[en] Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.
FNS - Fonds National Suisse de la Recherche scientifique [CH] FEDER - Fonds Européen de Développement Régional [BE] MIUR - Ministero dell'Istruzione, dell'Università e della Ricerca [IT] Wallonia [BE]
Barka E.A. Vatsa P. Sanchez L. Gaveau-Vaillant N. Jacquard C. Klenk H.-P. Clément C. Ouhdouch Y. van Wezel G.P. Taxonomy, Physiology, and Natural Products of Actinobacteria Microbiol. Mol. Biol. Rev. 2015 80 1 43 10.1128/MMBR.00019-15
Hopwood D.A. Streptomyces in Nature and Medicine: The Antibiotic Makers Oxford University Press Oxford, NY, USA 2007 978-0-19-515066-7
van der Meij A. Worsley S.F. Hutchings M.I. van Wezel G.P. Chemical Ecology of Antibiotic Production by Actinomycetes FEMS Microbiol. Rev. 2017 41 392 416 10.1093/femsre/fux005 28521336
Hui M.L.-Y. Tan L.T.-H. Letchumanan V. He Y.-W. Fang C.-M. Chan K.-G. Law J.W.-F. Lee L.-H. The Extremophilic Actinobacteria: From Microbes to Medicine Antibiot. Basel Switz. 2021 10 682 10.3390/antibiotics10060682 34201133
Axenov-Gibanov D.V. Voytsekhovskaya I.V. Tokovenko B.T. Protasov E.S. Gamaiunov S.V. Rebets Y.V. Luzhetskyy A.N. Timofeyev M.A. Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds PLoS ONE 2016 11 e0149216 10.1371/journal.pone.0149216
Maciejewska M. Całusińska M. Cornet L. Adam D. Pessi I.S. Malchair S. Delfosse P. Baurain D. Barton H.A. Carnol M. et al. High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits Antibiotics 2018 7 E27 10.3390/antibiotics7020027 29561792
Park S. Cho Y.-J. Jung D. Jo K. Lee J.-S. Microbial Diversity of Baeg-Nyong Cave and Characterization of the Antibiotics Extracted from Streptomyces Exfoliatus FASEB J. 2019 33 637.4 10.1096/fasebj.2019.33.1_supplement.637.4
Rangseekaew P. Pathom-Aree W. Cave Actinobacteria as Producers of Bioactive Metabolites Front. Microbiol. 2019 10 387 10.3389/fmicb.2019.00387
Adam D. Maciejewska M. Naômé A. Martinet L. Coppieters W. Karim L. Baurain D. Rigali S. Isolation, Characterization, and Antibacterial Activity of Hard-to-Culture Actinobacteria from Cave Moonmilk Deposits Antibiotics 2018 7 E28 10.3390/antibiotics7020028
Maciejewska M. Adam D. Martinet L. Naômé A. Całusińska M. Delfosse P. Carnol M. Barton H.A. Hayette M.-P. Smargiasso N. et al. A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits Front. Microbiol. 2016 7 1455 10.3389/fmicb.2016.01455
Borsato A. Frisia S. Jones B. Van Der Borg K. Calcite Moonmilk: Crystal Morphology and Environment of Formation in Caves in the Italian Alps J. Sediment. Res. 2000 70 1171 1182 10.1306/032300701171
Cañaveras J.C. Cuezva S. Sanchez-Moral S. Lario J. Laiz L. Gonzalez J.M. Saiz-Jimenez C. On the Origin of Fiber Calcite Crystals in Moonmilk Deposits Naturwissenschaften 2006 93 27 32 10.1007/s00114-005-0052-3
Reinbacher W.R. Is It Gnome, Is It Berg, Is It Mont, It It Mond? An Updated View of the Origin and Etymology of Moonmilk Bull. Natl. Speleol. Soc. 1994 56 1 13
Miller A.Z. Dionísio A. Jurado V. Cuezva S. Sanchez-Moral S. Cañaveras J.C. Saiz-Jimenez C. Biomineralization by cave dwelling microorganisms Advances in Geochemistry Research Sanjurjo Sanchéz J. Nova Science Publishers Hauppauge, NY, USA 2013 Volume 5 77 105
Maciejewska M. Adam D. Naômé A. Martinet L. Tenconi E. Całusińska M. Delfosse P. Hanikenne M. Baurain D. Compère P. et al. Assessment of the Potential Role of Streptomyces in Cave Moonmilk Formation Front. Microbiol. 2017 8 1181 10.3389/fmicb.2017.01181
Cirigliano A. Tomassetti M.C. Di Pietro M. Mura F. Maneschi M.L. Gentili M.D. Cardazzo B. Arrighi C. Mazzoni C. Negri R. et al. Calcite Moonmilk of Microbial Origin in the Etruscan Tomba Degli Scudi in Tarquinia, Italy Sci. Rep. 2018 8 15839 10.1038/s41598-018-34134-y 30367083
Seifan M. Berenjian A. Microbially Induced Calcium Carbonate Precipitation: A Widespread Phenomenon in the Biological World Appl. Microbiol. Biotechnol. 2019 103 4693 4708 10.1007/s00253-019-09861-5 31076835
Portillo M.C. Gonzalez J.M. Moonmilk Deposits Originate from Specific Bacterial Communities in Altamira Cave (Spain) Microb. Ecol. 2011 61 182 189 10.1007/s00248-010-9731-5
Spötl C. Moonmilk as a Human and Veterinary Medicine: Evidence of Past Artisan Mining in Caves of the Austrian Alps Int. J. Speleol. 2018 47 127 135 10.5038/1827-806X.47.2.2174
Maciejewska M. Pessi I.S. Arguelles-Arias A. Noirfalise P. Luis G. Ongena M. Barton H. Carnol M. Rigali S. Streptomyces Lunaelactis Sp. Nov., a Novel Ferroverdin A-Producing Streptomyces Species Isolated from a Moonmilk Speleothem Antonie Van Leeuwenhoek 2015 107 519 531 10.1007/s10482-014-0348-4
Naômé A. Maciejewska M. Calusinska M. Martinet L. Anderssen S. Adam D. Tenconi E. Deflandre B. Coppieters W. Karim L. et al. Complete Genome Sequence of Streptomyces Lunaelactis MM109T, Isolated from Cave Moonmilk Deposits Genome. Announc. 2018 6 e00435-18 10.1128/genomeA.00435-18
Martinet L. Naômé A. Baiwir D. De Pauw E. Mazzucchelli G. Rigali S. On the Risks of Phylogeny-Based Strain Prioritization for Drug Discovery: Streptomyces Lunaelactis as a Case Study Biomolecules 2020 10 E1027 10.3390/biom10071027 32664387
Martinet L. Baiwir D. Mazzucchelli G. Rigali S. Structure of New Ferroverdins Recruiting Unconventional Ferrous Iron Chelating Agents Biomolecules 2022 12 752 10.3390/biom12060752 35740878
Martinet L. Naômé A. Deflandre B. Maciejewska M. Tellatin D. Tenconi E. Smargiasso N. de Pauw E. van Wezel G.P. Rigali S. A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators mBio 2019 10 e01230-19 10.1128/mBio.01230-19
Ma J. Wang Z. Huang H. Luo M. Zuo D. Wang B. Sun A. Cheng Y.-Q. Zhang C. Ju J. Biosynthesis of Himastatin: Assembly Line and Characterization of Three Cytochrome P450 Enzymes Involved in the Post-Tailoring Oxidative Steps Angew. Chem. Int. Ed. Engl. 2011 50 7797 7802 10.1002/anie.201102305 21726028
Xie Y. Li Q. Qin X. Ju J. Ma J. Enhancement of Himastatin Bioproduction via Inactivation of Atypical Repressors in Streptomyces Hygroscopicus Metab. Eng. Commun. 2019 8 e00084 10.1016/j.mec.2018.e00084 30671346
Kautsar S.A. Blin K. Shaw S. Navarro-Muñoz J.C. Terlouw B.R. van der Hooft J.J.J. van Santen J.A. Tracanna V. Suarez Duran H.G. Pascal Andreu V. et al. MIBiG 2.0: A Repository for Biosynthetic Gene Clusters of Known Function Nucleic Acids Res. 2020 48 D454 D458 10.1093/nar/gkz882 31612915
Fujimori D.G. Hrvatin S. Neumann C.S. Strieker M. Marahiel M.A. Walsh C.T. Cloning and Characterization of the Biosynthetic Gene Cluster for Kutznerides Proc. Natl. Acad. Sci. USA 2007 104 16498 16503 10.1073/pnas.0708242104
Liu W.-T. Lamsa A. Wong W.R. Boudreau P.D. Kersten R. Peng Y. Moree W.J. Duggan B.M. Moore B.S. Gerwick W.H. et al. MS/MS-Based Networking and Peptidogenomics Guided Genome Mining Revealed the Stenothricin Gene Cluster in Streptomyces Roseosporus J. Antibiot. 2014 67 99 104 10.1038/ja.2013.99
Zhao H. Wang L. Wan D. Qi J. Gong R. Deng Z. Chen W. Characterization of the Aurantimycin Biosynthetic Gene Cluster and Enhancing Its Production by Manipulating Two Pathway-Specific Activators in Streptomyces Aurantiacus JA 4570 Microb. Cell Factories 2016 15 160 10.1186/s12934-016-0559-7
Broberg A. Menkis A. Vasiliauskas R. Kutznerides 1−4, Depsipeptides from the Actinomycete Kutzneria Sp. 744 Inhabiting Mycorrhizal Roots of Picea Abies Seedlings J. Nat. Prod. 2006 69 97 102 10.1021/np050378g
Gräfe U. Schlegel R. Ritzau M. Ihn W. Dornberger K. Stengel C. Fleck W.F. Gutsche W. Härtl A. Paulus E.F. Aurantimycins, New Depsipeptide Antibiotics from Streptomyces Aumntiacus IMET 43917 Production, Isolation, Structure Elucidation, and Biological Activity J. Antibiot. 1995 48 119 125 10.7164/antibiotics.48.119
Leet J.E. Schroeder D.R. Golik J. Matson J.A. Doyle T.W. Lam K.S. Hill S.E. Lee M.S. Whitney J.L. Krishnan B.S. Himastatin, a New Antitumor Antibiotic from Streptomyces Hygroscopicus. III. Structural Elucidation J. Antibiot. 1996 49 299 311 10.7164/antibiotics.49.299
Hasenböhler A. Kneifel H. König W.A. Zähner H. Zeiler H.J. [Metabolic products of microorganisms. 134. Stenothricin, a new inhibitor of the bacterial cell wall synthesis (author’s transl)] Arch. Microbiol. 1974 99 307 321 10.1007/BF00696245 4215397
Neumann C.S. Jiang W. Heemstra J.R. Jr. Gontang E.A. Kolter R. Walsh C.T. Biosynthesis of Piperazic Acid via N5-Hydroxy-Ornithine in Kutzneria Spp. 744 ChemBioChem 2012 13 972 976 10.1002/cbic.201200054
Du Y.-L. He H.-Y. Higgins M.A. Ryan K.S. A Heme-Dependent Enzyme Forms the Nitrogen–Nitrogen Bond in Piperazate Nat. Chem. Biol. 2017 13 836 838 10.1038/nchembio.2411
Morgan K.D. Andersen R.J. Ryan K.S. Piperazic Acid-Containing Natural Products: Structures and Biosynthesis Nat. Prod. Rep. 2019 36 1628 1653 10.1039/C8NP00076J 30949650
Wei Z.-W. Niikura H. Morgan K.D. Vacariu C.M. Andersen R.J. Ryan K.S. Free Piperazic Acid as a Precursor to Nonribosomal Peptides J. Am. Chem. Soc. 2022 144 13556 13564 10.1021/jacs.2c03660 35867963
Chevrette M.G. Aicheler F. Kohlbacher O. Currie C.R. Medema M.H. SANDPUMA: Ensemble Predictions of Nonribosomal Peptide Chemistry Reveal Biosynthetic Diversity across Actinobacteria Bioinformatics 2017 33 3202 3210 10.1093/bioinformatics/btx400 28633438
Du Y. Wang Y. Huang T. Tao M. Deng Z. Lin S. Identification and Characterization of the Biosynthetic Gene Cluster of Polyoxypeptin A, a Potent Apoptosis Inducer BMC Microbiol. 2014 14 30 10.1186/1471-2180-14-30
Pohlmann V. Marahiel M.A. δ-Amino Group Hydroxylation of L-Ornithine during Coelichelin Biosynthesis Org. Biomol. Chem. 2008 6 1843 1848 10.1039/b801016a
Wei H. Lin Z. Li D. Gu Q. Zhu T. [OSMAC (one strain many compounds) approach in the research of microbial metabolites—A review] Wei Sheng Wu Xue Bao 2010 50 701 709
Rigali S. Anderssen S. Naômé A. van Wezel G.P. Cracking the Regulatory Code of Biosynthetic Gene Clusters as a Strategy for Natural Product Discovery Biochem. Pharmacol. 2018 153 24 34 10.1016/j.bcp.2018.01.007 29309762
Rigali S. Titgemeyer F. Barends S. Mulder S. Thomae A.W. Hopwood D.A. van Wezel G.P. Feast or Famine: The Global Regulator DasR Links Nutrient Stress to Antibiotic Production by Streptomyces EMBO Rep. 2008 9 670 675 10.1038/embor.2008.83 18511939
Świątek M.A. Urem M. Tenconi E. Rigali S. van Wezel G.P. Engineering of N-Acetylglucosamine Metabolism for Improved Antibiotic Production in Streptomyces Coelicolor A3(2) and an Unsuspected Role of NagA in Glucosamine Metabolism Bioengineered 2012 3 280 285 10.4161/bioe.21371 22892576
Shimada N. Morimoto K. Naganawa H. Takita T. Hamada M. Maeda K. Takeuchi T. Umezawa H. ANTRIMYCIN, A NEW PEPTIDE ANTIBIOTIC J. Antibiot. (Tokyo) 1981 34 1613 1614 10.7164/antibiotics.34.1613
Jiang L. Huang P. Ren B. Song Z. Zhu G. He W. Zhang J. Oyeleye A. Dai H. Zhang L. et al. Antibacterial Polyene-Polyol Macrolides and Cyclic Peptides from the Marine-Derived Streptomyces Sp. MS110128 Appl. Microbiol. Biotechnol. 2021 105 4975 4986 10.1007/s00253-021-11226-w
Moumbock A.F.A. Gao M. Qaseem A. Li J. Kirchner P.A. Ndingkokhar B. Bekono B.D. Simoben C.V. Babiaka S.B. Malange Y.I. et al. StreptomeDB 3.0: An Updated Compendium of Streptomycetes Natural Products Nucleic. Acids Res. 2021 49 D600 D604 10.1093/nar/gkaa868
van Santen J.A. Poynton E.F. Iskakova D. McMann E. Alsup T.A. Clark T.N. Fergusson C.H. Fewer D.P. Hughes A.H. McCadden C.A. et al. The Natural Products Atlas 2.0: A Database of Microbially-Derived Natural Products Nucleic Acids Res. 2022 50 D1317 D1323 10.1093/nar/gkab941
Peoples A.S. Ling L.L. Lewis K. Zhang Z. Novel Antibiotics Patent US-2011136752-A1 PubChem Available online: https://pubchem.ncbi.nlm.nih.gov/patent/US-2011136752-A1 (accessed on 31 October 2022)
Tenconi E. Rigali S. Self-Resistance Mechanisms to DNA-Damaging Antitumor Antibiotics in Actinobacteria Curr. Opin. Microbiol. 2018 45 100 108 10.1016/j.mib.2018.03.003
Besier S. Ludwig A. Zander J. Brade V. Wichelhaus T.A. Linezolid Resistance in Staphylococcus Aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances Antimicrob. Agents Chemother. 2008 52 1570 1572 10.1128/AAC.01098-07
Tedim A.P. Lanza V.F. Rodríguez C.M. Freitas A.R. Novais C. Peixe L. Baquero F. Coque T.M. Fitness Cost of Vancomycin-Resistant Enterococcus Faecium Plasmids Associated with Hospital Infection Outbreaks J. Antimicrob. Chemother. 2021 76 2757 2764 10.1093/jac/dkab249
Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics John Innes Foundation Norwich, UK 2000
Blin K. Shaw S. Kloosterman A.M. Charlop-Powers Z. van Wezel G.P. Medema M.H. Weber T. AntiSMASH 6.0: Improving Cluster Detection and Comparison Capabilities Nucleic Acids Res. 2021 49 W29 W35 10.1093/nar/gkab335 33978755
Röttig M. Medema M.H. Blin K. Weber T. Rausch C. Kohlbacher O. NRPSpredictor2--a Web Server for Predicting NRPS Adenylation Domain Specificity Nucleic Acids Res. 2011 39 W362 W367 10.1093/nar/gkr323 21558170
Humphries R.M. Ambler J. Mitchell S.L. Castanheira M. Dingle T. Hindler J.A. Koeth L. Sei K. CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests PMC Available online: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869819/ (accessed on 27 November 2022)
European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Dieases (ESCMID) EUCAST Definitive Document E.Def 1.2, May 2000: Terminology Relating to Methods for the Determination of Susceptibility of Bacteria to Antimicrobial Agents Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 2000 6 503 508 10.1046/j.1469-0691.2000.00149.x
Gubbens J. Wu C. Zhu H. Filippov D.V. Florea B.I. Rigali S. Overkleeft H.S. van Wezel G.P. Intertwined Precursor Supply during Biosynthesis of the Catecholate-Hydroxamate Siderophores Qinichelins in Streptomyces Sp. MBT76 ACS Chem. Biol. 2017 12 2756 2766 10.1021/acschembio.7b00597 28945067
