Nucleotide sequence of the <i>secY</i> gene from <i>Lactococcus lactis</i> and identification of conserved regions by comparison of four SecY proteins

Koivula, Teija; Palva, Ilkka; Hemilä, Harri

doi:10.1016/0014-5793(91)81015-z

Cited by 30 publications

(13 citation statements)

References 29 publications

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“…Protein translocation has been extensively studied in Escherichia coli and Bacillus subtilis, which are gram-negative and gram-positive bacteria, respectively (50). Homologues of SecA and SecYEG are also present in L. lactis (3,17). Azide, a known SecA inhibitor (30,33), blocks Sec-dependent translocation of proteins in L. lactis (10).…”

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confidence: 99%

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Kuipers¹,

Wierenga²,

Rink³

et al. 2006

Appl Environ Microbiol

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Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.

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confidence: 99%

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Kuipers¹,

Wierenga²,

Rink³

et al. 2006

Appl Environ Microbiol

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“…The Sec translocase transports unfolded proteins, which is driven by the ATPase SecA. Homologues of SecYEG and SecA are also found in L. lactis (2,9). In vitro studies demonstrated that the translocon SecYEG of E. coli can also translocate the polypeptide proOmpA with a disulfide-bridge, which can have a loop of 18 amino acids or smaller (22).…”

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confidence: 99%

Translocation of a Thioether-Bridged Azurin Peptide Fragment via the Sec Pathway in Lactococcus lactis

Kuipers¹,

Rink²,

Moll³

2009

Appl Environ Microbiol

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This study demonstrates for the first time that a thioether-containing peptide, an azurin fragment, can be translocated via the Sec pathway. This methyl-lanthionine was introduced by the nisin modification enzymes. The Sec pathway can therefore be a successful alternative for those cyclized peptides that are inefficiently transported via NisT.Azurin, a cupredoxin produced by Pseudomonas aeruginosa, can selectively enter human cancer cells and induce apoptosis (24) via binding to the tumor suppressor protein p53 (1). The azurin peptide fragment p28, containing amino acids 50 to 77 (LSTAADMQGVVTDGMASGLDKDYLKPDD), still enters human cancer cells and inhibits tumor proliferation (20). Importantly, novel cancer treatments can be based on azurin peptide fragments and derivatives thereof (T. Das Gupta and A. Chakrabarty, 20 March 2008, patent application WO2008033820). Although the pharmacokinetic property of therapeutic peptides is promising, lack of biostability is the major hurdle for their successful application. Consequently, it is very relevant to explore the possibilities for enhancing biostability of these peptides.In our group, we developed a technology to improve the stability of therapeutic peptides by exploiting the nisin synthetase enzymes NisB and NisC for the introduction of thioether bridges. We applied a two-plasmid expression system (7,8,12), in which the NisBTC-encoding plasmid is compatible with the substrate-peptide-encoding plasmid. Lactococcus lactis containing this expression system can secrete nonlantibiotic peptides which are dehydrated or stabilized by a thioether ring (8, 16). NisB dehydrates serines and threonines in substrate peptides, NisC couples dehydrated residues stereo-and regioselectively to cysteines, and NisT, the ABC transporter, translocates the modified peptides out of the cell (10,11,13,15). The leader peptide is essential for targeting and modification of the propeptides (23).When transport via NisT is impaired or is less efficient, the Sec pathway of L. lactis is a successful alternative in translocation of dehydrated peptides. When the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively, modification by NisB and NisC still occurs (12; G. N. Moll, A. Kuipers, R. Rink, A. J. M. Driessen, and O. P. Kuipers, 15 June 2006, patent application WO 2006062398). However, NisC-cyclized prenisin was not translocated via the Sec system (12). This is likely due to the dimensions of fully modified nisin (3), which is too large to fit in the SecY pore (12, 21). Here, we report for the first time that the Sec pathway of L. lactis can translocate a p28 azurin fragment analog with a thioether ring.

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“…Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.Bacterial protein secretion is an attractive target for antimicrobial chemotherapy because the secretion machinery is highly conserved among bacterial species but is distinct from its eukaryotic counterparts (10,11,14,15,27,42,43). In Escherichia coli, approximately 20% of the total cellular protein is secreted across the cytoplasmic membrane (30).…”

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confidence: 99%

“…Bacterial protein secretion is an attractive target for antimicrobial chemotherapy because the secretion machinery is highly conserved among bacterial species but is distinct from its eukaryotic counterparts (10,11,14,15,27,42,43). In Escherichia coli, approximately 20% of the total cellular protein is secreted across the cytoplasmic membrane (30).…”

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confidence: 99%

Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

Alksne

Burgio

et al. 2000

Antimicrob Agents Chemother

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Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be <128 g/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.Bacterial protein secretion is an attractive target for antimicrobial chemotherapy because the secretion machinery is highly conserved among bacterial species but is distinct from its eukaryotic counterparts (10,11,14,15,27,42,43). In Escherichia coli, approximately 20% of the total cellular protein is secreted across the cytoplasmic membrane (30). The Sec-dependent pathway of secretion is a multicomponent system consisting of at least seven proteins, five of which are essential for cell viability (for a review, see reference 8). Homologs for several of the components have been identified in both gramnegative and gram-positive bacteria, and therefore it is considered likely that an inhibitor of this pathway would have broadspectrum activity.Secreted proteins are produced as precursors containing signal sequences. Those which are secreted via the Sec pathway are accompanied to the membrane by SecB, a chaperone molecule (Fig. 1). SecA, an ATPase required for translocation at the multisubunit translocase SecY-SecE-SecG, is found associated with both the signal sequence of secretable proteins, and also with the membrane at SecE-SecY-SecG. During translocation ATP is hydrolyzed, the protein is inserted across the membrane, the signal sequence is cleaved, and the protein is released.Expression of SecA appears to be a focal point of regulation for this process (20,24,33) (Fig. 2). SecA translation is autogenously regulated in response to changes in secretion levels. secA is transcribed as the second gene in an operon after geneX, an open reading frame of unknown function. Under normal secretion conditions, SecA binds to its own mRNA, blocking the ribosomal binding site, thus inhibiting initiation of translation. If secretion is blocked, SecA releases its mRNA and translation levels increase (25).An E. coli cell-bas...

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Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins

Cited by 30 publications

References 29 publications

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Translocation of a Thioether-Bridged Azurin Peptide Fragment via the Sec Pathway in Lactococcus lactis

Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

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