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

Abstract

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.

FIG. 1.

Schematic representations of preproproteinase PrtP from L. lactis (A) and PrtB from L. bulgaricus and four truncated forms (PrtBδ99, PrtBδ168, PrtBδ247, and PrtBδ806) (B). The different domains of the proteinases and the amino acids (D, H, N, and S for PrtB) involved in the active site are indicated. Small bent arrows correspond to primers designed for gene amplification. The thick horizontal bar in the catalytic domain indicates the peptide (amino acids 280 to 467) used for preparation of the anti-PrtB serum. The dashed thick horizontal bars within the C-terminal region correspond to the two imperfect repeats of 59 residues surrounding the degenerated sorting signal LPKKT. The motifs of the B domain are indicated (B2 to B5) to position the truncated PrtBδ806. Amino acids are numbered starting from the amino end of the mature proteinase.

FIG. 2.

Growth kinetics in milk of L. lactis NCDO 712, L. lactis MG1363, and L. lactis MG1363 expressing the full-length proteinase of L. bulgaricus, PrtB, and the truncated forms PrtBδ99 and PrtBδ806.

FIG. 3.

SDS-PAGE analysis of beta-casein (ca) hydrolyzed for the times indicated (in minutes) by L. bulgaricus (Ldb) and L. lactis MG1363 expressing PrtB (Ll-PrtB). Hydrolysis products (a to f) and molecular mass standards in kilodaltons are indicated.

FIG. 4.

Amino acid sequence of the H and W domains located at the carboxy end of L. bulgaricus PrtB. The last amino acids of truncated proteinases (PrtBδ247, PrtBδ168, and PrtBδ99) are boxed. Dashed bars underline both alpha-helix structures of the W domain. The 4-amino-acid repeats (KSDX) are underlined (thin lines), and lysine residues are shown in boldface type. Brackets surround the 59-amino-acid imperfect repeats, and identical amino acids are underlined (thick lines). The LPKKT degenerated sorting signal is indicated by a horizontal bracket above the sequence.

FIG. 5.

Immunodetection of cells (A) or culture supernatants (B) of L. lactis MG1363 (Ll) synthesizing the full-length PrtB or its truncated forms (PrtBδ99, PrtBδ168, PrtBδ247, and PrtBδ806). PrtB and truncated derivatives were revealed by using anti-PrtB serum. Molecular mass markers in kilodaltons are indicated.

FIG. 6.

SDS-PAGE analysis of beta-casein (ca) hydrolyzed for the times indicated (in minutes) by whole cells of L. lactis MG1363 producing full-length PrtB, PrtBδ99, or PrtBδ806. Beta-casein was also hydrolyzed by culture supernatants of L. lactis MG1363 synthesizing full-length PrtB or PrtBδ99.