Post‐translational modification of nisin

Sen, Asuman Karakas; Narbad, Arjan; Horn, Nikki; Dodd, Helen M.; Parr, Adrian; Colquhoun, Ian J.; Gasson, Michael J.

doi:10.1046/j.1432-1327.1999.00303.x

Cited by 85 publications

(78 citation statements)

References 42 publications

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“…This is likely due to an increased activity caused by elevated expression of NisB as verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining of cell lysates (Fig. 3) (also see reference 14).…”

Section: Resultsmentioning

confidence: 74%

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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Section: Resultsmentioning

confidence: 74%

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Kuipers¹,

Wierenga²,

Rink³

et al. 2006

Appl Environ Microbiol

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“…Primers pAD1 (5Ј-CCTGAATAATATAG AGATAGGTT-3Ј) and pAD3 (5Ј-AAATTTAATTGATTTT TCATTTTGAGTGCCTCC-3Ј) were used to amplify a 270-bp fragment (fragment 1) containing P nisA . Plasmid pFI1003 (14) was used as template. The 17 nucleotides forming a tail at the 5Ј end of primer pAD3 (underlined) are complementary to the amino-terminal sequence of the lactococcin leader.…”

Section: Construction Of Pfi2391 and Pfi2436mentioning

confidence: 99%

Nisin-Controlled Production of Pediocin PA-1 and Colicin V in Nisin- and Non-Nisin-Producing Lactococcus lactis Strains

Horn

Fernández

Dodd

et al. 2004

Appl Environ Microbiol

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The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.The production of the anti-Listeria bacteriocin pediocin PA-1 by strains of dairy origin is a desirable objective, since Listeria monocytogenes represents a major biological hazard in the dairy industry (19). Although the ability of a recombinant Lactococcus lactis starter culture containing a ped operon-encoding plasmid to control L. monocytogenes in coinoculated cheeses has been demonstrated (1), an alternative approach for the production of pediocin PA-1 in heterologous hosts is based on the amino acid homologies shared by leader peptides of class II bacteriocins (7,8,21). Recently, we reported the production of pediocin PA-1 in L. lactis as a result of the introduction of a chimeric gene containing a fusion of sequences encoding the lactococcin A leader and mature pediocin PA-1 under the control of the lactococcin A promoter, along with the genes lcnC and lcnD, which encode the lactococcin A secretion apparatus (12). In this study, the production of pediocin PA-1 in L. lactis was enhanced by placing the chimeric structural gene under the control of the nisin-inducible nisA promoter (P nisA ). A similar strategy was followed to achieve production of the Escherichia coli bacteriocin colicin V in L. lactis.Bacterial strains and growth conditions. Lactococcal strains (Table 1) were grown in M17 medium (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) supplemented with 0.5% (wt/ vol) glucose (GM17 medium) at 30°C without agitation. Pediococcus acidilactici 347 (18) was grown in MRS medium at 30°C without agitation. E. coli strains were grown in L broth at 37°C on an orbital shaker. Agar plates were made by adding 1.5% agar to broth media. Antibiotics (Sigma) were added as selective agents when appropriate (chloramphenicol at 5 g ml Ϫ1for lactococci and at 15 g ml Ϫ1 for E. coli, ampicillin at 200 g ml Ϫ1 , and erythromycin at 5 g ml Ϫ1 ). For induction purposes, nisin A (Aplin and Barret, Trowbridge, United Kingdom) was added to the media used for lactococcal growth at a concentration of 100 ng ml Ϫ1 . Construction of pFI2391 and pFI2436.The technique of spliced overlap extension was used in the construction of a P nisA -controlled hybrid gene consisting of a fusion of sequences encoding the lactococcin A leader and mature pediocin PA-1. This technique involved the amplification of two DNA fragments by using the polymerase BIO-X-ACT (Bioline, London, United Kingdom). Primers pAD1 (5Ј-CCTGAATAATATAG AGATAGGTT-3Ј) and pAD3 (5Ј-AAATTTAATTGATTTT TCATTTTGAGTGCCTCC-3Ј) were used to amplify a 270-bp fragment (fragment 1) containing P nisA . Plasmid pFI1003 (14) was used as template. The 17 nucleotides forming a tail at the 5Ј end of prim...

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“…Although the microcystin and nodularin biosynthesis pathways may involve serine/ threonine dehydration reactions, it is unlikely that these reactions are catalyzed by McyI and NdaH, as originally predicted. McyI and NdaH are homologous to 2-hydroxy-acid dehydrogenases, and this family of enzymes is neither structurally nor functionally related to the family of dehydratase enzymes known to convert serine to dehydroalanine in other secondary metabolite pathways (8). Whereas dehydrogenases such as PGDH catalyze redox reactions (i.e.…”

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confidence: 99%

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

Pearson

Barrow

Neilan

2007

Journal of Biological Chemistry

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The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thiotemplate function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of D-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce ␣-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.The hepatotoxic microcystins compose the largest and most structurally diverse group of cyanobacterial toxins. Over 65 isoforms of microcystin varying by degree of methylation, hydroxylation, epimerization, peptide sequence, and toxicity have been identified (1). Underlying the extraordinary heterogeneity present among the microcystins is their common cyclic structure and possession of several rare nonproteinogenic amino acid moieties. Collectively, the microcystins may be described as monocyclic heptapeptides containing both D-and L-amino acids plus N-methyldehydroalanine and a unique ␤-amino acid side group, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Fig. 1) (2). Although various amino acid substitutions can occur within the microcystin cyclic structure, the most common toxin isoform, microcystin LR, contains lysine and arginine at positions 2 and 4, respectively (Fig. 1).The microcystin biosynthesis (mcy) gene cluster was the first complex metabolite gene cluster to be fully sequenced from a cyanobacterium. In Microcystis aeruginosa PCC7806, the mcy gene cluster spans 55 kb and comprises 10 genes arranged in two divergently transcribed operons, mcyA-C and mcyD-J. Although most open reading frames within the mcy gene cluster have been assigned a function based on homologous entries in the data bases, no obvious function could be assigned to the 1014-bp gene mcyI. Preliminary sequence analysis of the inferred primary peptide seq...

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Post‐translational modification of nisin

Cited by 85 publications

References 42 publications

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Nisin-Controlled Production of Pediocin PA-1 and Colicin V in Nisin- and Non-Nisin-Producing Lactococcus lactis Strains

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

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