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Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.
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The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.
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Biotransformação , Pseudomonas , Metabolismo Secundário , Microbiologia do Solo , Pseudomonas/metabolismo , Pseudomonas/genética , Rizosfera , Microbiota , Interações Microbianas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Fenóis , Floroglucinol/análogos & derivados , PirróisRESUMO
Actinomycetes are a phylogenetically diverse bacterial group which are widely distributed across terrestrial and aquatic ecosystems. Within this order, the genus Pseudonocardia and their specialised metabolites have been the focus of previous ecological studies due to their antagonistic interactions with other microorganisms and their mutualistic interactions with insects. However, the chemical ecology of free-living Pseudonocardia remains understudied. This study applies a multi-omics approach to investigate the chemical ecology of free-living actinomycetes from the genus Pseudonocardia. In a comparative genomics analysis, it was observed that the biosynthetic gene cluster family distribution was influenced mainly by phylogenetic distance rather than the geographic or ecological origin of strains. This finding was also observed in the mass spectrometry-based metabolomic profiles of nine Pseudonocardia species isolated from marine sediments and two terrestrial species. Antagonist interactions between these 11 species were examined, and matrix-assisted laser desorption/ionisation-mass spectrometry imaging was used to examine in situ chemical interactions between the Southern Ocean strains and their phylogenetically close relatives. Overall, it was demonstrated that phylogeny was the main predictor of antagonistic interactions among free-living Pseudonocardia. Moreover, two features at m/z 441.15 and m/z 332.20 were identified as metabolites related to these interspecies interactions.
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Ecossistema , Metabolômica , Filogenia , Pseudonocardia , Antibiose , Genômica , Sedimentos Geológicos/microbiologia , Família Multigênica , Multiômica , Pseudonocardia/genéticaRESUMO
Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.
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Adaptação Fisiológica , Aspergillus niger , Bacillus subtilis , Lipopeptídeos , Bacillus subtilis/fisiologia , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Aspergillus niger/metabolismo , Aspergillus niger/fisiologia , Aspergillus niger/crescimento & desenvolvimento , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Interações Microbianas/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Técnicas de Cocultura , Mutação , Parede Celular/metabolismoRESUMO
Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RNâN+(O-)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here, we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knockout using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A-like clusters are widely distributed across the phylogenetic tree.
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Streptomyces , Espectrometria de Massas em Tandem , Filogenia , Streptomyces/genética , Streptomyces/metabolismo , Família MultigênicaRESUMO
Siderophores have long been implicated in sociomicrobiology as determinants of bacterial interrelations. For plant-associated genera, like Bacillus and Pseudomonas, siderophores are well known for their biocontrol functions. Here, we explored the functional role of the Bacillus subtilis siderophore bacillibactin (BB) in an antagonistic interaction with Pseudomonas marginalis. The presence of BB strongly influenced the outcome of the interaction in an iron-dependent manner. The BB producer B. subtilis restricts colony spreading of P. marginalis by repressing the transcription of histidine kinase-encoding gene gacS, thereby abolishing production of secondary metabolites such as pyoverdine and viscosin. By contrast, lack of BB restricted B. subtilis colony growth. To explore the specificity of the antagonism, we cocultured B. subtilis with a collection of fluorescent Pseudomonas spp. and found that the Bacillus-Pseudomonas interaction is conserved, expanding our understanding of the interplay between two of the most well-studied genera of soil bacteria.
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Bacillus subtilis , Ferro , Ferro/metabolismo , Bacillus subtilis/genética , Sideróforos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismoRESUMO
In nature, secondary metabolites mediate interactions between microorganisms residing in complex microbial communities. However, the degree to which community dynamics can be linked to secondary metabolite potential remains largely unknown. In this study, we address the relationship between community succession and secondary metabolism variation. We used 16S and 18S rRNA gene and adenylation domain amplicon sequencing, genome-resolved metagenomics, and untargeted metabolomics to track the taxons, biosynthetic gene clusters, and metabolome dynamics in situ of microorganisms during marine biofilm succession over 113 days. Two phases were identified during the community succession, with a clear shift around Day 29, where the alkaloid secondary metabolites, pseudanes, were also detected. The microbial secondary metabolite potential changed between the phases, and only a few community members, including Myxococotta spp., were responsible for the majority of the biosynthetic gene cluster potential in the early succession phase. In the late phase, bryozoans and benthic copepods were detected, and the microbial nonribosomal peptide potential drastically decreased in association with a reduction in the relative abundance of the prolific secondary metabolite producers. Conclusively, this study provides evidence that the early succession of the marine biofilm community favors prokaryotes with high nonribosomal peptide synthetase potential. In contrast, the late succession is dominated by multicellular eukaryotes and a reduction in bacterial nonribosomal peptide synthetase potential.
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Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS, and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.
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Microbial mats are microbial communities capable of recycling the essential elements of life and considered to be the oldest evidence of microbial communities on Earth. Due to their uniqueness and limited sampling material, analyzing their metabolomic profile in different seasons or conditions is challenging. In this study, microbial mats from a small pond in the Cuatro Cienegas Basin in Coahuila, Mexico, were collected in wet and dry seasons. In addition to these samples, mesocosm experiments from the wet samples were set. These mats are elastic and rise after heavy rainfall by forming gas domes structures known as "Archean domes", by the outgassing of methanogenic bacteria, archaea, and sulfur bacteria. Extracts from all mats and mesocosms were subjected to untargeted mass spectrometry-based metabolomics and molecular networking analysis. Interestingly, each mat showed high chemical diversity that may be explained by the temporal dynamic processes in which they were sampled.
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In the marine environment, surface-associated bacteria often produce an array of antimicrobial secondary metabolites, which have predominantly been perceived as competition molecules. However, they may also affect other hallmarks of surface-associated living, such as motility and biofilm formation. Here, we investigate the ecological significance of an antibiotic secondary metabolite, tropodithietic acid (TDA), in the producing bacterium, Phaeobacter piscinae S26. We constructed a markerless in-frame deletion mutant deficient in TDA biosynthesis, S26ΔtdaB. Molecular networking demonstrated that other chemical sulfur-containing features, likely related to TDA, were also altered in the secondary metabolome. We found several changes in the physiology of the TDA-deficient mutant, ΔtdaB, compared to the wild type. Growth of the two strains was similar; however, ΔtdaB cells were shorter and more motile. Transcriptome and proteome profiling revealed an increase in gene expression and protein abundance related to a type IV secretion system, and to a prophage, and a gene transfer agent in ΔtdaB. All these systems may contribute to horizontal gene transfer (HGT), which may facilitate adaptation to novel niches. We speculate that once a TDA-producing population has been established in a new niche, the accumulation of TDA acts as a signal of successful colonization, prompting a switch to a sessile lifestyle. This would lead to a decrease in motility and the rate of HGT, while filamentous cells could form the base of a biofilm. In addition, the antibiotic properties of TDA may inhibit invading competing microorganisms. This points to a role of TDA in coordinating colonization and adaptation. IMPORTANCE Despite the broad clinical usage of microbial secondary metabolites with antibiotic activity, little is known about their role in natural microbiomes. Here, we studied the effect of production of the antibiotic tropodithietic acid (TDA) on the producing strain, Phaeobacter piscinae S26, a member of the Roseobacter group. We show that TDA affects several phenotypes of the producing strain, including motility, cell morphology, metal metabolism, and three horizontal gene transfer systems: a prophage, a type IV secretion system, and a gene transfer agent. Together, this indicates that TDA participates in coordinating the colonization process of the producer. TDA is thus an example of a multifunctional secondary metabolite that can mediate complex interactions in microbial communities. This work broadens our understanding of the ecological role that secondary metabolites have in microbial community dynamics.
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Rhodobacteraceae , Sistemas de Secreção Tipo IV , Sistemas de Secreção Tipo IV/metabolismo , Rhodobacteraceae/genética , Antibacterianos/metabolismoRESUMO
Covering: up to the end of 2021Aspergilli are biosynthetically 'talented' micro-organisms and therefore the natural products community has continually been interested in the wealth of biosynthetic gene clusters (BGCs) encoding numerous secondary metabolites related to these fungi. With the rapid increase in sequenced fungal genomes combined with the continuous development of bioinformatics tools such as antiSMASH, linking new structures to unknown BGCs has become much easier when taking retro-biosynthetic considerations into account. On the other hand, in most cases it is not as straightforward to prove proposed biosynthetic pathways due to the lack of implemented genetic tools in a given fungal species. As a result, very few secondary metabolite biosynthetic pathways have been characterized even amongst some of the most well studied Aspergillus spp., section Nigri (black aspergilli). This review will cover all known biosynthetic compound families and their structural diversity known from black aspergilli. We have logically divided this into sub-sections describing major biosynthetic classes (polyketides, non-ribosomal peptides, terpenoids, meroterpenoids and hybrid biosynthesis). Importantly, we will focus the review on metabolites which have been firmly linked to their corresponding BGCs.
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Aspergillus , Genoma Fúngico , Humanos , Aspergillus/genética , Biologia Computacional , Metabolismo Secundário/genética , Família Multigênica , Vias Biossintéticas/genéticaRESUMO
Society faces the challenge of storing energy from sustainable sources in inexpensive, nontoxic ways that do not deplete the limited resources of Earth. In this regard, quinone redox flow batteries have been proposed as ideal; however, industrially used quinones have traditionally been synthesized from fossil fuels. Therefore, we investigated the production of phoenicin (compound 1), a deep violet dibenzoquinone produced by certain Penicillium species, for its industrial potential. Strains grew as surface cultures on customized growth media with varying production parameters, and phoenicin production was assessed by ultrahigh-performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOF MS) analysis of the supernatant. Phoenicin production was reliant on the sucrose concentration, and by varying that, we produced 4.94 ± 0.56 g/L phoenicin on a Czapek yeast autolysate broth (CY)-based medium with Penicillium phoeniceum (CBS 249.32) as the production host, with 71.91% phoenicin purity in the resulting medium broth. Unexpectedly, metabolites corresponding to phoenicin polymers were tentatively identified in P. phoeniceum, of which the dimer (diphoenicin) was a major chromatographic peak. An MS-based metabolomics study was conducted on P. atrosanguineum using feature-based molecular networking and multivariate statistics, and it was found that few or no known secondary metabolites besides phoenicin were secreted into the growth medium. Finally, the effects of sucrose, sodium nitrate, and yeast extract (YE) in the growth medium were investigated in a 23 full factorial design. The results indicated an optimal sucrose concentration of 92.87 g/L on CY when NaNO3 and YE were fixed at 3 and 5 g/L, respectively. IMPORTANCE This work was undertaken to explore the production of fungal quinones in wild-type strains for use as electrolytes in redox flow batteries. As society converts energy production in a more sustainable direction, it becomes increasingly more important to store sustainable energy in smart ways. Conventional battery technologies imply the use of highly toxic, expensive, and rare metals; thus, quinone redox flow batteries have been proposed to be a desirable alternative. In this study, we explored the possibility of producing the fungal quinone phoenicin in Penicillium spp. by changing the growth parameters. The production of other secondary metabolites and known mycotoxins was also investigated in a metabolomics study. It was shown that phoenicin production was activated by optimizing the carbon concentration of the medium, resulting in high titers and purity of the single metabolite.
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Micotoxinas , Penicillium , Benzoquinonas , Espectrometria de Massas/métodos , Micotoxinas/metabolismo , Penicillium/metabolismo , Sacarose/metabolismoRESUMO
Covering: 2016 up to 2021Mass spectrometry (MS) is an essential technology in natural products research with MS fragmentation (MS/MS) approaches becoming a key tool. Recent advancements in MS yield dense metabolomics datasets which have been, conventionally, used by individual labs for individual projects; however, a shift is brewing. The movement towards open MS data (and other structural characterization data) and accessible data mining tools is emerging in natural products research. Over the past 5 years, this movement has rapidly expanded and evolved with no slowdown in sight; the capabilities of today vastly exceed those of 5 years ago. Herein, we address the analysis of individual datasets, a situation we are calling the '2021 status quo', and the emergent framework to systematically capture sample information (metadata) and perform repository-scale analyses. We evaluate public data deposition, discuss the challenges of working in the repository scale, highlight the challenges of metadata capture and provide illustrative examples of the power of utilizing repository data and the tools that enable it. We conclude that the advancements in MS data collection must be met with advancements in how we utilize data; therefore, we argue that open data and data mining is the next evolution in obtaining the maximum potential in natural products research.
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Produtos Biológicos/química , Mineração de Dados , Espectrometria de Massas em Tandem/métodos , Produtos Biológicos/metabolismo , Análise de Dados , MetabolômicaRESUMO
Siderophores are iron-chelating compounds that aid iron uptake, one of the key strategies for microorganisms to carve out ecological niches in microbially diverse environments. Desferrioxamines are the principal siderophores produced by Streptomyces spp. Their biosynthesis has been well studied and as a consequence, the chemical potential of the pathway continues to expand. With all of this in mind, our study aimed to explore extremotolerant and lupine rhizosphere-derived Streptomyces sp. S29 for its potential antifungal capabilities. Cocultivation of isolate S29 was carried out with Aspergillus niger and Botrytis cinerea, both costly fungal phytopathogens in the wine industry, to simulate their interaction within the rhizosphere. The results indicate that not only is Streptomyces sp. S29 extraordinary at producing hydroxamate siderophores but uses siderophore production as a means to 'starve' the fungi of iron. High resolution LC-MS/MS followed by GNPS molecular networking was used to observe the datasets for desferrioxamines and guided structure elucidation of new desferrioxamine analogues. Comparing the new chemistry, using tools like molecular networking and MS2LDA, with the known biosynthesis, we show that the chemical potential of the desferrioxamine pathway has further room for exploration.
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Desferroxamina/metabolismo , Ferro/metabolismo , Lupinus/microbiologia , Rizosfera , Streptomyces/metabolismo , Antifúngicos/química , Antifúngicos/farmacologia , Cromatografia Líquida , Desferroxamina/química , Desferroxamina/farmacologia , Redes e Vias Metabólicas , Espectrometria de Massas em TandemRESUMO
Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T.
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Espectrometria de Massas/métodos , Peptídeos Cíclicos/genética , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Peptídeos Cíclicos/químicaRESUMO
We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.
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Bases de Dados de Compostos Químicos , Espectrometria de Massas , Metabolômica/métodos , Software , Metadados , Modelos QuímicosRESUMO
Whole genome sequences of four bacterial strains Dietzia maris SST1, Pseudomonas zhaodongensis SST2, Pseudomonas sp. SST3 and Halomonas sulfidaeris SST4, recovered from the South Shetland Trench sediment in Antarctica were analyzed using Ion Torrent sequencing technology. The respective sizes of their genomes (3.88, 4.99, 5.60 and 4.25 Mb) and GC contents (70.0, 60.3, 59.9 and 53.8%) are in agreement with these values of other strains of the species. The bacterial strains displayed promising antimicrobial activity against a number of pathogenic bacterial and fungal species. Whole genomes have been assembled and biosynthetic gene clusters (BGCs) have been identified using the antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) web platform. Comparative analysis of the genome sequences revealed that the strains host abundant BGCs encoding for terpenes, siderophores, arylpolyene, bacteriocins, and lassopeptides. Furthermore, the key stress-related genes were identified and their distribution provided an insight into how these isolates adapt to key marine environmental conditions. This comprehensive study is a contribution to understanding the nature of life on the deep-sea environments.
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Actinobacteria/genética , Genoma Bacteriano , Halomonas/genética , Pseudomonas/genética , Regiões Antárticas , Sedimentos Geológicos , Sequenciamento de Nucleotídeos em Larga Escala , Oceanos e Mares , Sequenciamento Completo do GenomaRESUMO
A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.
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Peptídeos , Streptomyces , Família Multigênica , Streptomyces/genéticaRESUMO
Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.