A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

Gilbert, Christophe; Atlan, Danièle; Blanc, Baptiste; Portailer, R; Germond, Jacques‐Edouard; Lapierre, Luciane; Mollet, Beat

doi:10.1128/jb.178.11.3059-3065.1996

Cited by 102 publications

(81 citation statements)

References 51 publications

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“…Here the presence of an extracellular protease, several amino acid transport systems, two complete peptide transport systems, and numerous peptidases (Tables 9 and 10, which are published as supporting information on the PNAS web site) would render most amino acid biosynthesis pathways superfluous. As a consequence, the extracellular protease has become essential for growth in milk (32).…”

Section: Regulatory Functions: Two Siga Homologues and Relatively Fewmentioning

confidence: 99%

The complete genome sequence ofLactobacillus bulgaricusreveals extensive and ongoing reductive evolution

Guchte

Penaud

Grimaldi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

366

300

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Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.bulgaricus) is one of the economically most important representatives of the heterogeneous group of lactic acid bacteria, with a worldwide application in yogurt production. Yogurt has long been recognized as a nutritious, natural, and safe component of a healthy diet and is at the basis of the concept of probiotics (1, 2). A well documented health benefit of the consumption of yogurt containing live L. bulgaricus and Streptococcus thermophilus is an attenuation of lactose intolerance (3). In addition, immune modulation and diarrhea-alleviating effects have been reported (4), and both L. bulgaricus and S. thermophilus have been implicated in these effects (3,5). During yogurt fermentations protocooperation between these two bacteria results in an accelerated acidification, but the mechanisms involved are not completely understood (6).Among the lactic acid bacteria, L. bulgaricus belongs to the acidophilus complex, a group of lactobacilli related to Lactobacillus acidophilus, Lactobacillus johnsonii, and Lactobacillus gasseri, which have been used as probiotic cultures. Although within this group L. bulgaricus is considered unique because of its atypical GC content, until recently the lack of tools for genetic manipulation has severely hampered a more detailed analysis of this organism (7, 8, † †).Here we present the genome sequence of L. bulgaricus strain ATCC11842, originally isolated from bulgarian yogurt by S. OrlaJensen in 1919 (unpublished work). The analysis of this genome and comparison to other members of the acidophilus complex and S. thermophilus (9) have contributed to a more complete understanding of its p...

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Section: Regulatory Functions: Two Siga Homologues and Relatively Fewmentioning

confidence: 99%

The complete genome sequence ofLactobacillus bulgaricusreveals extensive and ongoing reductive evolution

Guchte

Penaud

Grimaldi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

366

300

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“…Only S-layer proteins and surface-located enzymes have been extensively characterized (2,4,11,12,33). We are currently investigating the surface of the guinea pig female genital tract isolate Lactobacillus fermentum BR11 (26).…”

Section: Extraction Ofmentioning

confidence: 99%

Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11

et al. 1997

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Extraction ofCell surface polypeptides of gram-positive bacteria may be divided into a number of classes on the basis of the way in which they are anchored to the cell envelope. In two such classes, the LPXTG-containing proteins and the lipoproteins, attachment is covalent. The former are anchored to cell wall peptidoglycan via the threonine residue in the C-terminally located LPXTG motif (20), while the latter are anchored to cytoplasmic membrane lipids via the cysteine residue in the N-terminally located LXXC motif (29). Many lipoproteins of gram-positive bacteria are homologs of periplasmic solutebinding proteins of gram-negative bacteria, and it is currently thought that lipid anchoring of these proteins is necessary in gram-positive bacteria because there is no outer membrane to prevent their loss to the environment (29).A number of other surface-located proteins are anchored via charge interactions. Examples include S-layer proteins which form regular arrays covering the cell surface and the protein monomers that constitute polymeric structures such as fimbriae and flagella. A characteristic of S-layer proteins from lactobacilli is that they can be extracted from the cell surface by charge-occupying agents such as concentrated salt or guanidine hydrochloride (17, 18).The cell surfaces of lactobacilli are poorly understood in comparison to those of the related streptococci. Only S-layer proteins and surface-located enzymes have been extensively characterized (2,4,11,12,33). We are currently investigating the surface of the guinea pig female genital tract isolate Lactobacillus fermentum BR11 (26). Here we report the sequence of BspA, a surface-located polypeptide of this organism. BspA is selectively removed by extraction with 5 M LiCl and is clearly homologous to family III of the bacterial solute-binding proteins (30). Unlike solute-binding proteins from gram-positive bacteria, BspA does not contain a lipoprotein consensus sequence. It has a highly alkaline isoelectric point, and it appears likely that it is anchored by electrostatic interaction with negatively charged groups on the cell surface. MATERIALS AND METHODSStrains and plasmids. L. fermentum BR11 is a guinea pig vaginal isolate that has been previously described (26). It was grown in liquid or solid MRS medium (Oxoid) for 18 to 24 h at 37°C in the presence of 5% CO 2 . Escherichia coli JM109 (36) and recombinant derivatives were grown at 37°C in Luria-Bertani medium (27) supplemented with ampicillin (100 g/ml), isopropyl-␤-D-thiogalactopyranoside (IPTG) (0.25 mM), and 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) (40 l of a 20-mg/ml solution) as appropriate. Plasmid pUC18 has been described previously (36), while plasmids pMFT1, pMFT2, and pMFT3 are pUC18 derivatives described more fully in Results.L. fermentum BR11 extractions. Cell surface extractions were made from 35-ml stationary-phase cultures. The cells were harvested by centrifugation (10,000 ϫ g, 5 min), washed once with an equal volume of 0.15 M NaCl, resuspended in 500 l of ...

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“…The PrtB of L. bulgaricus strongly differs from other CSPs in its specificity of cleavage and the structure of the long Cextension domains (ϳ1,100 residues) (13,23). The cleavage specificity of CSP is mainly dependent on two regions: a substrate binding site located in the catalytic domain and a short region of the A domain (35,36).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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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

Germond

Delley

Gilbert

et al. 2003

Appl Environ Microbiol

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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 (...

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A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

Cited by 102 publications

References 51 publications

The complete genome sequence ofLactobacillus bulgaricusreveals extensive and ongoing reductive evolution

The complete genome sequence ofLactobacillus bulgaricusreveals extensive and ongoing reductive evolution

Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11

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

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