Characterization of the <i>Lactococcus lactis pepN</i> gene encoding an aminopeptidase homologous to mammalian aminopeptidase N

Tan, Paris S. T.; Alen-Boerrigter, Ingrid J. van; Poolman, Bert; Siezen, Roland J.; Vos, Willem M. de; Konings, W. N.

doi:10.1016/0014-5793(92)80827-4

Cited by 76 publications

(49 citation statements)

References 36 publications

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“…This could be due to the inactivation or low catalytic activity of PepC under cheese physicoche mi cal conditions. These observations suggest that the other general aminopeptidase PepN found in lactococci (Tan et al, 1992) might be the main enzyme with broad specificity which is active in cheese.…”

mentioning

confidence: 82%

Phage-induced lysis of Lactococcus lactis during Saint-Paulin cheese ripening and its impact on proteolysis

Kawabata

Vassal

Bars

et al. 1997

Lait

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Summary -The autolysis of starter bacteria during chee se ripening results in the release of intracellular enzymes, especially peptidases, into the curd and in consequence is expected to accelerate and enhance the development of cheese flavour. In the course of a series of cheese assays with the nonautolytic Lactococcus lactis NC00763 strain, the presence of a homologous bacteriophage was encountered. The phage was present at different levels which allowed us to evaluate the impact of the extent of starter bacteria Iysis on Saint-Paulin-type cheese proteolysis during ripening. We estimated bacterial Iysis by viable cell enumeration and electron microscopy observations over a 2-month ripening period. On the same cheeses, we have estimated proteolysis by measuring soluble nitrogen (SN) and phosphotun~stic acid-soluble nitrogen (PT A-N) and by quantifying free amino acids. For phage levels up to 10 0 pfu/g of cheese, the pH and dry matter values were in the standard range, whereas for a higher phage level incorrect acidification was observed. The ex te nt of starter Iysis increased with the phage level and it was also correlated with the rate of formation of small peptides and free amino acids. After 2 months of ripening, the PTA-N value was three-fold higher and the total free amino acid content was seven-fold higher in the cheese with a phage level of 10 10 pfu/g than in the control cheeses. These results confirm that the early Iysis of starter bacteria allows the release of intracellular peptidases which are active in cheese, and that the extent of starter Iysis can be correlated with the proteolysis rate.Lactococcus lactis / cheese ripening / Iysis / phage / proteolysis Résumé -Lyse de Lactococcus lactis induite par des phages au cours de l'affinage de fromages de type Saint-Paulin et impact sur la protéolyse.L'autolyse des bactéries lactiques du levain résulte en la libération des enzymes intracellulaires, en particulier des peptidases, dans le

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mentioning

confidence: 82%

Phage-induced lysis of Lactococcus lactis during Saint-Paulin cheese ripening and its impact on proteolysis

Kawabata

Vassal

Bars

et al. 1997

Lait

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“…Among the other overexpressed genes with known functions were mtlARF, belonging to the mtl operon, responsible for mannitol transport and metabolism (39), and the gene encoding the PepN peptidase (40). Among the down-regulated genes that may be related to the lysozyme-resistant phenotype were the main lactococcal autolysin acmA and the genes belonging to the opp operon, B. subtilis homologs of which were reported to import PG degradation products (41 Fig.…”

Section: Overexpression Of Pgda and Spxb (Yneh) Confers Lactococcalmentioning

confidence: 99%

SpxB Regulates O-Acetylation-dependent Resistance of Lactococcus lactis Peptidoglycan to Hydrolysis

Veiga

Bulbarela-Sampieri

Furlan

et al. 2007

Journal of Biological Chemistry

105

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Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis. Peptidoglycan (PG)7 is the major and essential component of the bacterial cell envelope, the main function of which is to preserve cell integrity by withstanding internal osmotic pressure (1, 2). It is also responsible for cell shape, participates in cell division, and serves as support for attachment of other cell wall molecules such as teichoic acids, proteins, and exopolysaccharides (3). The bacterial PG is a giant multilayer polymer that envelops the cell as a rigid sacculus. It is composed of N-acetylglucosamine-N-acetylmuramic acid disaccharide pentapeptide blocks that are synthesized intracellularly and transported through the cytoplasmic membrane as lipid-disaccharide pentapeptides. These blocks are covalently linked to the pre-existing PG polymers by high molecular weight penicillin-binding proteins (4).To allow cell division and surface expansion, the rigid PG sacculus has to be relaxed. This is achieved by PG ruptures, which could be introduced in several ways. First, not all possible covalent bonds are formed during PG synthesis; for example, only 36% of possible PG cross-links between stem peptides are formed in Lactococcus lactis (5). Bacteria also possess a number of endogenous bacterial enzymes (collectively called autolysins) that disrupt PG and can result in cell lysis. According to their hydrolytic bond specificity and products, autolysins are classified as muramidases, lytic transglycosylases, glucosaminidases, amidases, and peptidases (6). Bacteria often possess several autolysins, e.g. L. lactis encodes a main autolysin (N-acetylglucosaminidase AcmA) (7, 8) and four minor PG hydrolases (9, 10) as well as prophage-encoded bacteriolytic enzymes (11).In the human or animal host, antimicrobial PG lytic enzymes such as lysozyme constitute a first line of defense against infection. Bactericidal propertie...

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“…A number of proteolytic enzymes have been characterized mainly in Lactococcus lactis Pritchard and Coolbear, 1993). A serine-type peptidase of the subtilisin family associated with the cell envelope (commonly named cell-wall proteinase) is involved in the first step of casein breakdown (Kok, 1990) and peptidases with various specificities, localized in the cytoplasm, cleave peptides into free amino acids (Mayo et al, 1991;Nardi et al, 1991, Van Allen-Boerrigter et al, 1991Tan et al, 1992b;Stroman, 1992;Chapot-Chartier et al, 1993;Mierau et al, 1993;Monnet et al, 1993). The aminopeptidase PepC was the only cysteine peptidase found in this bacteria.…”

Section: )mentioning

confidence: 99%

Gene Cloning and Characterization of PepC, a Cysteine Aminopeptidase from Streptococcus thermophilus, with sequence Similarity to the Eucaryotic Bleomycin Hydrolase

Chapot‐Chartier

Rul

Nardi

et al. 1994

European Journal of Biochemistry

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Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe-P-naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid. The wild-type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe-P-naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC-like enzyme on the basis of cross-reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame coding for a 445-residue (50414 Da) polypeptide was identified. 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase. In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and was purified from a cellular extract. Purification to homogeneity was achieved by two-step ion-exchange chromatography. Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity. The enzyme has a molecular mass of 300kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC-type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases.

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Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N

Cited by 76 publications

References 36 publications

Phage-induced lysis of Lactococcus lactis during Saint-Paulin cheese ripening and its impact on proteolysis

Phage-induced lysis of Lactococcus lactis during Saint-Paulin cheese ripening and its impact on proteolysis

SpxB Regulates O-Acetylation-dependent Resistance of Lactococcus lactis Peptidoglycan to Hydrolysis

Gene Cloning and Characterization of PepC, a Cysteine Aminopeptidase from Streptococcus thermophilus, with sequence Similarity to the Eucaryotic Bleomycin Hydrolase

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