MOLECULAR-CLONING AND ANALYSIS OF NUCLEOTIDE-SEQUENCE OF THE BACILLUS-SUBTILIS LYSA-GENE REGION USING BACILLUS-SUBTILIS PHAGE VECTORS AND A MULTI-COPY PLASMID, PUB110

A 3.8-kb EcoRI-fragment containing the lysA gene [diaminopimelate (DAP) decarboxylase] of Bacillus subtilis has been cloned into B. subtilis phage phi-105 and its nucleotides sequenced. The nucleotide sequence of a 3,762 bp stretch contained three open reading frames (ORF1, ORF2, and ORF3) in one orientation and another open reading frame (ORF4) in the opposite orientation. ORF2 coded for the lysA gene based on the complementation of a B. subtilis lys auxotroph and on the fact that the predicted amino acid sequence (440 amino acids with a molecular weight of 48,876) of ORF2 shared a 29.7%, 38.3%, and 32.9% identity with the sequences of Escherichia coli, Corynebacterium glutamicum and Pseudomonas aeruginosa lysA genes, respectively. ORF1, ORF3, and ORF4 did not correspond to E. coli lysR. Based on the comparison of the B. subtilis lysA sequence with a sequence of the DAP-decarboxylase gene cloned into pUB110 (Yamamoto et al., Nucleic Acids Res., 17, 10105 (1989)), it was found that the lys gene in the plasmid was fused with the dnaN gene in its COOH-terminal region.