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Insights Into the Lifestyle of Uncultured Bacterial Natural Product Factories

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Insights Into the Lifestyle of Uncultured Bacterial Natural Product Factories Insights into the lifestyle of uncultured bacterial PNAS PLUS natural product factories associated with marine sponges Gerald Lacknera,b, Eike Edzard Petersa, Eric J. N. Helfricha, and Jörn Piela,1 aInstitute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, 8093 Zurich, Switzerland; and bJunior Research Group Synthetic Microbiology, Friedrich Schiller University at the Leibniz Institute for Natural Product Research and Infection Biology–Hans Knöll Institute, 07745 Jena, Germany Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved December 7, 2016 (received for review September 29, 2016) The as-yet uncultured filamentous bacteria “Candidatus Entotheonella anticancer drug candidate discodermolide (9, 10), but their chemical factor” and “Candidatus Entotheonella gemina” live associated with role remains unclear (11). Despite repeated efforts (12, 13), the vast themarinespongeTheonella swinhoei Y, the source of numerous majority of T. swinhoei symbionts, including Ca. Entotheonella, re- unusual bioactive natural products. Belonging to the proposed candi- main uncultured to date, except for a report on the detection of date phylum “Tectomicrobia,” Candidatus Entotheonella members are Ca. Entotheonella in a mixed culture (7). There is some pros- only distantly related to any cultivated organism. The Ca.E.factorhas pect, however, of overcoming this challenge by genomics-based been identified as the source of almost all polyketide and modified targeted cultivation (14). peptides families reported from the sponge host, and both Ca. Ento- By metagenomic, single-particle genomic, and functional studies, we theonella phylotypes contain numerous additional genes for as-yet and collaborators recently provided evidence that almost all known unknown metabolites. Here, we provide insights into the biology of bioactive polyketides and modified peptides previously reported from these remarkable bacteria using genomic, (meta)proteomic, and chem- a Japanese chemotype of T. swinhoei (termed “chemotype Y”)are ical methods. The data suggest a metabolic model of Ca. Entotheonella produced by a single member of the complex microbiome named as facultative anaerobic, organotrophic organisms with the ability to “Candidatus Entotheonella factor” TSY1 (15–18). In this sponge, the use methanol as an energy source. The symbionts appear to be auxo- bacterium co-occurs with a second Ca. Entotheonella symbiont (15) MICROBIOLOGY trophic for some vitamins, but have the potential to produce most that, based on average nucleotide identity values, is a distinct candi- amino acids as well as rare cofactors like coenzyme F420.Thelatter date species and was termed “Candidatus Entotheonella gemina” likely accounts for the strong autofluorescence of Ca. Entotheonella TSY2 (21). Disentangling the metagenomic sequence data by binning filaments. A large expansion of proteinfamiliesinvolvedinregulation analysis revealed a striking number of natural product gene clusters in and conversion of organic molecules indicates roles in host–bacterial both phylotypes, but assigned all clusters for attributable T. swinhoei interaction. In addition, a massive overrepresentation of members of Y compounds (onnamides, polytheonamides, keramamides, pseudo- the luciferase-like monooxygenase superfamily points toward an im- theonamides, cyclotheonamides, and nazumamides) to Ca.E.factor portant role of these proteins in Ca. Entotheonella. Furthermore, we (15). In addition, multiple clusters for as-yet cryptic natural products performed mass spectrometric imaging combined with fluorescence in were identified in both phylotypes,suggestinganevenhigherbio- situ hybridization to localize Ca. Entotheonella and some of the bio- synthetic capacity. Both phylotypes remain as-yet uncultivated; are active natural products in the sponge tissue. These metabolic insights only distantly related to any cultivated bacterium; and, on the basis of into a new candidate phylum offer hints on the targeted cultivation of phylogenomic data, belong to a novel candidate phylum that was the chemically most prolific microorganisms known from microbial termed “Tectomicrobia” (15). dark matter. Recently, we presented evidence that another Ca. Entotheonella phylotype, “Candidatus Entotheonella serta,” present in the chemically uncultivated bacteria | natural products | genomics | proteomics | symbiosis Significance arine sponges are prolific sources of bioactive natural products The candidate genus “Candidatus Entotheonella” belongs to a Mand of great interest for drug development (1). Besides their recently proposed bacterial candidate phylum with largely un- pharmacological potential, sponges are among the oldest metazoans known properties due to the lack of cultivated members. Among – and have attracted attention as ancient models of animal bacterial the few known biological properties is an association of Ca. symbioses. Many sponges harbor highly abundant bacterial commu- Entotheonella with marine sponges and an extraordinarily rich nities that exhibit a similar biological complexity as the human genomic potential for bioactive natural products with unique microbiome (2, 3), but the ecological roles of these mostly un- structures and unprecedented biosynthetic enzymology. In- cultivated microbes remain largely elusive. One of the functions, for creasing evidence suggests that Ca. Entotheonella are wide- which evidence is accumulating, is the production of toxic natural spread key producers of sponge natural products with a chemical products that might contribute to host defense (4, 5). Significant ef- richness comparable to soil actinomycetes. Given the unusual forts have been made to connect the chemistry of sponges to possible biology and exceptional pharmacological potential of Ca.Ento- bacterial producers, largely motivated by the prospect of developing theonella, the bioinformatic and functional insights into their sustainable production systems for drug development. One of the lifestyle presented here provide diverse avenues for marine important sponge models that has emerged in these studies is The- natural product research, biotechnology, and microbial ecology. onella swinhoei, a chemically exceptionally rich complex of distinct chemotypes. Pioneering work on a variant from Palau revealed the Author contributions: J.P. designed research; G.L., E.E.P., and E.J.N.H. performed research; presence of filamentous, multicellular bacteria that could be G.L. and J.P. analyzed data; and G.L. and J.P. wrote the paper. mechanically enriched and contained elevated amounts of theopa- The authors declare no conflict of interest. lauamide-type antifungal peptides (6). The symbiont was assigned to This article is a PNAS Direct Submission. a new candidate genus and named “Candidatus Entotheonella pal- 1To whom correspondence should be addressed. Email: [email protected]. ” auensis (7). Related Candidatus Entotheonella bacteria were also This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. detected in the sponge Discodermia dissoluta (8), the source of the 1073/pnas.1616234114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1616234114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 distinct Japanese sponge T. swinhoei WA, is the producer of (at a false discovery rate of 1%). Considering the challenging the actin-binding polyketide misakinolide (19). Additional Ca. sample and large genome size of ca. 9Mb,thisproteomecoverage Entotheonella variants were also detected by PCR in a wide range of is respectable. The lower number of identifications for Ca.E. other sponge species (15) and were connected to natural product gemina is most likely due to the lower abundance of Ca.E.gemina biosynthesis in the sponge Discodermia calyx (20, 21). These data and in the sample as determined by genome sequencing (15). the previous Ca. Entotheonella studies mentioned above (6–8) suggest a more general relevance for the production of sponge- Central Carbon and Energy Metabolism. One important aspect of derived compounds. Biosynthetic pathways from Ca. Entotheonella genomic research on uncultured bacteria is the deduction of meta- exhibit a high frequency of unusual enzymatic features, and their bolic pathways that might deliver valuable information for targeted chemistry shows little similarity to the chemistry of cultivated cultivation experiments. Important clues for successful cultivation bacteria (17, 22–24). Being the first example of a chemically pro- are, for instance, potential energy sources, carbon sources, and tol- lific taxon among uncultivated bacteria, these producers offer ex- erance to oxygen. Concerning oxygen, the DNA-based data suggest citing opportunities for pharmaceutical applications, for systematic that Ca.E.factorandCa. E. gemina perform aerobic metabolism studies on the ecology of sponge-associated bacteria, and for in- and are likely facultative anaerobes. Both strains harbor genes vestigating functional properties of elusive candidate phyla. encoding a respiratory chain, including cytochrome c oxidase Since our first release of the Ca. Entotheonella genomes from (expressed) as well as catalase (expressed) and superoxide dismutase T. swinhoei Y (15), we have made several attempts to close gaps in (Dataset S1). In addition, anaerobic growth might be promoted, as the metagenome by rounds of PacBio and Illumina sequencing. suggested by putative fermentation pathways to D-lactate, to acetate, Unfortunately, these attempts did
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