Evolution of lanthipeptide synthetasesQi Zhanga, Yi Yub, Juan E. Vélasqueza, and Wilfred A. van der Donka,b,c,1

aDepartment of Chemistry, bDepartment of Biochemistry, and cHoward Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana,IL 61801

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved September 24, 2012 (received for review June 19, 2012)

Lanthionine-containing peptides (lanthipeptides) are a family ofribosomally synthesized and posttranslationally modified peptidescontaining (methyl)lanthionine residues. Here we present a phylo-genomic study of the four currently known classes of lanthipep-tide synthetases (LanB and LanC for class I, LanM for class II, LanKCfor class III, and LanL for class IV). Although they possess verysimilar cyclase domains, class II–IV synthetases have evolved inde-pendently, and LanB and LanC enzymes appear to not always havecoevolved. LanM enzymes from various phyla that have three cys-teines ligated to a zinc ion (as opposed to the more common Cys-Cys-His ligand set) cluster together. Most importantly, the phylo-genomic data suggest that for some scaffolds, the ring topology ofthe final lanthipeptides may be determined in part by the se-quence of the precursor peptides and not just by the biosyntheticenzymes. This notion was supported by studies with two chimericpeptides, suggesting that the nisin and prochlorosin biosyntheticenzymes can produce the correct ring topologies of epilancin 15Xand lacticin 481, respectively. These results highlight the potentialof lanthipeptide synthetases for bioengineering and combinatorialbiosynthesis. Our study also demonstrates unexplored areas ofsequence space that may be fruitful for genome mining.

molecular evolution | natural products | phylogeny |posttranslational modification | lantibiotics

Peptide antibiotics represent a large and diverse group of bio-active natural products with a wide range of applications. Most

of these compounds are produced via two distinct biosyntheticparadigms. The nonribosomal peptide synthetases are responsiblefor the biosynthesis of many clinically important antibiotics (1, 2).A different strategy involves posttranslational modifications oflinear ribosomally synthesized peptides (3, 4). This biosyntheticstrategy is also widely distributed and found in all three domains oflife. Although the building blocks used by ribosomes are generallyconfined to the 20 proteinogenic amino acids, the structural di-versity generated by posttranslational modifications is vast (5).Among the best-studied ribosomally synthesized and posttransla-

tionally modified peptides are the lanthipeptides, a class of com-pounds distinguished by the presence of sulfur-to-β–carbon thioethercross-links named lanthionines andmethyllanthionines (Fig. 1) (6–9).Many lanthipeptides, such as the commercially used food pre-servative nisin, have potent antimicrobial activity and are termedlantibiotics. Maturation of lanthipeptides involves posttranslationalmodifications of a C-terminal core region of a precursor peptide andsubsequent proteolytic removal of an N-terminal leader sequencethat is not modified (3). Their thioether bridges are installed by theinitial dehydration of Ser and Thr residues, followed by stereo-selective intramolecular Michael-type addition of Cys thiols to thenewly formed dehydroamino acids (Fig. 1A). In some cases, this re-action is coupledwith a secondMichael-type addition of the resultingenolate to a second dehydroalanine to produce a labionin structure(10). Genetic and biochemical studies have revealed four distinctclasses of lanthipeptides according to their biosyntheticmachinery (7,11, 12) (Fig. 1B). Class I lanthipeptides are synthesized by two dif-ferent enzymes, a dehydratase LanB, and a cyclase LanC (Lan isa generic designation for lanthipeptide biosynthetic proteins). Forclass II lanthipeptides, the reactions are carried out by a single lan-thipeptide synthetase, LanM, containing an N-terminal dehydratasedomain that bears no homology toLanB, and aC-terminal LanC-like

cyclase domain. Class III and class IV lanthipeptides are synthesizedby trifunctional enzymesLanKC (10, 13) andLanL (12), respectively.These enzymes contain an N-terminal lyase domain and a centralkinase domain but differ in their C termini. LanC and the C-terminaldomains of LanM and LanL contain a conserved zinc-binding motif(Cys-Cys-His/Cys),whereas theC-terminal cyclase domain ofLanKClacks these conserved residues (13) (Fig. 1B).Recent genomemining studies have revealed that lanthipeptide

biosynthetic genes are present in a wide range of bacteria (14–16).The widespread occurrence may not be surprising, given the po-tential benefits of gene-encoded natural products with respect tofacile evolution of new structures and biological function. Here wepresent a systematic analysis of the phylogenetic distribution oflanthipeptide synthetases. We correlate the taxonomy of the bac-terial host and the structure of the final products with the enzymephylogenies. Implications for biosynthetic engineering of the lan-thipeptide family and for genome mining are discussed.

Results and DiscussionEvolution of LanC enzymes. LanCs have about 400 amino acid res-idues, and possess a double α–barrel-fold topology (17) and astrictly conservedCys-Cys-His triad near their C termini for bindingof a zinc ion. In vitro reconstitution of the nisin cyclase activity ofNisC and solution of its crystal structure have supported a zinc-dependent mechanism (17, 18). The zinc ion is believed to activatethe Cys thiols of the precursor peptide for nucleophilic attack onthe dehydroamino acids. LanC or LanC-like enzymes are not onlyfound as stand-alone cyclases and as cyclase domains in LanM andLanL enzymes, their encoding genes are also present in eukaryotes,although the detailed functions of these LanC-like (LanCL) pro-teins remain to be determined (19).The phylogeny of LanC from different bacterial lineages, in-

cluding the C-terminal domains of LanMs and LanLs, and theLanCL proteins from human that can serve as the outgroup, wasconstructed using both Bayesian Markov chain Monte Carlo(MCMC) (20) andmaximum-likelihood inferences (21). To obviatecodon bias, the trees were constructed based on amino acidsequences instead of nucleic acid sequences. The overall BayesianMCMC tree shown in Fig. 2A andB is almost exactly the same withthat prepared by the maximum-likelihood method (SI Appendix,Fig. S1), strongly supporting the reliability of both trees.TheC-terminal domains of LanMandLanL fall into two distinct

clades. These two clades group into a larger clade and are sepa-rated from a sister LanC clade and the eukaryotic LanCL clade(Fig. 2A and SI Appendix, Fig. S1), suggesting that LanMandLanLevolved independently from LanC. If LanM and LanL originatefrom hybridization of an ancestral LanC with a dehydratase orkinase, this event likely occurred only once. Gene recombination

Author contributions: Q.Z., Y.Y., J.E.V., and W.A.v.d.D. designed research; Q.Z., Y.Y., andJ.E.V. performed research; Q.Z., Y.Y., J.E.V., and W.A.v.d.D. analyzed data; and Q.Z. andW.A.v.d.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: vddonk@illinois.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210393109/-/DCSupplemental.

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between the C-terminal domains of LanM and LanL is not sup-ported by our analysis of the currently available sequences.LanCs from different bacterial phyla group into three groups

with strong statistical support (Fig. 2B and SI Appendix, Fig. S1).Group 1 consists of enzymes from bacteroidetes and proteobac-teria. Although proteobacteria are a prolific source of LanMenzymes (vide infra), only very few LanCs are found in this phylum.Group 2 and group 3 consist of proteins from actinobacteria andfirmicutes, respectively, which group into a larger clade that is sisterto group 1, indicating that LanC enzymes from these two phyla aremore closely related with each other than with their counterpartsfrom bacteroidetes and proteobacteria. Group 3 consists of theenzymes from firmicutes, with only one exception of an enzymefrom Bifidobacterium longum, which belongs to the actinobacteria.Taken together, these results indicate that LanCs from differentphyla have evolved independently, and that interphylum horizon-tal gene transfer generally did not occur during LanC evolution.Class I lanthipeptides have been grouped into nisin-like, epi-

dermin-like, and Pep5-like groups (8) (SI Appendix, Figs. S2 andS3). Although LanC enzymes for producing structurally similarlanthipeptides usually cluster together (Fig. 2B), this is not alwaysthe case. Nisin and subtilin have similar precursor peptide sequen-ces and exactly the same ring topologies (6) (Fig. 3), but their as-sociated LanC enzymes have relatively distant relationships (Fig.2B). Moreover, microbisporicin has a similar N-terminal structureto that of nisin, but is produced by Actinobacteria (22, 23), and itsLanCdoes not even belong to group 3, which harborsmost enzymesfor nisin-like lanthipeptides. These results demonstrate convergentevolution to arrive at nisin-like architectures. When also taking intoconsideration the distinct C-terminal ring topologies of subtilin (6),ericin A (24), and geobacillin (25) (Fig. 3), and their closely relatedLanCs (Fig. 2B), the ring topology of some lanthipeptides may bedetermined as much by the sequence of their precursor peptides asby their cyclase sequences. This hypothesis could have importantimplications for bioengineering (vide infra). Conversely, it is puz-zling that thus far the potent lipid II binding topology of theA andBrings of the nisin-like peptides has only been observed in class Icompounds and not in the other classes of lanthipeptides.

Evolution of LanB enzymes. LanB proteins usually have about 1,000residues, consisting of a lanthipeptide dehydratase domain anda so-called SpaB_C (SpaB C-terminal) domain (26) (Fig. 1B).

A few lanthipeptide biosynthetic gene clusters contain genesencoding a C-terminally truncated LanB and a stand-aloneSpaB_C-like protein, suggesting that the two domains of LanBmight be able to act both in cis and in trans. Similarly, most ofthe thiopeptide biosynthetic gene clusters encode two proteinsfor dehydration: a putative dehydratase that shares low se-quence similarity with LanBs and a SpaB_C-like protein (27). Inthis study, only full-length LanB sequences were used.The phylogenetic trees of LanB enzymes from the same gene

cluster as the LanC proteins in Fig. 2B were constructed by bothBayesian MCMC and maximum-likelihood methods, and theresulting two trees are almost identical (SI Appendix, Figs. S4 andS5). Interestingly, the topology of the LanB tree is distinct fromthat of LanC. The enzymes from bacteroidetes and proteobac-teria are possibly derived from firmicutes because they are deeplynested within a group that mainly consists of LanBs from firmi-cutes (SI Appendix, Fig. S4). This finding is in distinct contrast tothe LanC tree in which the enzymes of group 1 are distantly re-lated to those of group 3 (Fig. 2B). It appears therefore that forthese clusters, LanBs have evolved independently from LanCs, orthat the LanBs or LanCs may have been recruited from otherorganisms and have formed a functional pair. In general, how-ever, the trees show that LanB and LanC enzymes from the sameorganism fall into similar clades (Fig. 2B and SI Appendix, Fig.S4), suggesting that they have coevolved.Genomemining for new class I lanthipeptidesmight be facilitated

by phylogenetic categorization of LanC and LanB enzymes. Forexample, we note that Streptococcus pasteurianus contains genes forLanB and LanC that are phylogenetically closely related to NisBand NisC (Fig. 2). Examining their associated lanA gene suggeststhat this strainmay be able to produce a nisin analog similar to nisinU (designated nisin P in Fig. 3). Similarly, Actinomyces sp. oralmayproduce a microbisporicin analog (designated microbisporicin B inFig. 3), and Streptococcus sanguinis may produce a streptin analog(designated streptin B in SI Appendix, Fig. S6). Given the vast se-quence space of uncharacterized enzymes from strains other thanfirmicutes (e.g., group 1 and group 2 in Fig. 2B), these datamay alsoserve as a guideline for future genome mining efforts.

Evolution of LanM Enzymes. LanMs are bifunctional enzymes of900–1,200 residues containing an N-terminal dehydratase anda C-terminal LanC-type cyclization domain (7, 28). LanM pro-teins use ATP to phosphorylate Ser/Thr residues in their sub-strates and they subsequently eliminate the resulting phosphateester to yield the dehydroamino acids (29). The C-terminal do-main then catalyzes the cyclization reaction in a similar mannerto LanC enzymes. Unlike LanBs and LanCs, LanMs are preva-lent in proteobacteria and have also been characterized fromcyanobacteria (30, 31). However, in our analysis they are notfound in bacteroidetes, again indicating that different classes oflanthipeptides likely evolved independently.Bayesian MCMC and maximum-likelihood trees of 91 LanM

sequences from different bacterial phyla were constructed (Fig.2C and SI Appendix, Fig. S7). The Bayesian MCMC inference ofLanM phylogeny shown in Fig. 2C is similar to that of LanC andLanB, in that enzymes are usually grouped according to theirproducing organisms and form three major subdivisions. Group 1is a polyphyletic clade mainly consisting of enzymes from pro-teobacteria and cyanobacteria. Because the base of group 1consists exclusively of proteobacterial enzymes (Fig. 2C), mem-bers of group 1 from other phyla possibly evolved from proteo-bacterial ancestors. Groups 2 and 3 are made up of enzymes fromactinobacteria and firmicutes, respectively, with the only excep-tions enzymes from Bifidobacteria (Fig. 2C), which fall into thefirmicutes clade similar to what was observed for LanC (Fig. 2B).Notably, the enzymes that synthesize type IIA compounds (SI

Appendix, Fig. S8) (8, 32) are grouped into a subclade with strongsupport (Fig. 2C). Type IIA compounds are structurally similar

Fig. 1. Biosynthesis of lanthipeptides, showing the mechanism of (methyl)lanthionine formation (A), and the four classes of synthetases (B). Xn rep-resents a peptide linker. The conserved zinc-binding motifs are highlightedby the purple lines in the cyclase domains. SpaB_C is an in silico defineddomain currently found as a stand-alone protein for thiopeptide bio-synthesis and as the C-terminal domain of LanB enzymes. The N-terminaldomain of LanB enzymes is made up of two subdomains according to theConserved Domain Database (26).

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to the prototypical peptide lacticin 481, which possesses a linearN terminus and a globular C-terminal scaffold (SI Appendix, Fig.S8) (6, 32). The close correlation of the enzyme phylogeny andthe structure of the final products demonstrate that theseproducts evolved from the same ancestors, in contrast to theapparent convergent evolution of several members of the nisin-

like peptides discussed above. Enzymes for synthesizing the two-component lantibiotics are distributed in different subclades ofgroup 3 (Fig. 2C). These systems are potent antimicrobial agentsthat display strong synergism between the two posttranslationallymodified peptides (7–9). Although the two peptides of lacticin3147 (Ltnα and Ltnβ) have similar ring topologies as haloduracin-α

Fig. 2. Bayesian MCMC phylogeny of LanC and LanM enzymes. (A) Tree of LanC and LanC-like enzymes. LanM_C and LanL_C represent the C-terminal cyclasedomains of LanM and LanL, respectively. (B) The LanC clade of A. (C) Phylogenetic tree of LanM enzymes. As no suitable outgroup protein can be found (LanLscannot serve as outgroup, because their N-terminal domains are not homologous to those of LanMs), the trees were rooted by using all members of a sisterclade as the outgroup, an approach previously suggested as optimal in such instances (44). Bayesian inferences of posterior probabilities are indicated by linewidth. Lanthipeptides in each tree are shown by different colored boxes according to structural types. Two-component lantibiotics are in red font, andlanthipeptides proposed in this study are in yellow font.

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and -β, particularly in their C-terminal regions (SI Appendix, Fig.S9), their corresponding LanMs are phylogenetically distantlyrelated (Fig. 2C). These results reinforce the idea that the ringtopologies of lanthipeptides might be determined to a larger extentthan previously anticipated by the sequence of the precursorpeptide, as discussed above.The biosynthesis of the prochlorosins from cyanobacteria rep-

resents a remarkable example of natural combinatorial bio-synthesis (31): up to 29 different ProcA peptides with highlyvariable sequences in the core region but highly conserved leadersequences serve as substrates of a single enzyme ProcM, resultingin a library of structurally diverse lanthipeptides (30, 31). Thissystem is a prime example of the highly evolvable nature of ribo-somal biosynthesis to access high structural diversity of naturalproducts at low genetic cost. Analysis of the ProcM sequencerevealed that this enzyme contains a “CCG”motif (30) rather thana “CHG”motif (17, 18) found in all LanCs and most of the LanMsknown to date, indicating that ProcM likely uses three Cys residuesrather than a Cys-Cys-His triad for binding of the active site zincion. Model studies of activation of thiolate nucleophiles by Zn2+

have demonstrated increased reactivity with an increased numberof thiolate ligands (33), suggesting that ProcM may derive itspromiscuity in part from a highly active zinc ion (30). Intriguingly,all LanM proteins containing the “CCG”motif cluster together toform a distinct subgroup within group 1 (Fig. 2C). This phyloge-netic distribution is not merely a consequence of the “CCG” or“CHG” motifs, because artificially changing Cys to His or His toCys in the motif for five representative group 1 enzymes did notalter their position in the tree or greatly affect the statistic supportof the phylogenetic trees (SI Appendix, Fig. S10). Rather, theseresults suggest that LanMs containing the conserved “CCG”motifhave evolved independently from the other LanM proteins. Somebut not all of the members of the CCG clade have multiple pre-cursor genes nearby and at other loci of their genomes similarto ProcM.

Evolution of LanKC and LanL enzymes. LanKC and LanL both con-tain an N-terminal lyase domain and a central kinase domain, withboth enzymes generating dehydroamino acids via independentphosphorylation and elimination steps (7, 12). The C-terminalcyclase domain of LanKCenzymes lacks the conserved residues forbinding of a zinc ion. Consistent with this observation, AciKC

involved in catenulipeptin biosynthesis binds neither zinc nor othermetals, indicating a different cyclization mechanism of LanKCenzymes, but domain deletions support the hypothesis that theC-terminal domain is responsible for cyclization (34). Many, butnot all, LanKCs synthesize labionins (SI Appendix, Fig. S11), whichare thus far found exclusively for class III lanthipeptides (34–36).Bayesian MCMC and maximum-likelihood inferences of 39

LanKC sequences and 15 LanL sequences were constructed (SIAppendix, Figs. S12 and S13). Clearly, LanKC and LanL enzymesgroup into different clades, indicating that, despite the significantsequence similarities and the similar domain structure, these twoenzyme classes have evolved independently. The possibility thatLanKCs are derived from LanLs by loss of the zinc-binding site istherefore unlikely. Interestingly, some LanLs contain the “CCG”

instead of the “CHG” motif and may have three Cys residues thatcoordinate to the zinc ion. As was observed for LanMs, theseenzymes fall into adistinct subgroup (SIAppendix, Figs. S12 andS13).Phylogenetically, therearenoobviousdistinctionsbetweenLanKC

enzymes that generate lanthionine and labionin structures. For ex-ample, catenulipeptin and labyrinthopeptin contain only labionins(34, 35) and SapB contains only lanthionines (13) (SI Appendix, Fig.S11), but catenulipeptide synthetaseAciKC is phylogenetically closerto the SapB synthetase RamC than the labyrinthopeptin synthetaseLabKC (SI Appendix, Fig. S12). In addition, recently characterizedclass III peptides contain both lanthionine and labionin (36), and thebiosynthetic enzymes of these peptides are distributed in differentsubclades of the LanKC clade (SI Appendix, Fig. S12). Thus, it ispossible that the cyclization mode for class III may again be de-pendent on the precursor peptide sequences.

Determinants of Lanthipeptide Ring Topologies. The ring systems oflanthipeptides are very diverse, ranging from simple nonover-lapping rings to highly complex, intertwined rings (SI Appendix,Figs. S2, S3, and S9). The possibility to form labionin rings fur-ther diversifies lanthipeptide ring systems. The manner by whichring topology is determined from precursor peptides containingmultiple Cys and Ser/Thr residues is at present entirely unclear.As discussed above, the trees provide several examples ofenzymes that produce structurally similar lanthipeptides fallinginto different phylogenetic clades, as well as phylogeneticallyclosely related enzymes generating products with distinct rings.These results suggest that in some cases, the substrate sequencesmay be as important to determine the ring topologies of the finalproduct as the cyclization enzymes.The notion of substrate-directed ring patterns is supported by

comparative analysis of the nisin-group (Fig. 3). All members ofthis group contain a conserved N-terminal ring system, but theirbiosynthetic enzymes fall into three different clades (Fig. 2B).Interestingly, ericin S and A, despite possessing very differentC-terminal ring topologies, are produced by the same biosyntheticenzymes (24). The D-ring of ericin S is linked to the E-ring insimilar fashion as in nisin, whereas the D-ring of ericin A isintertwined with the C-ring, similar to that found in micro-bisporicin (Fig. 3). This analysis supports a more prominent role ofthe substrate sequences. Remarkably, Kuipers and colleagues haverecently shown that the precursors of a class II two-componentlantibiotic can be modified by the nisin biosynthetic enzymes toform antimicrobially active products (37). Although the structuresare currently unknown, the products likely have the same or verysimilar ring topologies as the wild-type peptides, supporting theimportance of substrate sequence in the ring-pattern formation.To further address this hypothesis, we generated a construct

encoding a chimeric peptide NisA-ElxA, in which the leadersequence of the nisin precursor peptide (NisA) was fused to thecore peptide of epilancin 15X separated by an engineered glu-tamic acid residue for proteolytic removal of the leader peptide.Nisin and epilancin 15X have very different N termini but similarC termini (SI Appendix, Figs. S2 and S3). Coexpression of NisA-

Fig. 3. Comparative analysis of the nisin-like peptides. A–E denote differentrings. The conserved N termini are highlighted by the orange box. Ser andThr that are involved in ring formation are shown by red highlighted font.Ser and Thr that are dehydrated but not involved in ring formation areshown in green and purple highlighted font. New compounds proposed inthis study are shown in blue font.

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ElxA with NisB and NisC in Escherichia coli resulted in a seriesof products that were dehydrated up to six times compared witheight dehydrations in epilancin 15X (Fig. 4A). Electron sprayionization (ESI)-MS/MS analysis suggested that the two N-ter-minal Ser residues escaped dehydration in the sixfold dehydratedpeptide and that it very likely has the same ring pattern as that ofepilancin 15X (Fig. 4B). Indeed, proteolytic removal of theleader peptide with endoproteinase GluC and subsequent use ofthe product for well-diffusion assays clearly demonstrated a zoneof growth inhibition of the indicator strain Staphylococcus car-nosus (Fig. 4B). The product from a parallel experiment in whichNisC was not coexpressed lacked antibacterial activity.To extend these studies to chimera of lanthipeptides that have

no structural similarity, we fused the lacticin 481 core peptide tothe ProcA3.2 leader sequence to afford a chimeric peptideProcA-LctA. This peptide was coexpressed in E. coli with thehighly promiscuous enzyme ProcM. MS analysis showed that thepeptide was dehydrated up to five times (SI Appendix, Fig.S14A). Iodoacetamide alkylation assays and ESI-MS/MS analysisindicated that the products contained a mixture of peptides thatwere partially and fully cyclized (SI Appendix, Fig. S14 B and C).Intriguingly, after removal of the ProcA leader peptide, the re-sultant product was active against the indicator strain Lacto-coccus lactis HP (SI Appendix, Fig. S14D), collectively suggestingthat the correct rings of lacticin 481 were produced. Theseresults support a model in which the Cys residues lodge onto theZn2+ ion in the active site and that subsequently the precursorpeptide sequence determines the site selectivity of cyclization. Inother words, the cyclase does not appear to enforce the rings tobe generated, nor do initially formed rings govern the site se-lectivity of subsequently formed rings. The latter point is alsosupported by previous studies on single-ring disruptions of hal-oduracin (38). To emphasize that not all synthetases can make

every lanthipeptide, ProcM did not process a chimeric peptideconsisting of the ProcA leader and NisA core peptides to a bio-active product (SI Appendix, Fig. S15). Possibly, incomplete de-hydration precluded formation of the correct rings.

Base Composition and Codon Use of Lanthipeptide Synthetase Genes.Base composition analysis is a common strategy to investigategene history and potential horizontal gene transfer events (39,40). If a gene is a vertical descendent, it should have a similarbase composition as the host genome, whereas if it does not, thegene is likely acquired relatively recently from another organism.We calculated the GC content of every lanthipeptide synthetasegene for which complete genome sequences are available andcompared the results with the GC content of their genomes (Fig.5). The analysis shows that enzymes from actinobacteria usuallyhave nucleotide composition similar to their genomes, withexceptions again found for Bifidobacteria. The GC content oflanC and lanKC genes from this genus strongly deviate from theirgenomes, indicating these genes were most likely acquired byrecent horizontal gene transfer. Genes of lanthipeptide synthe-tases from firmicutes show substantial variations of nucleotidebase composition (Fig. 5). A notable example involves the genesfrom Geobacillus, with GC contents that are decreased signifi-cantly compared with their genomes. Taken together with thephylogenetic results, these genes were likely acquired horizon-tally from Bacillus strains (Fig. 2 B and C). Application of othermethods to investigate nucleotide use in lanthipeptide bio-synthetic genes, including GC3s and the effective number ofcodons (Nc) (41), indicates that our analysis is not biased by thecodon preference of different organisms (SI Appendix, Fig. S16).All lanthipeptide biosynthetic genes from Streptococci have

relatively low GC contents (Fig. 5). To evaluate the significanceof these high deviations, for every Streptococcus strain of Fig. 5,we selected 25 genes believed to be less prone to horizontal genetransfer (42) and calculated their GC composition and overallSDs (σ) (SI Appendix, Table S1). This analysis indicates that theσ values of the core genes for all strains are lower than 3% (SIAppendix, Table S1), whereas the majority of the streptococcallanthipeptide synthetase genes have GC contents decreased bymore than 4σ from their genome, suggesting that they were likelyacquired recently by horizontal gene transfer.

ConclusionThe knowledge of the chemical and biosynthetic diversity of ri-bosomal natural products has been greatly expanded in recentyears. These compounds are distinct from other well-establishednatural products because they are gene-encoded and theirstructures can be easily changed by simple permutation of theprecursor peptide sequences. Using lanthipeptide synthetases

Fig. 4. Generation of a bioactive epilancin 15X analog with the nisin bio-synthetic enzymes. (A) MALDI-MS analysis of NisA-ElxA modified in E. coli byNisB and NisC and treated with GluC protease. (B) ESI-MS/MS analysis of thesixfold dehydrated peptide. The proposed structure, the MS/MS fragmen-tation pattern, and the in vitro bioassay against S. carnosus are shown. Spots1 and 2 on the bioassay plate are assay and negative control.

Fig. 5. Base composition analysis of the lanthipeptide synthetase genes andthe associated genomes. Genome sequences are from the same species asthe synthetase genes but the subspecies are different in some cases.

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as a model system, the phylogenomic studies represented hereinindicate a complex, dynamic, and sometimes convergent evolu-tion mechanism of the biosynthetic enzymes. Several interestingobservations are made, such as mostly phylum-dependent group-ings, the clustering of subgroups of enzymes that differ in theidentity of a single metal ligand, and the possibility that precursorpeptide sequence may be a larger determinant of final ring to-pology than previously recognized. This hypothesis is supportedby experimental studies with chimeric peptides showing thatphylogenetically distantly related enzymes can produce the samering topologies given the same peptide sequence. The phyloge-netic trees described herein can also direct future genome miningstudies, as they show that some biosynthetic sequence space hasnot been tapped at all, whereas other areas have been heavilysampled. These findings may also serve as an entry point for un-derstanding the evolutionary mechanism of other ribosomalnatural products.

Materials and MethodsBayesian MCMC inference analyses were performed using the programMrBayes (version 3.2) (43). Final analyses consisted of two sets of eight chainseach (one cold and seven heated), run for about 2–10 million generationswith trees saved and parameters sampled every 100 generations. Analyseswere run to reach a convergence with SD of split frequencies < 0.01. Pos-terior probabilities were averaged over the final 75% of trees (25% burn in).For additional details of phylogenetic analysis, procedures for the studieswith the chimeric peptides, expression and purification of modified peptideproducts, MS and bioactivity assays, and nucleotide base composition andcodon use analysis, please see the SI Appendix. The SI Appendix also containsthe accession number and the source organism of the synthetases used (SIAppendix, Tables S2–S5).

ACKNOWLEDGMENTS. We thank Dr. Taras Pogorelov and Mike Hallock(University of Illinois at Urbana–Champaign) for providing computation andnetwork assistance. This work was supported by the National Institutes ofHealth Grants GM58822 (to W.A.v.d.V.) and T32 GM070421 (to J.E.V.).

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