Abstract
Caves are considered to be extreme and challenging environments. It is believed that the ability of microorganisms to produce secondary metabolites enhances their survivability and adaptiveness in the energy-starved cave environment. Unfortunately, information on the genetic potential for the production of secondary metabolites, such as polyketides and nonribosomal peptides, is limited. In the present study, we aimed to identify and characterize genes responsible for the production of secondary metabolites in the microbial community of one of the deepest caves in the world, Krubera-Voronja Cave (43.4184 N 40.3083 E, Western Caucasus). The analysed sample materials included sediments, drinkable water from underground camps, soil and clay from the cave walls, speleothems and coloured spots from the cave walls. The type II polyketide synthases (PKSs) ketosynthases α and β and the adenylation domains of nonribosomal peptide synthetases (NRPSs) were investigated using a metagenomic approach. Taxonomic diversity analysis showed that most PKS sequences could be attributed to Actinobacteria followed by unclassified bacteria and Acidobacteria, while the NRPS sequences were more taxonomically diverse and could be assigned to Proteobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Chloroflexi, etc. Only three putative metabolites could be predicted: an angucycline group polyketide, a massetolide A-like cyclic lipopeptide and a surfactin-like lipopeptide. The absolute majority of PKS and NRPS sequences showed low similarity with the sequences of the reference biosynthetic pathways, suggesting that these sequences could be involved in the production of novel secondary metabolites.
Similar content being viewed by others
References
Ghosh S, Kuisiene N, Cheeptham N (2017) The cave microbiome as a source for drug discovery: reality or pipe dream? Biochem. Pharmacol. 134:18–34. https://doi.org/10.1016/j.bcp.2016.11.018
Adam D, Maciejewska M, Naȏme A, Martinet L, Coppieters W, Karim L, Baurain D, Rigali S (2018) Isolation, characterization, and antibacterial activity of hard-to-culture actinobacteria from cave moonmilk deposits. Antibiotics 7:28. https://doi.org/10.3390/antibiotics7020028
Belyagoubi L, Belyagoubi-Benhammou N, Jurado V, Dupont J, Lacoste S, Djebbah F, Ounadjela FZ, Benaissa S, Habi S, Abdelouahid DE, Saiz-Jimenez C (2018) Antimicrobial activities of culturable microorganisms (actinomycetes and fungi) isolated from Chaabe Cave, Algeria. Int. J. Speleol. 47:189–199. https://doi.org/10.5038/1827-806X.47.2.2148
Duo JL, Cha QY, Zhou XK, Zhang TK, Qin SC, Yang PX, Zhu ML, Mo MH, Duan YQ (2019) Aquabacter cavernae sp. nov., a bacterium isolated from cave soil. Int. J. Syst. Evol. Microbiol. 69:3716–3722. https://doi.org/10.1099/ijsem.0.003585
Wiseschart A, Mhuanthong W, Tangphatsornruang S, Chantasingh D, Pootanakit K (2019) Shotgun metagenomic sequencing from Manao-Pee cave, Thailand, reveals insight into the microbial community structure and its metabolic potential. BMC Microbiol. 19:144. https://doi.org/10.1186/s12866-019-1521-8
Fang BZ, Han MX, Jiao JY, Xie YG, Zhang XT, Liu L, Zhang ZT, Xiao M, Li WJ (2020) Streptomyces cavernae sp. nov., a novel actinobacterium isolated from a karst cave sediment sample. Int. J. Syst. Evol. Microbiol. 70:120–125. https://doi.org/10.1099/ijsem.0.003724
Narsing Rao MP, Dong ZY, Kan Y, Zhang K, Fang BZ, Xiao M, Kang YQ, Li WJ (2020) Description of Paenibacillus antri sp. nov. and Paenibacillus mesophilus sp. nov., isolated from cave soil. Int. J. Syst. Evol. Microbiol. 70:1048–1054. https://doi.org/10.1099/ijsem.0.003870
Zhou XK, Huang Y, Li M, Zhang XF, Wei YQ, Qin SC, Zhang TK, Wang XJ, Liu JJ, Wang L, Liu ZY, Mo MH (2020) Asticcacaulis tiandongensis sp. nov., a new member of the genus Asticcacaulis, isolated from a cave soil sample. Int. J. Syst. Evol. Microbiol. 70:687–692. https://doi.org/10.1099/ijsem.0.003818
Avguštin JA, Petrič P, Pašić L (2019) Screening the cultivable cave microbial mats for the production of antimicrobial compounds and antibiotic resistance. Int. J. Speleol. 48:295–303. https://doi.org/10.5038/1827-806X.48.3.2272
De Mandal S, Chatterjee R, Kumar NS (2017) Dominant bacterial phyla in caves and their predicted functional roles in C and N cycle. BMC Microbiol. 17:90. https://doi.org/10.1186/s12866-017-1002-x
D’Auria G, Artacho A, Rojas RA, Bautista JS, Méndez R, Gamboa MT, Gamboa JR, Gómez-Cruz R (2018) Metagenomics of bacterial diversity in Villa Luz caves with sulfur water springs. Genes 9:55. https://doi.org/10.3390/genes9010055
Busquets A, Fornós JJ, Zafra F, Lalucat J, Merino A (2014) Microbial communities in a coastal cave: Cova des Pas de Vallgornera (Mallorca, Western Mediterranean). Int. J. Speleol. 43:205–216. https://doi.org/10.5038/1827-806X.43.2.8
Kumaresan D, Wischer D, Stephenson J, Hillebrand-Voiculescu A, Murrell JC (2014) Microbiology of Movile Cave - a chemolithoautotrophic ecosystem. Geomicrobiol J. 31:186–193. https://doi.org/10.1080/01490451.2013.839764
Ortiz M, Legatzki A, Neilson JW, Fryslie B, Nelson WM, Wing RA, Soderlund CA, Pryor BM, Maier RM (2014) Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave. ISME J 8:478–491. https://doi.org/10.1038/ismej.2013.159
Zepeda Mendoza ML, Lundberg J, Ivarsson M, Campos P, Nylander JAA, Sallstedt T, Dalen L (2016) Metagenomic analysis from the interior of a speleothem in Tjuv-Ante’s Cave, Northern Sweden. PLoS One 11:e0151577. https://doi.org/10.1371/journal.pone.0151577
Waring CL, Hankin SI, Griffith DWT, Kertesz MA, Kobylski V, Wilson NL, Coleman NV, Kettlewell G, Zlot R, Bosse M, Bell G (2017) Seasonal total methane depletion in limestone caves. Sci. Rep. 7:8314. https://doi.org/10.1038/s41598-017-07769-6
Montano ET, Henderson LO (2013) Studies of antibiotic production by cave bacteria. In: Cheeptham N (ed) Cave microbiomes: a novel resource for drug discovery. SpringerBriefs in Microbiology, vol 1. Springer, New York, pp 109–130. https://doi.org/10.1007/978-1-4614-5206-5_6
Bauer MA, Kainz K, Carmona-Gutierrez D, Madeo F (2018) Microbial wars: competition in ecological niches and within the microbiome. Microb Cell 5:215–219. https://doi.org/10.15698/mic2018.05.628
Sengupta S, Chattopadhyay MK, Grossart HP (2013) The multifaceted roles of antibiotics and antibiotic resistance in nature. Front. Microbiol. 4:47. https://doi.org/10.3389/fmicb.2013.00047
Stubbendieck RM, Straight PD (2016) Multifaceted interfaces of bacterial competition. J. Bacteriol. 98:2145–2155. https://doi.org/10.1128/JB.00275-16
Chevrette MG, Currie CR (2019) Emerging evolutionary paradigms in antibiotic discovery. J. Ind. Microbiol. Biotechnol. 46:257–271. https://doi.org/10.1007/s10295-018-2085-6
Jiang ZK, Guo L, Chen C, Liu SW, Zhang L, Dai SJ, He QY, You XF, Hu XX, Tuo L, Jiang W, Sun CH (2015) Xiakemycin A, a novel pyranonaphthoquinone antibiotic, produced by the Streptomyces sp. CC8-201 from the soil of a karst cave. J Antibiot. 68:771–774. https://doi.org/10.1038/ja.2015.70
Axenov-Gribanov DV, Voytsekhovskaya IV, Tokovenko BT, Protasov ES, Gamaiunov SV, Rebets YV, Luzhetskyy AN, Timofeyev MA (2016) 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 11:e0149216. https://doi.org/10.1371/journal.pone.0152957
Ghosh S, Kam G, Nijjer M, Stenner C, Cheeptham N (2020) Culture dependent analysis of bacterial diversity in Canada’s Raspberry Rising Cave revealed antimicrobial properties. Int. J. Speleol. 49:43–53. https://doi.org/10.5038/1827-806X.49.1.2291
Bhullar K, Waglechner N, Pawlowski A, Koteva K, Banks ED, Johnston MD, Barton HA, Wright GD (2012) Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS One 7:e34953. https://doi.org/10.1371/journal.pone.0034953
Gaálová B, Donauerová A, Seman M, Bujdáková H (2014) Identification and β-lactam resistance in aquatic isolates of Enterobacter cloacae and their status in microbiota of Domica Cave in Slovak Karst (Slovakia). Int. J. Speleol. 43:69–77. https://doi.org/10.5038/1827-806X.43.1.7
Pawlowski AC, Wang W, Koteva K, Barton HA, McArthur AG, Wright GD (2016) A diverse intrinsic antibiotic resistome from a cave bacterium. Nat. Commun. 7:13803. https://doi.org/10.1038/ncomms13803
Lavoie K, Ruhumbika T, Bawa A, Whitney A, de Ondarza J (2017) High levels of antibiotic resistance but no antibiotic production detected along a gypsum gradient in Great Onyx Cave, KY, USA. Diversity 9:42. https://doi.org/10.3390/d9040042
Miller IJ, Chevrette MG, Kwan JC (2017) Interpreting microbial biosynthesis in the genomic age: biological and practical considerations. Mar Drugs 15:165. https://doi.org/10.3390/md15060165
Libis V, Antonovsky N, Zhang M, Shang Z, Montiel D, Maniko J, Ternei MA, Calle PY, Lemetre C, Owen JG, Brady SF (2019) Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences. Nat. Commun. 10:3848. https://doi.org/10.1038/s41467-019-11658-z
Maciejewska M, Adam D, Martinet L, Naômé A, Całusińska M, Delfosse P, Carnol M, Barton HA, Hayette MP, Smargiasso N, De Pauw E, Hanikenne M, Baurain D, Rigali S (2016) A phenotypic and genotypic analysis of the antimicrobial potential of cultivable Streptomyces isolated from cave moonmilk deposits. Front. Microbiol. 7:1455. https://doi.org/10.3389/fmicb.2016.01455
Bukelskis D, Dabkeviciene D, Lukoseviciute L, Bucelis A, Kriaučiūnas I, Lebedeva J, Kuisiene N (2019) Screening and transcriptional analysis of polyketide synthases and non-ribosomal peptide synthetases in bacterial strains from Krubera-Voronja cave. Front. Microbiol. 10:2149. https://doi.org/10.3389/fmicb.2019.02149
Gosse JT, Ghosh S, Sproule A, Overy D, Cheeptham N, Boddy CN (2019) Whole genome sequencing and metabolomic study of cave Streptomyces isolates ICC1 and ICC4. Front. Microbiol. 10:1020. https://doi.org/10.3389/fmicb.2019.01020
Wiseschart A, Mhuanthong W, Thongkam P, Tangphatsornruang S, Chantasingh D, Pootanakit K (2018) Bacterial diversity and phylogenetic analysis of type II polyketide synthase gene from Manao-Pee Cave, Thailand. Geomicrobiol J. 35:518–527. https://doi.org/10.1080/01490451.2017.1411993
Riquelme C, Enes Dapkevicius ML, Miller AZ, Charlop-Powers Z, Brady S, Mason C, Cheeptham N (2017) Biotechnological potential of Actinobacteria from Canadian and Azorean volcanic caves. Appl. Microbiol. Biotechnol. 101:843–857. https://doi.org/10.1007/s00253-016-7932-7
Cuadrat RRC, Ionescu D, Dávila AMR, Grossart H-P (2018) Recovering genomics clusters of secondary metabolites from lakes using genome-resolved metagenomics. Front. Microbiol. 9:251. https://doi.org/10.3389/fmicb.2018.00251
Klusaite A, Vickackaite V, Vaitkeviciene B, Karnickaite R, Bukelskis D, Kieraite-Aleksandrova I, Kuisiene N (2016) Characterization of antimicrobial activity of culturable bacteria isolated from Krubera-Voronja Cave. Int. J. Speleol. 45:275–287. https://doi.org/10.5038/1827-806X.45.3.1978
Chevrette MG, Handelsman J (2020) From metagenomes to molecules: innovations in functional metagenomics unlock hidden chemistry in the human microbiome. Biochemistry 59:729–730. https://doi.org/10.1021/acs.biochem.0c00033
Kieraite-Aleksandrova I, Aleksandrovas V, Kuisiene N (2015) Down into the earth: microbial diversity of the deepest cave of the world. Biologia 70:989–1002. https://doi.org/10.1515/biolog-2015-0127
Studholme DJ, Jackson RA, Leak DJ (1999) Phylogenetic analysis of transformable strains of thermophilic Bacillus species. FEMS Microbiol. Lett. 172:85–90. https://doi.org/10.1111/j.1574-6968.1999.tb13454.x
Kuisiene N, Jomantiene R, Valiunas D, Chitavichius D (2002) Characterization of thermophilic proteolytic spore-forming bacteria from a geothermal site in Lithuania based on 16S rDNA RFLP and ITS-PCR analyses. Microbiology 71:712–716. https://doi.org/10.1023/A:1021440208887
Tambadou F, Lanneluc I, Sablé S, Klein GL, Doghri I, Sopéna V, Didelot S, Barthélémy C, Thiéry V, Chevrot R (2014) Novel nonribosomal peptide synthetase (NRPS) genes sequenced from intertidal mudflat bacteria. FEMS Microbiol. Lett. 357:123–130. https://doi.org/10.1111/1574-6968.12532
Ayuso-Sacido A, Genilloud O (2005) New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: detection and distribution of these biosynthetic gene sequences in major taxonomic groups. Microb. Ecol. 49:10–24. https://doi.org/10.1007/s00248-004-0249-6
Wood SA, Kirby BM, Goodwin CM, Le Roes M, Meyers PR (2007) PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16. J. Appl. Microbiol. 102:245–253. https://doi.org/10.1111/j.1365-2672.2006.03043.x
Metsä-Ketelä M, Salo V, Halo L, Hautala A, Hakala J, Mäntsälä P, Ylihonko K (1999) An efficient approach for screening minimal PKS genes from Streptomyces. FEMS Microbiol. Lett. 180:1–6. https://doi.org/10.1111/j.1574-6968.1999.tb08770.x
Jenke-Kodama H, Dittmann E (2009) Evolution of metabolic diversity: insights from microbial polyketide synthases. Phytochemistry 70:1858–1866. https://doi.org/10.1016/j.phytochem.2009.05.021
Chevrette MG, Gutiérrez-García K, Selem-Mojica N, Aguilar-Martínez C, Yañez-Olvera A, Ramos-Aboites HE, Hoskisson PA, Barona-Gómez F (2020) Evolutionary dynamics of natural product biosynthesis in bacteria. Nat. Prod. Rep. 37:566–599. https://doi.org/10.1039/c9np00048h
Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/bty560
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199
Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200. https://doi.org/10.1093/bioinformatics/btr381
Kautsar SA, Blin K, Shaw S, Navarro-Muñoz JC, Terlouw BR, van der Hooft JJJ, van Santen JA, Tracanna V, Suarez Duran HG, Pascal Andreu V, Selem-Mojica N, Alanjary M, Robinson SL, Lund G, Epstein SC, Sisto AC, Charkoudian LK, Collemare J, Linington RG, Weber T, Medema MH (2020) MIBiG 2.0: a repository for biosynthetic gene clusters of known function. Nucleic Acids Res. 48:D454–D458. https://doi.org/10.1093/nar/gkz882
Conway KR, Boddy CN (2013) ClusterMine360: a database of microbial PKS/NRPS biosynthesis. Nucleic Acids Res. 41:D402–D407. https://doi.org/10.1093/nar/gks993
Meyer F, Paarmann D, D’Souza M, Olson R, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J, Edwards RA (2008) The metagenomics RAST sever – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9:386. https://doi.org/10.1186/1471-2105-9-386
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Letunic I, Bork P (2019) Interactive Tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47:W256–W259. https://doi.org/10.1093/nar/gkz239
Ziemert N, Podell S, Penn K, Badger JH, Allen E, Jensen PR (2012) The natural product domain seeker NaPDoS: a phylogeny based bioinformatics tool to classify secondary metabolite gene diversity. PLoS One 7:e34064. https://doi.org/10.1371/journal.pone.0034064
Prieto C, García-Estrada C, Lorenzana D, Martín JF (2012) NRPSsp: non-ribosomal peptide synthase substrate predictor. Bioinformatics 28:426–427. https://doi.org/10.1093/bioinformatics/btr659
Della Sala G, Hochmuth T, Teta R, Costantino V, Mangoni A (2014) Polyketide synthases in the microbiome of the marine sponge Plakortis halichondrioides: a metagenomic update. Mar Drugs 12:5425–5440. https://doi.org/10.3390/md12115425
Müller CA, Oberauner-Wappis L, Peyman A, Amos GCA, Wellington EMH, Berg G (2015) Mining for nonribosomal peptide synthetase and polyketide synthase genes revealed a high level of diversity in the Sphagnum bog metagenome. Appl. Environ. Microbiol. 81:5064–5072. https://doi.org/10.1128/AEM.00631-15
Wei Y, Zhang L, Zhou Z, Yan X (2018) Diversity of gene clusters for polyketide and nonribosomal peptide biosynthesis revealed by metagenomic analysis of the Yellow Sea sediment. Front. Microbiol. 9:295. https://doi.org/10.3389/fmicb.2018.00295
Hershey OS, Barton HA (2018) The microbial diversity of caves. In: Moldovan OT, Kováč L, Halse S (eds). Springer International Publishing, Cave ecology, pp 69–90. https://doi.org/10.1007/978-3-319-98852-8
Oliveira C, Gunderman L, Coles CA, Lochmann J, Parks M, Ballard E, Glazko G, Rahmatallah Y, Tackett AJ, Thomas DJ (2017) 16S rRNA gene-based metagenomic analysis of Ozark Cave bacteria. Diversity (Basel) 9:31. https://doi.org/10.3390/d9030031
Zhu H-Z, Zhang Z-F, Zhou N, Jiang C-Y, Wang B-J, Cai L, Liu S-J (2019) Diversity, distribution and co-occurrence patterns of bacterial communities in a karst cave system. Front. Microbiol. 10:1726. https://doi.org/10.3389/fmicb.2019.01726
Ziemert N, Jensen PR (2012) Phylogenetic approaches to natural product structure prediction. Methods Enzymol. 517:161–182. https://doi.org/10.1016/b978-0-12-404634-4.00008-5
Gui C, Liu Y, Zhou Z, Zhang S, Hu Y, Gu YC, Huang H, Ju J (2018) Angucycline glycosides from mangrove-derived Streptomyces diastaticus subsp. SCSIO GJ056. Mar Drugs 16:185. https://doi.org/10.3390/md16060185
Crits-Christoph A, Diamond S, Butterfield CN, Thomas BC, Banfield JF (2018) Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis. Nature 558:440–444. https://doi.org/10.1038/s41586-018-0207-y
Kielak AM, Barreto CC, Kowalchuk GA, van Veen JA, Kuramae EE (2016) The ecology of Acidobacteria: moving beyond genes and genomes. Front. Microbiol. 7:744. https://doi.org/10.3389/fmicb.2016.00744
Radjasa OK, Wiese J, Sabdono A, Imhoff JF (2008) Corals as source of bacteria with antimicrobial activity. J Coast Dev 11:121–130
McErlean M, Overbay J, Van Lanen S (2019) Refining and expanding nonribosomal peptide synthetase function and mechanism. J. Ind. Microbiol. Biotechnol. 46:493–513. https://doi.org/10.1007/s10295-018-02130-w
Khayatt BI, Overmars L, Siezen R, Francke C (2013) Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific Hidden Markov Models. PLoS One 8:e62136. https://doi.org/10.1371/journal.pone.0062136
Flissi A, Ricart E, Campart C, Chevalier M, Dufresne Y, Michalik J, Jacques P, Flahaut C, Lisacek F, Leclère V, Pupin M (2020) Norine: update of the nonribosomal peptide resource. Nucleic Acids Res. 48:D465–D469. https://doi.org/10.1093/nar/gkz1000
Hou J, Robbel L, Marahiel MA (2011) Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture. Chem. Biol. 18:655–664. https://doi.org/10.1016/j.chembiol.2011.02.012
Ringel MT, Brüser T (2018) The biosynthesis of pyoverdines. Microb Cell 5:424–437. https://doi.org/10.15698/mic2018.10.649
Sendra A, Reboleira ASPS (2012) The world’s deepest subterranean community Krubera-Voronja Cave (Western Caucasus). Int. J. Speleol. 41:221–230. https://doi.org/10.5038/1827-806X.41.2.9
Funding
This work was supported by the Research Council of Lithuania (grant no. S-MIP-17-21).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Lukoseviciute, L., Lebedeva, J. & Kuisiene, N. Diversity of Polyketide Synthases and Nonribosomal Peptide Synthetases Revealed Through Metagenomic Analysis of a Deep Oligotrophic Cave. Microb Ecol 81, 110–121 (2021). https://doi.org/10.1007/s00248-020-01554-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00248-020-01554-1