Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.
The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.
Law
J
J
Department of Food Microbiology, University College, Cork, Ireland.
Vos
P
P
Hayes
F
F
Daly
C
C
de Vos
W M
WM
Fitzgerald
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002364:Caseins / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D006868:Hydrolysis; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D002364:Caseins; D010450:Endopeptidases