Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

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Abstract
Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of S. cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed and the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli, but could be cloned in 2 parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in B. subtilis on plasmid pGKv2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies 2 proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient S. lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis (pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.