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Hans-georg Sahl - One of the best experts on this subject based on the ideXlab platform.
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family of class i Lantibiotics from actinomycetes and improvement of their antibacterial activities
ACS Chemical Biology, 2015Co-Authors: Sonia I Maffioli, Hans-georg Sahl, Daniela Münch, Paolo Monciardini, Bruno Catacchio, Carlo Mazzetti, Cristina Brunati, Stefano DonadioAbstract:Lantibiotics, an abbreviation for "lanthionine-containing antibiotics", interfere with bacterial metabolism by a mechanism not exploited by the antibiotics currently in clinical use. Thus, they have aroused interest as a source for new therapeutic agents because they can overcome existing resistance mechanisms. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we isolated a series of related class I Lantibiotics produced by different genera of actinomycetes. Analytical techniques together with explorative chemistry have been used to establish their structures: the newly described compounds share a common 24 aa sequence with the previously reported Lantibiotic planosporicin (aka 97518), differing at positions 4, 6, and 14. All of these compounds maintain an overall -1 charge at physiological pH. While all of these Lantibiotics display modest antibacterial activity, their potency can be substantially modulated by progressively eliminating the negative charges, with the most active compounds carrying basic amide derivatives of the two carboxylates originally present in the natural compounds. Interestingly, both natural and chemically modified Lantibiotics target the key biosynthetic intermediate lipid II, but the former compounds do not bind as effectively as the latter in vivo. Remarkably, the basic derivatives display an antibacterial potency and a killing effect similar to those of NAI-107, a distantly related actinomycete-produced class I Lantibiotic which lacks altogether carboxyl groups and which is a promising clinical candidate for treating Gram-positive infections caused by multi-drug-resistant pathogens.
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expression of the Lantibiotic mersacidin in bacillus amyloliquefaciens fzb42
PLOS ONE, 2011Co-Authors: Anna Maria Herzner, Hans-georg Sahl, Jasmin Dischinger, Christiane Szekat, Michaele Josten, Stephanie Schmitz, Anja Yakéléba, Ricarda Reinartz, Andrea Jansen, Jörn PielAbstract:Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, Lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in Lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The Lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent. Methodology/Principal Findings The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.
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Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42
2011Co-Authors: Anna Maria Herzner, Hans-georg Sahl, Jasmin Dischinger, Christiane Szekat, Michaele Josten, Stephanie Schmitz, Anja Yakéléba, Ricarda Reinartz, Andrea Jansen, Jörn PielAbstract:Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, Lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in Lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The Lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent. Methodology/Principal Findings: The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. Conclusions/Significance: The Lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of th
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Lipid II and other bactoprenol-bound cell wall precursors as drug targets.
Current opinion in investigational drugs, 2010Co-Authors: Tanja Schneider, Hans-georg SahlAbstract:: Bacterial cell wall biosynthesis represents an antibiotic target pathway for therapeutic intervention. An increasing number of natural antibiotic compounds have been demonstrated to inhibit the membrane-associated steps of cell wall biosynthesis by targeting bactoprenol-mediated precursor cycling, particularly at the stage of the completed building block Lipid II. These antibiotic compounds belong to various chemical classes including glycopeptides, lipopeptides and lipodepsipeptides, and Lantibiotics and other antimicrobial peptides. The clinical success of vancomycin in the treatment of multiresistant Gram-positive bacteria has stimulated further development of glycopeptide antibiotics and research of other Lipid II-binding compounds. The state-of-the-art in the targeting of cell wall precursors is summarized in this review.
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production of the novel two peptide Lantibiotic lichenicidin by bacillus licheniformis dsm 13
PLOS ONE, 2009Co-Authors: Jasmin Dischinger, Hans-georg Sahl, Christiane Szekat, Michaele Josten, Gabriele BierbaumAbstract:Background Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in Lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. Methodology/Principal Findings Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative Lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in Lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the Lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. Conclusions/Significance In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide Lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.
Paul R Ross - One of the best experts on this subject based on the ideXlab platform.
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in silico analysis highlights the frequency and diversity of type 1 Lantibiotic gene clusters in genome sequenced bacteria
BMC Genomics, 2010Co-Authors: Paul D Cotter, Paul R Ross, Alan J Marsh, Orla Osullivan, Colin HillAbstract:Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 Lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential Lantibiotic-encoding gene clusters in genome sequenced bacteria. In total 49 novel type 1 Lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with Lantibiotic production. Multiple type 1 Lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.
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investigating the importance of charged residues in Lantibiotics
Bioengineered bugs, 2010Co-Authors: Srinivas Suda, Paul D Cotter, Colin Hill, Paul R RossAbstract:Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide Lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other Lantibiotics and cationic antimicrobial peptides.
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identification of a novel two peptide Lantibiotic lichenicidin following rational genome mining for lanm proteins
Applied and Environmental Microbiology, 2009Co-Authors: Paul D Cotter, Colin Hill, Paul R Ross, Maire BegleyAbstract:Lantibiotics are ribosomally synthesized peptide antimicrobials which contain considerable posttranslational modifications. Given their usually broad host range and their highly stable structures, there have been renewed attempts to identify and characterize novel members of the Lantibiotic family in recent years. The increasing availability of bacterial genome sequences means that in addition to traditional microbiological approaches, in silico screening strategies may now be employed to the same end. Taking advantage of the highly conserved nature of Lantibiotic biosynthetic enzymes, we screened publicly available microbial genome sequences for genes encoding LanM proteins, which are required for the posttranslational modification of type 2 Lantibiotics. By using this approach, 89 LanM homologs, including 61 in strains not known to be Lantibiotic producers, were identified. Of these strains, five (Streptococcus pneumoniae SP23-BS72, Bacillus licheniformis ATCC 14580, Anabaena variabilis ATCC 29413, Geobacillus thermodenitrificans NG80-2, and Herpetosiphon aurantiacus ATCC 23779) were subjected to a more detailed bioinformatic analysis. Four of the strains possessed genes potentially encoding a structural peptide in close proximity to the lanM determinants, while two, S. pneumoniae SP23-BS72 and B. licheniformis ATCC 14580, possess two potential structural genes. The B. licheniformis strain was selected for a proof-of-concept exercise, which established that a two-peptide Lantibiotic, lichenicidin, which exhibits antimicrobial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococcus strains tested, was indeed produced, thereby confirming the benefits of such a bioinformatic approach when screening for novel Lantibiotic producers.
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complete alanine scanning of the two component Lantibiotic lacticin 3147 generating a blueprint for rational drug design
Molecular Microbiology, 2006Co-Authors: Paul D Cotter, Colin Hill, Paul R Ross, Lucy H Deegan, Elaine M Lawton, Lorraine A Draper, Paula M OconnorAbstract:Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some Lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that Lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel Lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no Lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component Lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of Lantibiotic-based antimicrobial compounds.
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bacterial Lantibiotics strategies to improve therapeutic potential
Current Protein & Peptide Science, 2005Co-Authors: Paul D Cotter, Colin Hill, Paul R RossAbstract:Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of Lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies (intelligenetics) to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual Lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made Lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-Lantibiotic peptides. In the shorter term, the extensive characterisation of Lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.
Colin Hill - One of the best experts on this subject based on the ideXlab platform.
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Bioengineering of the model Lantibiotic nisin
Bioengineered, 2015Co-Authors: Des Field, Paul D Cotter, R. Paul Ross, Colin HillAbstract:The Lantibiotics are a class of bacterially produced antimicrobial peptides (bacteriocins) that contain several unusual amino acids resulting from enzyme-mediated post-translational modifications. They exhibit high specific activity against Gram-positive targets, including many antibiotic-resistant pathogens, and consequently have been investigated with a view to their application as antimicrobials in both the food and medical arenas. Importantly, the gene-encoded nature of Lantibiotics makes them more amenable to bioengineering strategies to further enhance their antimicrobial and physicochemical properties. However, although the bioengineering of Lantibiotics has been underway for over 2 decades, significant progress has only been reported in recent years. This review charts recent developments with regard to the implementation of bioengineering strategies to enhance the functional characteristics of the prototypical and most studied Lantibiotic nisin.
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in silico analysis highlights the frequency and diversity of type 1 Lantibiotic gene clusters in genome sequenced bacteria
BMC Genomics, 2010Co-Authors: Paul D Cotter, Paul R Ross, Alan J Marsh, Orla Osullivan, Colin HillAbstract:Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 Lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential Lantibiotic-encoding gene clusters in genome sequenced bacteria. In total 49 novel type 1 Lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with Lantibiotic production. Multiple type 1 Lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.
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investigating the importance of charged residues in Lantibiotics
Bioengineered bugs, 2010Co-Authors: Srinivas Suda, Paul D Cotter, Colin Hill, Paul R RossAbstract:Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide Lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other Lantibiotics and cationic antimicrobial peptides.
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production of the bsa Lantibiotic by community acquired staphylococcus aureus strains
Journal of Bacteriology, 2010Co-Authors: Karen Daly, Ralph W Jack, Friedrich Gotz, Lorraine A Draper, Paula M Oconnor, Mathew Upton, Stephanie K Sandiford, Philip A Wescombe, Angela Rossney, Colin HillAbstract:Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many Lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some Lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a Lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional Lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related Lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.
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identification of a novel two peptide Lantibiotic lichenicidin following rational genome mining for lanm proteins
Applied and Environmental Microbiology, 2009Co-Authors: Paul D Cotter, Colin Hill, Paul R Ross, Maire BegleyAbstract:Lantibiotics are ribosomally synthesized peptide antimicrobials which contain considerable posttranslational modifications. Given their usually broad host range and their highly stable structures, there have been renewed attempts to identify and characterize novel members of the Lantibiotic family in recent years. The increasing availability of bacterial genome sequences means that in addition to traditional microbiological approaches, in silico screening strategies may now be employed to the same end. Taking advantage of the highly conserved nature of Lantibiotic biosynthetic enzymes, we screened publicly available microbial genome sequences for genes encoding LanM proteins, which are required for the posttranslational modification of type 2 Lantibiotics. By using this approach, 89 LanM homologs, including 61 in strains not known to be Lantibiotic producers, were identified. Of these strains, five (Streptococcus pneumoniae SP23-BS72, Bacillus licheniformis ATCC 14580, Anabaena variabilis ATCC 29413, Geobacillus thermodenitrificans NG80-2, and Herpetosiphon aurantiacus ATCC 23779) were subjected to a more detailed bioinformatic analysis. Four of the strains possessed genes potentially encoding a structural peptide in close proximity to the lanM determinants, while two, S. pneumoniae SP23-BS72 and B. licheniformis ATCC 14580, possess two potential structural genes. The B. licheniformis strain was selected for a proof-of-concept exercise, which established that a two-peptide Lantibiotic, lichenicidin, which exhibits antimicrobial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococcus strains tested, was indeed produced, thereby confirming the benefits of such a bioinformatic approach when screening for novel Lantibiotic producers.
Gabriele Bierbaum - One of the best experts on this subject based on the ideXlab platform.
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Lantibiotics promising candidates for future applications in health care
International Journal of Medical Microbiology, 2014Co-Authors: Jasmin Dischinger, Shradha Basi Chipalu, Gabriele BierbaumAbstract:Abstract The immense potential of bacteria for production of antimicrobials represents an inexhaustible source of new antibiotics. An emerging class of natural products is constituted by ribosomally synthesized and posttranslationally modified peptides (RiPPs). “Lantibiotics” ( lan thionine and/or methyl-lanthionine containing an tibiotics ) belong to the earliest members of this class. The characteristic thioether amino acids are introduced into the precursor peptides by enzyme-mediated posttranslational modifications. The encouraging antimicrobial activity of Lantibiotics against multiresistant clinical pathogens, their stability against proteases, heat and oxidation make Lantibiotics interesting candidates for novel antimicrobial applications in many areas of the healthcare sector and associated industries. In addition to applications as alternatives to classical antibiotics, Lantibiotics can be used as probiotics, prophylactics or additives. Furthermore, the in vitro activity of the Lantibiotic modification machinery opens the possibility to generate either improved synthetic Lantibiotic peptides or to introduce thioether cross-links into existing therapeutics.
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production of the novel two peptide Lantibiotic lichenicidin by bacillus licheniformis dsm 13
PLOS ONE, 2009Co-Authors: Jasmin Dischinger, Hans-georg Sahl, Christiane Szekat, Michaele Josten, Gabriele BierbaumAbstract:Background Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in Lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. Methodology/Principal Findings Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative Lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in Lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the Lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. Conclusions/Significance In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide Lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.
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Lantibiotics mode of action biosynthesis and bioengineering
Current Pharmaceutical Biotechnology, 2009Co-Authors: Gabriele Bierbaum, Hans-georg SahlAbstract:Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues. The overwhelming majority of the Lantibiotics shows antibacterial activity. Some Lantibiotics, e.g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among Lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. In the two-component system of lacticin 3147, the two functions are performed by the two different peptides. The genetic information for production of Lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the Lantibiotic prepeptide. The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC). These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies. In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides. Furthermore, variant Lantibiotics with enhanced properties have been engineered and at least one promising new Lantibiotic with strong activity against multiresistant pathogens has been described.
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construction of an expression system for site directed mutagenesis of the Lantibiotic mersacidin
Applied and Environmental Microbiology, 2003Co-Authors: Christiane Szekat, Ralph W Jack, Dirk Skutlarek, Harald Farber, Gabriele BierbaumAbstract:The Lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of Lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related Lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.
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Protein engineering of Lantibiotics
Antonie van Leeuwenhoek, 1996Co-Authors: Oscar P. Kuipers, Gabriele Bierbaum, Birgit Ottenwälder, Helen M. Dodd, Nicky Horn, Jörg Metzger, Thomas Kupke, Volker Gnau, Roger Bongers, Patrick BogaardAbstract:Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the Lantibiotics, is just coming of age. The engineering of Lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active Lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural Lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these Lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the Lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of Lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of Lantibiotics.
Karl-dieter Entian - One of the best experts on this subject based on the ideXlab platform.
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autoinduction specificities of the Lantibiotics subtilin and nisin
Applied and Environmental Microbiology, 2015Co-Authors: Tobias Spies, Peter Kotter, Sophie Marianne Korn, Karl-dieter EntianAbstract:The biosynthesis of the Lantibiotics subtilin and nisin is regulated by autoinduction via two-component systems. Although subtilin is structurally closely related to nisin and contains the same lanthionine ring structure, both Lantibiotics specifically autoinduce their biosynthesis. Subtilin and also the subtilin-like Lantibiotics entianin and ericin autoinduce the two-component system SpaRK of Bacillus subtilis, whereas the biosynthesis of nisin is autoinduced via the two-component system NisRK of Lactococcus lactis. Autoinduction is highly specific for the respective Lantibiotic and therefore of major importance for the functional expression of genetically engineered subtilin-like Lantibiotics. To identify the structural features required for subtilin autoinduction, subtilin-nisin hybrids and specific point mutations of amino acid position 1 were generated. For subtilin autoinduction, the N-terminal tryptophan is the most important for full SpaK activation. The failure of subtilin to autoinduce the histidine kinase NisK mainly depends on the N-terminal tryptophan, as its single exchange to the aliphatic amino acid residues isoleucine, leucine, and valine provided NisK autoinduction. In addition, the production of subtilin variants which did not autoinduce their own biosynthesis could be rescued upon heterologous coexpression in B. subtilis DSM15029 by the autoinducing subtilin-like Lantibiotic entianin.
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structure function relationships of the lanthionine cyclase spac involved in biosynthesis of the bacillus subtilis peptide antibiotic subtilin
Biochemistry, 2007Co-Authors: Markus Helfrich, Karl-dieter Entian, Torsten SteinAbstract:Biosynthesis of the Lantibiotic subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the Lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a Lantibiotic precursor peptide (subtilin) by a hybrid Lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His231, Trp302, Cys303, Tyr304, Gly305, Cys349, and His350, as well as the conserved C-terminal motif Lys437-Ala438-Leu439-Leu440-Ile441 for subtilin biosynthesis. Assignment of these strictly conserved Lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464−1...
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two different Lantibiotic like peptides originate from the ericin gene cluster of bacillus subtilis a1 3
Journal of Bacteriology, 2002Co-Authors: Torsten Stein, Stefan Borchert, Birgit Conrad, Jorg Feesche, Brigitte Hofemeister, Jurgen Hofemeister, Karl-dieter EntianAbstract:A Lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two Lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured Lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.
