Lacticin 3147 displays activity in buffer against Gram‐positive bacterial pathogens which appear insensitive in standard plate assays

Galvin, Mary; Hill, Colin; Ross, R. Paul

doi:10.1046/j.1365-2672.1999.00550.x

Cited by 95 publications

(58 citation statements)

References 6 publications

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“…The in vitro reconstitution opens the door to new, intriguing possibilities involving antimicrobial peptide design and engineering because the therapeutic potential of two-component systems is very high (37). The in vitro biosynthesis system will allow detailed investigation of the substrate specificity of each individual LanM protein as site directed mutants are readily and rapidly accessible.…”

Section: Discussionmentioning

confidence: 99%

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

McClerren¹,

Cooper

Quan

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

235

321

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Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser͞Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substratespecific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Hal␣ and Hal␤ were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.lanthionine ͉ dehydroalanine ͉ antibiotic

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Section: Discussionmentioning

confidence: 99%

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

McClerren¹,

Cooper

Quan

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

235

321

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“…For quite some time, however, the presence of D-alanines in lacticin 3147 and lactocin S (13)(14)(15), members of a group of highly posttranslationally modified antimicrobial peptides called lantibiotics (class I bacteriocins), has been the subject of much interest because the chirality and the identity of the relevant amino acids are altered. Lacticin 3147 is a twocomponent lantibiotic produced by Lactococcus lactis, which is active at low concentrations against a wide range of Grampositive organisms, including a number of human pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (16). Lacticin 3147 is representative of an emerging subclass of lantibiotics, which also includes the enterococcal cytolytic toxin cytolysin (17) that functions through the combined activity of two lanthioninecontaining peptides.…”

mentioning

confidence: 99%

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Cotter

O’Connor²,

Draper³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

120

146

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As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA͞RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and͞or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).antimicrobial ͉ bacteriocin ͉ chirality T he presence of D-amino acids in ribosomally synthesized peptides was first observed in dermorphins, peptides found in the skin secretions of species of a subfamily of South American tree frogs, Phyllomedusinae. Subsequently, D-amino acids have been identified in a number of other ribosomally synthesized peptides of eukaryotic origin (1, 2). -Agatoxin IVb and bombinin H, produced by the funnel-web spider and the skin secretions of the frog Bombina variegata, respectively, represent the only instances in which the responsible enzymes, both peptide epimerases, have been identified (1, 2). For -agatoxin IVb the relevant L-serine to D-serine conversion is thought to involve a deprotonation͞reprotonation reaction (3). The presence of D-amino acids in many of these peptides is crucial, as demonstrated by the creation of synthetic analogues with Lrather than D-amino acids at the relevant locations, which are devoid of activity (4). The artificial incorporation of D-amino acids can also be beneficial. For example, a growing number of synthetic peptides͞peptide libraries containing D-amino acids have been assembled to capitalize on the ability of such residues to provide improved protease stability [enhanced pharmokinetic profile (5-7)], alter tertiary structure (new structural elements can be assembled that cannot be built solely from L-amino acids), and affect the activity of bioactive peptides [enhanced antibacterial activity with concomitant reduction in cell cytotoxicity (5, 6, 8-11)].In prokaryotic systems, there are only a few examples of L-to D-amino acid conversions. It...

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“…The enormous chemotherapeutic potential of these peptides stems from the fact that a number of lantibiotics have been found to be active at nanomolar concentrations (3,23), can inhibit multidrug-resistant pathogens (4,13,18), and are among a rare group of antibacterial compounds that target the lipid II component of the bacterial cell wall (2,3). However, the study and application of lantibiotics are often compromised by limited production of these peptides by the native producer.…”

mentioning

confidence: 99%

Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147

Cotter

Draper

Lawton

et al. 2006

Appl Environ Microbiol

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Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.

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Lacticin 3147 displays activity in buffer against Gram‐positive bacterial pathogens which appear insensitive in standard plate assays

Cited by 95 publications

References 6 publications

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147

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