Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring β-hydroxyaspartate (β-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of β-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe3+ to Fe2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread β-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, β-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Biology Centre of the CAS Institute of Hydrobiology České Budějovice Czech Republic

Centre Algatech Institute of Microbiology The Czech Academy of Sciences Třeboň Czech Republic

Dipartimento di Farmacia Università degli Studi di Napoli Federico 2 Naples Italy

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Innovative Genomics Institute University of California Berkeley California USA

Institute of Botany of the Czech Academy of Sciences Třeboň Czech Republic

Zobrazit více v PubMed

Sandy M, Butler A. 2009. Microbial iron acquisition: marine and terrestrial siderophores. Chem Rev 109:4580–4595. 10.1021/cr9002787. PubMed DOI PMC

Årstøl E, Hohmann-Marriott MF. 2019. Cyanobacterial siderophores—physiology, structure, biosynthesis, and applications. Mar Drugs 17:281. 10.3390/md17050281. PubMed DOI PMC

Morrissey J, Bowler C. 2012. Iron utilization in marine cyanobacteria and eukaryotic algae. Front Microbiol 3:43. 10.3389/fmicb.2012.00043. PubMed DOI PMC

Shih P, Wu D, Latifi A, Axen S, Fewer D, Talla E, Calteau A, Cai F, de Marsac N, Rippka R, Herdman M, Sivonen K, Coursin T, Laurent T, Goodwin L, Nolan M, Davenport K, Han C, Rubin E, Eisen J, Woyke T, Gugger M, Kerfeld C. 2013. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci U S A 110:1053–1058. 10.1073/pnas.1217107110. PubMed DOI PMC

Gross H. 2007. Strategies to unravel the function of orphan biosynthesis pathways: recent examples and future prospects. Appl Microbiol Biotechnol 75:267–277. 10.1007/s00253-007-0900-5. PubMed DOI

Butler A, Theisen RM. 2010. Iron(III)-siderophore coordination chemistry: reactivity of marine siderophores. Coord Chem Rev 254:288–296. 10.1016/j.ccr.2009.09.010. PubMed DOI PMC

Kreutzer MF, Kage H, Nett M. 2012. Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16. J Am Chem Soc 134:5415–5422. 10.1021/ja300620z. PubMed DOI

Rosconi F, Davyt D, Martínez V, Martínez M, Abin-Carriquiry JA, Zane H, Butler A, de Souza EM, Fabiano E. 2013. Identification and structural characterization of serobactins, a suite of lipopeptide siderophores produced by the grass endophyte Herbaspirillum seropedicae. Environ Microbiol 15:916–927. 10.1111/1462-2920.12075. PubMed DOI

Hardy CD, Butler A. 2019. Ambiguity of NRPS structure predictions: four bidentate chelating groups in the siderophore pacifibactin. J Nat Prod 82:990–997. 10.1021/acs.jnatprod.8b01073. PubMed DOI

Singh GM, Fortin PD, Koglin A, Walsh CT. 2008. beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae. Biochemistry 47:11310–11320. 10.1021/bi801322z. PubMed DOI PMC

Boiteau RM, Mende DR, Hawco NJ, McIlvin MR, Fitzsimmons JN, Saito MA, Sedwick PN, DeLong EF, Repeta DJ. 2016. Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean. Proc Natl Acad Sci U S A 113:14237–14242. 10.1073/pnas.1608594113. PubMed DOI PMC

Mareš J, Hájek J, Urajová P, Kust A, Jokela J, Saurav K, Galica T, Čapková K, Mattila A, Haapaniemi E, Permi P, Mysterud I, Skulberg OM, Karlsen J, Fewer DP, Sivonen K, Tønnesen HH, Hrouzek P. 2019. Alternative biosynthetic starter units enhance the structural diversity of cyanobacterial lipopeptides. Appl Environ Microbiol 85:e02675-18. 10.1128/AEM.02675-18. PubMed DOI PMC

Galica T, Hrouzek P, Mareš J. 2017. Genome mining reveals high incidence of putative lipopeptide biosynthesis NRPS/PKS clusters containing fatty acyl-AMP ligase genes in biofilm-forming cyanobacteria. J Phycol 53:985–998. 10.1111/jpy.12555. PubMed DOI

Manavalan B, Murugapiran SK, Lee G, Choi S. 2010. Molecular modeling of the reductase domain to elucidate the reaction mechanism of reduction of peptidyl thioester into its corresponding alcohol in non-ribosomal peptide synthetases. BMC Struct Biol 10:1. 10.1186/1472-6807-10-1. PubMed DOI PMC

González A, Angarica VE, Sancho J, Fillat MF. 2014. The FurA regulon in Anabaena sp. PCC 7120: in silico prediction and experimental validation of novel target genes. Nucleic Acids Res 42:4833–4846. 10.1093/nar/gku123. PubMed DOI PMC

Carroll CS, Moore MM. 2018. Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases. Crit Rev Biochem Mol Biol 53:356–381. 10.1080/10409238.2018.1476449. PubMed DOI

Reid RT, Live DH, Faulkner DJ, Butler A. 1993. A siderophore from a marine bacterium with an exceptional ferric ion affinity constant. Nature 366:455–458. 10.1038/366455a0. PubMed DOI

Sritharan M. 2016. Iron homeostasis in Mycobacterium tuberculosis: mechanistic insights into siderophore-mediated iron uptake. J Bacteriol 198:2399–2409. 10.1128/JB.00359-16. PubMed DOI PMC

Kem MP, Butler A. 2015. Acyl peptidic siderophores: structures, biosyntheses and post-assembly modifications. Biometals 28:445–459. 10.1007/s10534-015-9827-y. PubMed DOI

Barbeau K, Rue EL, Bruland KW, Butler A. 2001. Photochemical cycling of iron in the surface ocean mediated by microbial iron(III)-binding ligands. Nature 413:409–413. 10.1038/35096545. PubMed DOI

Medema MH, Kottmann R, Yilmaz P, Cummings M, Biggins JB, Blin K, de Bruijn I, Chooi YH, Claesen J, Coates RC, Cruz-Morales P, Duddela S, Düsterhus S, Edwards DJ, Fewer DP, Garg N, Geiger C, Gomez-Escribano JP, Greule A, Hadjithomas M, Haines AS, Helfrich EJN, Hillwig ML, Ishida K, Jones AC, Jones CS, Jungmann K, Kegler C, Kim HU, Kötter P, Krug D, Masschelein J, Melnik AV, Mantovani SM, Monroe EA, Moore M, Moss N, Nützmann H-W, Pan G, Pati A, Petras D, Reen FJ, Rosconi F, Rui Z, Tian Z, Tobias NJ, Tsunematsu Y, Wiemann P, Wyckoff E, Yan X, et al.. 2015. Minimum information about a biosynthetic gene cluster. Nat Chem Biol 11:625–631. 10.1038/nchembio.1890. PubMed DOI PMC

Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, Medema MH, Weber T. 2019. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res 47:W81–W87. 10.1093/nar/gkz310. PubMed DOI PMC

Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH. 2015. CDD: NCBI's conserved domain database. Nucleic Acids Res 43:D222–D226. 10.1093/nar/gku1221. PubMed DOI PMC

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. 10.1093/molbev/mst010. PubMed DOI PMC

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. 10.1093/nar/25.17.3389. PubMed DOI PMC

Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil P-A, Hugenholtz P. 2018. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol 36:996–1004. 10.1038/nbt.4229. PubMed DOI

Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2020. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics 36:1925–1927. 10.1093/bioinformatics/btz848. PubMed DOI PMC

Price MN, Dehal PS, Arkin AP. 2010. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490. 10.1371/journal.pone.0009490. PubMed DOI PMC