Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.
Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, 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 two 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 Bacillus 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 two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus 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.
Kok
J
J
van Dijl
J M
JM
van der Vossen
J M
JM
Venema
G
G
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D015247:Deoxyribonuclease HindIII; D004587:Electrophoresis, Agar Gel; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D007123:Immunoelectrophoresis, Two-Dimensional; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010957:Plasmids; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D010450:Endopeptidases