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.Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), a gram-positive, facultatively anaerobic, homofermentative lactic acid bacterium, plays an important role in the dairy industry because of its efficient and reliable utilization of milk constituents, mainly lactose and caseins, and its good resistance to low pH. Spontaneous mutants of L. bulgaricus, unable to grow on milk in the absence of peptide extract, were characterized by ISL3 transposition-induced deletions, including that of the prtB gene coding for the cell surface proteinase (CSP) (11). Actually, prtB is essential for L. bulgaricus growth in milk and is responsible for the first step of caseinolysis. Most L. bulgaricus strains are characterized by a high CSP activity resulting from the adaptation of this species to fast growth and rapid fermentation of milk (14).So far, four different types of genes encoding CSPs of dairy lactic acid bacteria have been cloned and sequenced: prtB from L. bulgaricus, prtP from Lactococcus lactis and Lactobacillus casei, prtS from Streptococcus thermophilus, and prtH from Lactobacillus helveticus (8,13,18,21,28,38). Comparative sequence analysis of CSPs revealed different domains associated with putative functions (35). CSPs are synthesized as long and inactive preproproteins (ϳ2,000 residues). For the N terminus of CSP, eight domains have been predicted (Fig. 1A). (i) The predomain (ϳ30 residues) corresponds to a signal sequence required for secretion and is removed by a specific signal peptidase during translocation through the cytoplasmic membrane. (ii) The prodomain (ϳ150 residues) is essential for enzyme maturation and is removed by autoproteolytic cleavage. (iii) The catalytic domain (...

A Multifunction ABC Transporter (Opt) Contributes to Diversity of Peptide Uptake Specificity within the Genus Lactococcus

“…M17Lac-grown bacterial cells (OD 600 ϭ 1.0) were collected, washed, and resuspended in 100 mM HEPES (pH 6.5) and 10 mM CaCl 2 to a final OD 600 of approximately 7.0. A 5.5 solution of dephosphorylated ␤-casein (Sigma) was hydrolyzed by cellular suspension as previously described (13). Hydrolysis products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 15% polyacrylamide gels and stained with Coomassie brillant blue R250 (Prolabo, Fontenaysous-Bois, France) as previously described (14).…”

Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis

“…Most CEPs require the presence of chaperones for maturation (lactococcal PrtP [69] and PrtH from Lactobacillus helveticus [53]), whereas others are capable of an autocatalytic process that substitutes the chaperone protein PrtM with an intramolecular chaperone (PrtB of L. delbrueckii spp. bulgaricus [25]). The identified CEP of L. johnsonii NCC533 is a unique gene among lactobacilli of human origins.…”

“…1b, XXXI). Cell-envelop proteinases (CEP) facilitate the proteolysis of casein in lactic acid bacteria [53,61] and are often essential for the growth in milk [25]. Furthermore, CEPs were also associated with periodontal disease in Bacteroides forsythes [64].…”

Amino Acid Biosynthesis and Proteolysis in <i>Lactobacillus Bulgaricus</i> Revisited: A Genomic Comparison

“…As is known that H domain is able to position the N-terminal of PrtB outside the cell wall, and W domain spans the cell wall [19]. The loss of this region might affect the attachment to cell wall and folding of PrtB, thus changing the cleavage pattern on substrate.…”