scholarly journals Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

2012 ◽  
Vol 78 (9) ◽  
pp. 3469-3472 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Dolores Gutiérrez ◽  
Beatriz Martínez ◽  
Ana Rodríguez ◽  
Pilar García
Keyword(s):  
Staphylococcus Aureus ◽  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Content Type ◽  
Inducible Promoters ◽  
Secretion Signal ◽  
Cell Extracts ◽  
Culture Supernatants ◽  
Secretion Signal Sequence

ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

scholarly journals Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

1998 ◽  
Vol 64 (12) ◽  
pp. 4857-4861 ◽  
Author(s):  
Toshiyuki Murai ◽  
Mitsuyoshi Ueda ◽  
Takashi Kawaguchi ◽  
Motoo Arai ◽  
Atsuo Tanaka
Keyword(s):  
Cell Surface ◽  
Rhizopus Oryzae ◽  
Signal Sequence ◽  
Hydrolytic Enzymes ◽  
Water Soluble ◽  
Aspergillus Aculeatus ◽  
Cellulosic Materials ◽  
Secretion Signal ◽  
Engineered Yeast ◽  
Secretion Signal Sequence

ABSTRACT Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungusAspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.

scholarly journals Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus, theagrSystem

2014 ◽  
Vol 80 (22) ◽  
pp. 7028-7035 ◽  
Author(s):  
Sébastien Nouaille ◽  
Lucie Rault ◽  
Sophie Jeanson ◽  
Pascal Loubière ◽  
Yves Le Loir ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Response Regulator ◽  
Food Poisoning ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Virulence Regulator ◽  
Reducing Properties

ABSTRACTStaphylococcus aureusis a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol ofS. aureususing lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability ofLactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence,agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties ofL. lactiscontribute toagrdownregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% releasedagrdownregulation in the presence ofL. lactis. This downregulation still occurred in anS. aureus srrAmutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability ofL. lactisreducing properties to interfere with the expression ofS. aureusvirulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.

scholarly journals Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

2012 ◽  
Vol 79 (1) ◽  
pp. 347-356 ◽  
Author(s):  
Daphne T. W. Ng ◽  
Casim A. Sarkar
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Biotechnological Production ◽  
Content Type ◽  
Amino Acid Mutagenesis ◽  
Native Signal Peptide

ABSTRACTLactococcus lactisis an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion fromL. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels ofStaphylococcus aureusnuclease and further evaluated them for secretion ofBacillus subtilisα-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineeredL. lactissignal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

scholarly journals NisI Maturation and Its Influence on Nisin Resistance in Lactococcus lactis

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Jiaheng Liu ◽  
Hui Xiong ◽  
Yuhui Du ◽  
Itsanun Wiwatanaratanabutr ◽  
Xiaofang Wu ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Decisive Role ◽  
Fold Increase ◽  
Signal Peptidase ◽  
Nisin Production ◽  
Content Type ◽  
Peptide Cleavage ◽  
Potential Strategy

ABSTRACT NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro. Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production. IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.

scholarly journals The Papain Inhibitor (SPI) of Streptomyces mobaraensis Inhibits Bacterial Cysteine Proteases and Is an Antagonist of Bacterial Growth

2013 ◽  
Vol 57 (7) ◽  
pp. 3388-3391 ◽  
Author(s):  
Stephan Zindel ◽  
Wendy E. Kaman ◽  
Sabrina Fröls ◽  
Felicitas Pfeifer ◽  
Anna Peters ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacillus Anthracis ◽  
Pathogenic Bacteria ◽  
Cysteine Proteases ◽  
Inhibitory Effect ◽  
Inhibitory Protein ◽  
Content Type ◽  
Cysteine Protease Activity ◽  
Streptomyces Mobaraensis ◽  
Culture Supernatants

ABSTRACTA novel papain inhibitory protein (SPI) fromStreptomyces mobaraensiswas studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureusSspB) and culture supernatants (Porphyromonas gingivalis,Bacillus anthracis). Further, growth ofBacillus anthracis,Staphylococcus aureus,Pseudomonas aeruginosa, andVibrio choleraewas completely inhibited by 10 μM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.

scholarly journals Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

2012 ◽  
Vol 79 (4) ◽  
pp. 1309-1315 ◽  
Author(s):  
Stefan Irmler ◽  
Tharmatha Bavan ◽  
Andrea Oberli ◽  
Alexandra Roetschi ◽  
René Badertscher ◽  
...  
Keyword(s):  
Pediococcus Acidilactici ◽  
Gene Encoding ◽  
Pediococcus Pentosaceus ◽  
Content Type ◽  
Cell Extracts ◽  
Serine Dehydratase ◽  
Mutant Strains ◽  
Amino Acid Profiles ◽  
Culture Supernatants ◽  
Dehydratase Activity

ABSTRACTThe ability to produce diacetyl from pyruvate andl-serine was studied in various strains ofPediococcus pentosaceusandPediococcus acidilacticiisolated from cheese. After being incubated on both substrates, onlyP. pentosaceusproduced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified inP. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functionaldsdAwas cloned fromP. pentosaceusFAM19132 and expressed inEscherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate froml- andd-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants fromdsdAwild-type anddsdAmutant strains ofP. pentosaceusdid not show differences in serine levels. In contrast,P. acidilacticidegraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

scholarly journals Inhibition of Staphylococcus aureus by Lysostaphin-Expressing Lactobacillus plantarum WCFS1 in a Modified Genital Tract Secretion Medium

2011 ◽  
Vol 77 (24) ◽  
pp. 8500-8508 ◽  
Author(s):  
Huanli Liu ◽  
Yuan Gao ◽  
Li-Rong Yu ◽  
Richard C. Jones ◽  
Christopher A. Elkins ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Lactobacillus Plantarum ◽  
Genital Tract ◽  
Signal Sequence ◽  
Promoter Sequence ◽  
Content Type ◽  
Neutral Ph ◽  
Vaginal Microflora ◽  
Tract Secretion ◽  
Ph Conditions

ABSTRACTLactobacillusspecies are a predominant member of the vaginal microflora and are critical in maintaining an acidic vaginal environment thought to contribute to the prevention of a number of urogenital diseases. However, during menstruation the pH of the vaginal environment increases to neutrality, a pH conducive forStaphylococcus aureusproliferation and the production of toxic shock syndrome toxin 1 (TSST-1) in susceptible women. In order to generateLactobacillusspecies capable of expressing lysostaphin (an endopeptidase that cleaves the cell wall ofS. aureus) in a modified genital tract secretion medium (mGTS) under neutral-pH conditions, six prominent proteins fromLactobacillus plantarumWCFS1 spent medium were identified by mass spectrometry. Sequences for promoters, signal peptides, and mature lysostaphin were used to construct plasmids that were subsequently transformed intoL. plantarumWCFS1. The promoter and signal sequences of Lp_3014 (putatively identified as a transglycosylase) or the promoter sequence of Lp_0789 (putatively identified as glyceraldehyde 3-phosphate dehydrogenase) with the signal sequence of Lp_3014 exhibited lysostaphin activity on buffered medium containing heat-killedS. aureus. The cassettes were integrated into the chromosome ofL. plantarumWCFS1, but only the cassette containing the promoter and signal sequence from Lp_3014 had integrated into the appropriate site. Coculture assays using buffered mGTS showed that lysostaphin expressed fromL. plantarumWCFS1 reduced the growth of TSST-1-producing strains ofS. aureusunder neutral-pH conditions. This study provides the basis for determining whether lysostaphin-producingLactobacillusstrains could potentially be used as a means to inhibit the growth ofS. aureusduring menstruation.

scholarly journals Functional Display of a Heterologous Protein on the Surface of Lactococcus lactis by Means of the Cell Wall Anchor of Staphylococcus aureus Protein A

1998 ◽  
Vol 64 (1) ◽  
pp. 342-345 ◽  
Author(s):  
Lothar Steidler ◽  
Jasmine Viaene ◽  
Walter Fiers ◽  
Erik Remaut
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Heterologous Protein ◽  
Protein A ◽  
Food Grade ◽  
Secretion Signal ◽  
Secretion Signal Peptide ◽  
Polystyrene Support

ABSTRACT In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.

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