lacZ expression was noted by change of base sequences in the - 22 region. Alterations in the -10 region are currently being assayed. The same technique was employed to make alterations in the B. subtilis gdh gene near the His 148 and Trp 154 region which is important for subunit binding in the Bacillus megaterium glucose dehydrogenase. An insertion of two amino acids (Val, Asn) into the B. subtilis gdh gene yielded extracts with only 5% of activity as compared to unaltered glucose dehydrogenase.