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1.  An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase† †Electronic supplementary information (ESI) available: General experimental procedures, primer sequences, and supporting figures. See DOI: 10.1039/c2cc34138g Click here for additional data file.  
Mutants of the highly promiscuous lantipeptide synthetase ProcM phosphorylate a wide range of peptides attached to ProcA leader peptides, both in vitro and in E. coli. As such, the ProcM mutants are useful enzymes for the enzymatic preparation of phosphorylated peptides.
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.
doi:10.1039/c2cc34138g
PMCID: PMC3475617  PMID: 23001385
2.  Chemical Synthesis of the Lantibiotic Lacticin 481 Reveals the Importance of Lanthionine Stereochemistry 
Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.
doi:10.1021/ja4014024
PMCID: PMC3736828  PMID: 23621626
3.  Chemical Synthesis of the Lantibiotic Lacticin 481 Reveals the Importance of Lanthionine Stereochemistry 
Lantibiotics are a family of antibacterial peptide natural products characterized by the posttranslational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single stereochemical configuration for each of these crosslinks was known in Nature. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing crosslinks with non-native stereochemical configuration. Biological evaluation revealed that these alterations abolished antibacterial activity in all analogues, revealing the critical importance of the enzymatically-installed stereochemistry for the biological activity of lacticin 481.
doi:10.1021/ja4014024
PMCID: PMC3736828  PMID: 23621626
4.  Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5. 
Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.
PMCID: PMC167809  PMID: 8593044
5.  The Importance of the Leader Sequence for Directing Lanthionine Formation in Lacticin 481† 
Biochemistry  2008;47(28):7342-7351.
Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for activity. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.
doi:10.1021/bi800277d
PMCID: PMC2574844  PMID: 18570437
Lantibiotic; leader peptide; lacticin 481; mutacin II; nukacin ISK-1
6.  Purification, Characterization, and Gene Sequence of Michiganin A, an Actagardine-Like Lantibiotic Produced by the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis 
Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.
doi:10.1128/AEM.00639-06
PMCID: PMC1563628  PMID: 16957199
7.  Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis# 
Biochemistry  2007;46(20):5991-6000.
Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dha), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether crosslinks lanthionine and methyllanthionine, respectively. In this study ten conserved residues have been mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in a mutant that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn lead to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.
doi:10.1021/bi602663x
PMCID: PMC2517115  PMID: 17455908
Lantibiotics; antibiotic; posttranslational modification; kinase; nisin
8.  An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase† 
Chemical communications (Cambridge, England)  2012;48(86):10.1039/c2cc34138g.
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.
doi:10.1039/c2cc34138g
PMCID: PMC3475617  PMID: 23001385
9.  Investigation of the Substrate Specificity of Lacticin 481 Synthetase using Nonproteinogenic Amino Acids 
Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semi-synthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing β-amino acids, D-amino acids, and N-alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide engineering applications, whereby nonproteinogenic amino acids may impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification.
doi:10.1002/cbic.200800752
PMCID: PMC2737179  PMID: 19222036
lantibiotic; antibiotic; post-translational modification; peptoid; peptide ligation
10.  Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1 
PLoS ONE  2013;8(12):e83037.
Background
Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.
Methodology/Findings
Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.
Conclusion/Significance
Apart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.
doi:10.1371/journal.pone.0083037
PMCID: PMC3857288  PMID: 24349428
11.  Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42 
PLoS ONE  2011;6(7):e22389.
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 the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.
doi:10.1371/journal.pone.0022389
PMCID: PMC3141056  PMID: 21811596
12.  Chemical Synthesis and Biological Activity of Analogues of the Lantibiotic Epilancin 15X 
Lantibiotics are a large family of antibacterial peptide natural products containing multiple post-translational modifications, including the thioether structures lanthionine and methyllanthionine. Efforts to probe structure–activity relationships and engineer improved pharmacological properties have driven the development of new methods to produce non-natural analogues of these compounds. In this study, solid-supported chemical synthesis was used to produce analogues of the potent lantibiotic epilancin 15X, in order to assess the importance of several N-terminal post-translational modifications for biological activity. Surprisingly, substitution of these moieties, including the unusual N-terminal d-lactyl moiety, resulted in relatively small changes in the antimicrobial activity and pore-forming ability of the peptides.
doi:10.1021/ja302435y
PMCID: PMC3349288  PMID: 22524291
13.  Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives 
PLoS ONE  2013;8(11):e79563.
Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.
doi:10.1371/journal.pone.0079563
PMCID: PMC3823697  PMID: 24244524
14.  Nine Post-translational Modifications during the Biosynthesis of Cinnamycin 
Journal of the American Chemical Society  2011;133(34):13753-13760.
Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19 amino acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-l-aspartic acid resulting from the hydroxylation of l-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli. CinX is an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of four Ser and Thr residues and subsequent cyclization of Cys residues to form the three (Me)Lan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity, but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli.
doi:10.1021/ja205783f
PMCID: PMC3163434  PMID: 21770392
15.  Site-Directed Mutations in the Lanthipeptide Mutacin 1140 
Applied and Environmental Microbiology  2013;79(13):4015-4023.
The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.
doi:10.1128/AEM.00704-13
PMCID: PMC3697549  PMID: 23603688
16.  Biosynthesis of the Class III Lantipeptide Catenulipeptin 
ACS Chemical Biology  2012;7(9):1529-1535.
Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn2+. Mutagenesis studies suggest that the labionin cyclization may proceed in a C-to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor.
doi:10.1021/cb3002446
PMCID: PMC3448297  PMID: 22725258
17.  Production of the Novel Two-Peptide Lantibiotic Lichenicidin by Bacillus licheniformis DSM 13 
PLoS ONE  2009;4(8):e6788.
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.
doi:10.1371/journal.pone.0006788
PMCID: PMC2727956  PMID: 19707558
18.  Versatile and Stereoselective Syntheses of Orthogonally Protected β-Methylcysteine and β-Methyllanthionine 
Organic letters  2005;7(13):2655-2658.
Lantibiotics are a class of lanthionine (and/or β-methyllanthionine)-containing peptides with antibioitic activity against Gram-positive bacteria. As part of our research effort directed toward the synthesis and mechanistic study of the lantibiotic peptide mersacidin (1), we report stereoselective syntheses of orthogonally protected β-methylcysteine (β-MeCys) and β-methyllanthionine (β-MeLan), two key nonnatural amino acid components of the mersacidin architecture.
doi:10.1021/ol0507930
PMCID: PMC1351053  PMID: 15957914
19.  Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis▿ †  
Applied and Environmental Microbiology  2006;72(12):7626-7633.
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.
doi:10.1128/AEM.01802-06
PMCID: PMC1694219  PMID: 17041158
20.  In Vitro Mutasynthesis of Lantibiotic Analogues Containing Nonproteinogenic Amino Acids 
Journal of the American Chemical Society  2009;131(34):12024-12025.
Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics containing the characteristic thioether cross-links lanthionine and methyllanthionine. To date, no analogues of lantibiotics that contain nonproteinogenic amino acids have been reported. In this study, in vitro-reconstituted lacticin 481 synthetase was used in conjunction with synthetic peptide substrates containing nonproteinogenic amino acids to generate 11 analogues of lacticin 481. These analogues contained sarcosine and aminocyclopropanoic acid in place of Gly5, d-valine at position 6, 4-cyanoaminobutyric acid in place of Glu13, β3-homoarginine at the position of Asn15, N-butylglycine and β-Ala at Met16, naphthylalanine (Nal) at Trp19, 4-pyridynylalanine (Pal) at Phe21, and homophenylalanine (hPhe) at Phe23. Of these analogues, the Trp19Nal and Phe23hPhe mutants provided zones of inhibition larger than the parent compound in agar diffusion assays against the indicator strains Lactococcus lactis HP and Bacillus subtilis 6633. These two compounds also demonstrated improved MIC values against liquid cultures of L. lactis HP.
doi:10.1021/ja903239s
PMCID: PMC2732204  PMID: 19655738
21.  The Sequence of the Enterococcal Cytolysin Imparts Unusual Lanthionine Stereochemistry 
Nature chemical biology  2013;9(3):157-159.
The enterococcal cytolysin is a two-component lantibiotic of unknown structure with hemolytic activity that is important for virulence. We prepared cytolysin by co-expression of each precursor peptide with the synthetase CylM in E. coli, and characterized its structure. Surprisingly, cytolysin is the first example of a lantibiotic containing lanthionine and methyllanthionine structures with different stereochemistries in the same peptide, which is determined by the sequence of the substrate peptide.
doi:10.1038/nchembio.1162
PMCID: PMC3578037  PMID: 23314913
22.  Non-proteinogenic Amino Acids in Lacticin 481 Analogues Result in More Potent Inhibition of Peptidoglycan Transglycosylation 
ACS Chemical Biology  2012;7(11):1791-1795.
Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.
doi:10.1021/cb300372b
PMCID: PMC3501146  PMID: 22920239
23.  In Vitro Selection of Functional Lantipeptides 
In this report we present a method to identify functional artificial lantipeptides. In vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 1011 different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.
doi:10.1021/ja302082d
PMCID: PMC3353655  PMID: 22545861
24.  ProPortal: a resource for integrated systems biology of Prochlorococcus and its phage 
Nucleic Acids Research  2011;40(D1):D632-D640.
ProPortal (http://proportal.mit.edu/) is a database containing genomic, metagenomic, transcriptomic and field data for the marine cyanobacterium Prochlorococcus. Our goal is to provide a source of cross-referenced data across multiple scales of biological organization—from the genome to the ecosystem—embracing the full diversity of ecotypic variation within this microbial taxon, its sister group, Synechococcus and phage that infect them. The site currently contains the genomes of 13 Prochlorococcus strains, 11 Synechococcus strains and 28 cyanophage strains that infect one or both groups. Cyanobacterial and cyanophage genes are clustered into orthologous groups that can be accessed by keyword search or through a genome browser. Users can also identify orthologous gene clusters shared by cyanobacterial and cyanophage genomes. Gene expression data for Prochlorococcus ecotypes MED4 and MIT9313 allow users to identify genes that are up or downregulated in response to environmental stressors. In addition, the transcriptome in synchronized cells grown on a 24-h light–dark cycle reveals the choreography of gene expression in cells in a ‘natural’ state. Metagenomic sequences from the Global Ocean Survey from Prochlorococcus, Synechococcus and phage genomes are archived so users can examine the differences between populations from diverse habitats. Finally, an example of cyanobacterial population data from the field is included.
doi:10.1093/nar/gkr1022
PMCID: PMC3245167  PMID: 22102570
25.  Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability 
The ISME Journal  2011;5(10):1580-1594.
Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron limitation of Prochlorococcus cell division rates in these regions has been shown. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency-induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.
doi:10.1038/ismej.2011.49
PMCID: PMC3176520  PMID: 21562599
cyanobacteria; iron; transcriptome

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