SEQUENCE-ANALYSIS OF FLANKING REGIONS OF THE PFOA GENE OF CLOSTRIDIUM-PERFRINGENS - BETA-GALACTOSIDASE GENE (PBG) IS LOCATED IN THE 3'-FLANKING REGION

The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfo A) of Clostridium perfringens was analyzed by chromosome walking. A to tal of 5,363 bp of the downstream region of the pfoA gene was sequence d and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter protei ns of other bacteria and the beta-galactosidase gene (bgaB) of Bacillu s stearothermophilus, respectively, ORF80 was named the pbg gene. Clon es which showed beta-galactosidase activities were selected from a lam bda FIXII genomic library of C. perfringens by blue plaque screening u sing X-Gal as a substrate. Four clones whose plaques showed blue appea rances were obtained. Two of the four clones hybridized with the pbg p robe but the others did not, indicating that there are two distinct be ta-galactosidase genes in C. perfringens. The pbg gene was subcloned i nto pBR322 and was successfully expressed in Escherichia coil, suggest ing that the pbg gene codes for a beta-galactosidase of C. perfringens .