Shipworms Play Critical Roles in Recycling Wood in the Sea
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bioRxiv preprint doi: https://doi.org/10.1101/826933; this version posted April 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Secondary metabolism in the gill microbiota of shipworms (Teredinidae) as revealed by 2 comparison of metagenomes and nearly complete symbiont genomes 3 4 Authors: Marvin A. Altamiaa,b*, Zhenjian Linc*, Amaro E. Trindade-Silvad,e, Iris Diana Uyb,f, J. 5 Reuben Shipwayg, Diego Veras Wilkee, Gisela P. Concepcionb,f, Daniel L. Distela, Eric W. 6 Schmidtc**, Margo G. Haygoodc** 7 8 9 aOcean Genome Legacy Center, Department of Marine and Environmental Science, 10 Northeastern University, Nahant, MA, USA 11 bThe Marine Science Institute, University of the Philippines Diliman, Quezon City 1101, 12 Philippines 13 cDepartment of Medicinal Chemistry, University of Utah 14 dBioinformatic and Microbial Ecology Laboratory - BIOME, Federal University of Bahia, Salvador, 15 Bahia, Brazil 16 eDrug Research and Development Center, Department of Physiology and Pharmacology, Federal 17 University of Ceara, 60430275, Ceara, Brazil 18 fPhilippine Genome Center, University of the Philippines Diliman, Quezon City 1101, Philippines 19 gInstitute of Marine Science, School of Biological Sciences, University of Portsmouth, UK 20 21 *authors contributed equally, author order was determined alphabetically 22 **co-corresponding authors 23 24 25 Abstract 26 Shipworms play critical roles in recycling wood in the sea. Symbiotic bacteria 27 supply enzymes that the organisms need for nutrition and wood degradation. 28 Some of these bacteria have been grown in pure culture and have the 29 capacity to make many secondary metabolites. However, little is known about 30 whether such secondary metabolite pathways are represented in the symbiont 31 communities within their hosts. In addition, little has been reported about the 32 patterns of host-symbiont co-occurrence. Here, we collected shipworms from 33 the United States, the Philippines, and Brazil, and cultivated symbiotic 34 bacteria from their gills. We analyzed sequences from 22 shipworm gill 35 metagenomes from seven shipworm species and from 23 cultivated symbiont 36 isolates. Using (meta)genome sequencing, we demonstrate that the cultivated 37 isolates represent all the major bacterial symbiont species and strains in 38 shipworm gills. We show that the bacterial symbionts are distributed among 39 shipworm hosts in consistent, predictable patterns. The symbiotic bacteria 40 encode many biosynthetic gene cluster families (GCFs) for bioactive 41 secondary metabolites, only <5% of which match previously described 42 biosynthetic pathways. Because we were able to cultivate the symbionts, and 1 bioRxiv preprint doi: https://doi.org/10.1101/826933; this version posted April 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 43 sequence their genomes, we can definitively enumerate the biosynthetic 44 pathways in these symbiont communities, showing that ~150 out of ~200 total 45 biosynthetic gene clusters (BGCs) present in the animal gill metagenomes are 46 represented in our culture collection. Shipworm symbionts occur in suites that 47 differ predictably across a wide taxonomic and geographic range of host 48 species, and collectively constitute an immense resource for the discovery of 49 new biosynthetic pathways to bioactive secondary metabolites. 50 51 52 Importance 53 We define a system in which the major symbionts that are important to host biology and to the 54 production of secondary metabolites can be cultivated. We show that symbiotic bacteria that 55 are critical to host nutrition and lifestyle also have an immense capacity to produce a multitude 56 of diverse and likely novel bioactive secondary metabolites that could lead to the discovery of 57 drugs, and that these pathways are found within shipworm gills. We propose that, by shaping 58 associated microbial communities within the host, the compounds support the ability of 59 shipworms to degrade wood in marine environments. Because these symbionts can be 60 cultivated and genetically manipulated, they provide a powerful model for understanding how 61 secondary metabolism impacts microbial symbiosis. 62 2 bioRxiv preprint doi: https://doi.org/10.1101/826933; this version posted April 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 63 Introduction 64 65 Shipworms (Family Teredinidae) are bivalve mollusks found throughout the world’s oceans (1, 66 2). Many shipworms eat wood, assisted by cellulases from intracellular symbiotic γ- 67 proteobacteria that inhabit their gills (Fig. 1) (3-6). Other shipworms use sulfide metabolism, 68 also relying on gill-dwelling γ-proteobacteria for sulfur oxidation (7). Shipworm gill symbionts of 69 several different species are thus essential to shipworm nutrition and survival. One of the most 70 remarkable features of the shipworm system is that wood digestion does not take place where 71 the bacteria are located, so that the bacterial cellulase products are transferred from the gill to 72 a nearly sterile cecum (8), where wood digestion occurs (Fig. 1) (9). This enables the host 73 shipworms to directly consume glucose and other sugars derived from wood lignocellulose and 74 hemicellulose, rather than the less energetic fermentation byproducts of cellulolytic gut 75 microbes as found in other symbioses. Shipworm symbionts are also essential for nitrogen 76 fixation that helps to offset the low nitrogen content of wood (10, 11). Thus, shipworms have 77 evolved structures and mechanisms enabling bacterial metabolism to support animal host 78 nutrition. 79 80 While in many nutritional symbioses the bacteria are difficult to cultivate, shipworm gill 81 symbiotic γ-proteobacteria have been brought into stable culture (5, 12, 13). This led to the 82 discovery that these bacteria are exceptional sources of secondary metabolites (14). Of bacteria 83 with sequenced genomes, the gill symbionts Teredinibacter turnerae T7901 and related strains 84 are among the richest sources of biosynthetic gene clusters (BGCs), comparable in content to 85 famous producers of commercial importance such as Streptomyces spp. (13-16). This implies 86 that shipworms might be a good source of new compounds for drug discovery. Of equal 87 importance, the symbiotic bacteria are crucial to survival of host shipworms, and bioactive 88 secondary metabolites might play a role in shaping those symbioses. 89 90 An early analysis of the turnerae T7901 genome revealed nine complex polyketide synthase 91 (PKS) and nonribosomal peptide synthetase (NRPS) BGCs (14). One of these was shown to 92 produce a novel catecholate siderophore, turnerbactin, which is crucial in obtaining iron and to 93 the survival of the symbiont in nature (17). A second BGC synthesizes the borated polyketide 94 tartrolons D/E, which are antibiotic and potently antiparasitic compounds (18). Both were 95 detected in the extracts of shipworms, implying a potential role in producing the remarkable 96 near sterility observed in the cecum (8). These data suggested specific roles for secondary 97 metabolism in shipworm ecology. 98 99 T. turnerae T7901 is just one of multiple strains and species of γ-proteobacteria living 100 intracellularly in shipworm gills (3, 12), and thus these analyses just begin to describe shipworm 101 secondary metabolism. Many shipworm species are generalists, consuming wood from a variety 102 of sources (1, 19). Other wood-eaters, such as Dicyathifer mannii, Bactronophorus thoracites, 103 and Neoteredo reynei, are specialists that live in the submerged branches, trunks and rhizomes 104 of mangroves (20, 21). There, they play an important role in ecological processes in mangrove 105 ecosystems, i.e. transferring large amount of carbon fixed by mangroves to the marine 106 environment (19). Several shipworm species, such as Kuphus polythalamius, live in other 3 bioRxiv preprint doi: https://doi.org/10.1101/826933; this version posted April 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 107 substrates. K. polythalamius often is found in sediment habitats (as well as in wood) where its 108 gill symbionts are crucial to sulfide oxidation and carry out carbon fixation (7). K. polythalamius 109 lacks significant amounts of cellulolytic symbionts such as T. turnerae, and instead contains 110 Thiosocius teredinicola, which oxidizes sulfide and generates energy for the host (22). Other 111 shipworms are found in solid rock and in seagrass (23, 24). Thus, gill symbionts vary, but in all 112 cases the symbionts appear to be essential to the survival of shipworms. 113 114 While the potential of T. turnerae as an unexplored producer of secondary metabolites has 115 been described (14, 16), the capacity of other shipworm symbionts is still largely unknown. 116 Moreover, several