Determination of the domain of the Lactobacillus delbrueckii subsp. bulgaricus cell surface proteinase PrtB involved in attachment to the cell wall after heterologous expression of the prtB gene in Lactococcus lactis.
Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP(-) PrtM(-)) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.
Germond
Jacques-Edouard
JE
Nestlé Research Centre, Nestec Ltd., CH-1000 Lausanne 26, Switzerland. jacques-edouard.germond@rdls.nestle.com
Delley
Michèle
M
Gilbert
Christophe
C
Atlan
Danièle
D
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D002364:Caseins / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D003470:Culture Media; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002364:Caseins; D003470:Culture Media; D010450:Endopeptidases; C099750:PrtB protein, bacteria