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 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 EcDBS1R6: A new broad-spectrum cationic antibacterial peptide derived from a signal peptide sequence

2019 ◽  
Author(s):  
William F. Porto ◽  
Luz N. Irazazabal ◽  
Vincent Humblot ◽  
Evan F. Haney ◽  
Suzana M. Ribeiro ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Broad Spectrum ◽  
Antimicrobial Agents ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Peptide Sequence ◽  
World Health ◽  
Signal Peptides ◽  
Microscopy Imaging ◽  
Signal Peptide Sequence

ABSTRACTBacterial infections represent a major worldwide health problem, with an special highlight on Gram-negative bacteria, which were assigned by the World Health Organization (WHO) as the most critical priority for development of novel antimicrobial compounds. Antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the broad-spectrum activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes predicted through the Joker algorithm. Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker made this peptide capable of killing bacteria. Membrane damage of the bacterial cells was observed by measuring membrane integrity using fluorescent probes and through scanning electron microscopy imaging. Structural analysis revealed that the C-terminus was unable to fold into an α-helix, indicating that the EcDBS1R6 antibacterial activity core was located at the N-terminus, corresponding to the signal peptide portion of the parent peptide. Therefore, the strategy of transforming signal peptides into AMPs seems to be promising and could be used for producing novel antimicrobial agents.

scholarly journals Lysis of a Lactococcus lactis Dipeptidase Mutant and Rescue by Mutation in the Pleiotropic Regulator CodY

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Chenxi Huang ◽  
Jhonatan A. Hernandez-Valdes ◽  
Oscar P. Kuipers ◽  
Jan Kok
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Nitrogen Metabolism ◽  
Lactococcus Lactis ◽  
Cell Shape ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Acid Uptake ◽  
Cell Wall Synthesis ◽  
Content Type

ABSTRACT Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔpepV) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔpepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔpepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala–d-Ala ends, which confirmed that MGΔpepV has an abnormal PG composition. A mutant of MGΔpepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔpepVcodYR218C. Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔpepVcodYR218C. A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed. IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis.

scholarly journals Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

2013 ◽  
Vol 79 (17) ◽  
pp. 5179-5185 ◽  
Author(s):  
Avelino Alvarez-Ordóñez ◽  
Máire Begley ◽  
Tanya Clifford ◽  
Thérèse Deasy ◽  
Kiera Considine ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Peptide Sequence ◽  
Content Type ◽  
Charged Amino Acids ◽  
Food Borne ◽  
Relationship Of ◽  
Positively Charged

ABSTRACTTemplate-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogensListeria monocytogenesandCronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness againstC. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency againstL. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

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 A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomycesoris

2016 ◽  
Vol 198 (15) ◽  
pp. 2064-2073 ◽  
Author(s):  
Sara D. Siegel ◽  
Chenggang Wu ◽  
Hung Ton-That
Keyword(s):  
Experimental Model ◽  
Signal Peptide ◽  
Biofilm Formation ◽  
Signal Peptidase ◽  
Type I ◽  
Signal Peptides ◽  
Gram Positive ◽  
Content Type ◽  
Lpxtg Motif ◽  
Gram Positive Bacteria

ABSTRACTThe Gram-positive bacteriumActinomycesoris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria,A. orisexpresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortasesrtAis genetically disrupted. Consistent with this finding, we show here that a mutant lackinglepB2andsrtAwas unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neitherlepB1norlepB2abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, thelepB2deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, thelepB2mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely,lepB1was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.IMPORTANCESec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria,A. orisexpresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortasesrtAis genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus,A. orisprovides an experimental model with which to investigate the specificity mechanism of type I SPases.

scholarly journals Unexpected Diversity of Signal Peptides in Prokaryotes

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Samuel H. Payne ◽  
Stefano Bonissone ◽  
Si Wu ◽  
Roslyn N. Brown ◽  
Dmitry N. Ivankov ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Protein Localization ◽  
Cellular Protein ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Proteomics Data ◽  
Detailed Model ◽  
Signal Peptide Sequence ◽  
Peptide Motifs ◽  
Computational Analyses

ABSTRACT Signal peptides are a cornerstone mechanism for cellular protein localization, yet until now experimental determination of signal peptides has come from only a narrow taxonomic sampling. As a result, the dominant view is that Sec-cleaved signal peptides in prokaryotes are defined by a canonical AxA motif. Although other residues are permitted in the motif, alanine is by far the most common. Here we broadly examine proteomics data to reveal the signal peptide sequences for 32 bacterial and archaeal organisms from nine phyla and demonstrate that this alanine preference is not universal. Discoveries include fundamentally distinct signal peptide motifs from Alphaproteobacteria, Spirochaetes, Thermotogae and Euryarchaeota. In these novel motifs, alanine is no longer the dominant residue but has been replaced in a different way for each taxon. Surprisingly, divergent motifs correlate with a proteome-wide reduction in alanine. Computational analyses of ~1,500 genomes reveal numerous major evolutionary clades which have replaced the canonical signal peptide sequence with novel motifs. IMPORTANCE This article replaces a widely held general model with a more detailed model describing phylogenetically correlated variation in motifs for Sec secretion.

scholarly journals Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

2001 ◽  
Vol 67 (2) ◽  
pp. 713-720 ◽  
Author(s):  
Wataru Hashimoto ◽  
Hikaru Miki ◽  
Noriaki Tsuchiya ◽  
Hirokazu Nankai ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Signal Peptide ◽  
Streptomyces Griseus ◽  
Peptide Sequence ◽  
Terminal Amino Acid ◽  
Signal Peptide Sequence ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT When grown on xanthan as a carbon source, the bacteriumBacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus(identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) fromBacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

scholarly journals Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

2013 ◽  
Vol 80 (4) ◽  
pp. 1463-1468 ◽  
Author(s):  
Laxmi Krishnappa ◽  
Carmine G. Monteferrante ◽  
Jolanda Neef ◽  
Annette Dreisbach ◽  
Jan Maarten van Dijl
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Protein Secretion ◽  
Secreted Proteins ◽  
Biotechnological Production ◽  
Content Type ◽  
Wide Range ◽  
Mutant Strains

ABSTRACTThe general protein secretion pathway ofBacillus subtilishas a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins ofB. subtilisare at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

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