Secondary Metabolism in the Gill Microbiota of Shipworms (Teredinidae) as Revealed by Comparison of Metagenomes and Nearly Complete Symbiont Genomes

Abstract

Shipworms play critical roles in recycling wood in the sea. Symbiotic bacteria supply enzymes that the organisms need for nutrition and wood degradation. Some of these bacteria have been grown in pure culture and have the capacity to make many secondary metabolites. However, little is known about whether such secondary metabolite pathways are represented in the symbiont communities within their hosts. In addition, little has been reported about the patterns of host-symbiont co-occurrence. Here, we collected shipworms from the United States, the Philippines, and Brazil and cultivated symbiotic bacteria from their gills. We analyzed sequences from 22 shipworm gill metagenomes from seven shipworm species and from 23 cultivated symbiont isolates. Using (meta)genome sequencing, we demonstrate that the cultivated isolates represent all the major bacterial symbiont species and strains in shipworm gills. We show that the bacterial symbionts are distributed among shipworm hosts in consistent, predictable patterns. The symbiotic bacteria harbor many gene cluster families (GCFs) for biosynthesis of bioactive secondary metabolites, only <5% of which match previously described biosynthetic pathways. Because we were able to cultivate the symbionts and to sequence their genomes, we can definitively enumerate the biosynthetic pathways in these symbiont communities, showing that ∼150 of ∼200 total biosynthetic gene clusters (BGCs) present in the animal gill metagenomes are represented in our culture collection. Shipworm symbionts occur in suites that differ predictably across a wide taxonomic and geographic range of host species and collectively constitute an immense resource for the discovery of new biosynthetic pathways corresponding to bioactive secondary metabolites.IMPORTANCE We define a system in which the major symbionts that are important to host biology and to the production of secondary metabolites can be cultivated. We show that symbiotic bacteria that are critical to host nutrition and lifestyle also have an immense capacity to produce a multitude of diverse and likely novel bioactive secondary metabolites that could lead to the discovery of drugs and that these pathways are found within shipworm gills. We propose that, by shaping associated microbial communities within the host, the compounds support the ability of shipworms to degrade wood in marine environments. Because these symbionts can be cultivated and genetically manipulated, they provide a powerful model for understanding how secondary metabolism impacts microbial symbiosis.

Keywords: biosynthesis; metagenomics.

FIG 1

(Top) Diagram of generic shipworm anatomy. Insets are from Fig. 2, panels B and D, in Betcher et al. (8). Bars, 20 μm. Red, signal from a fluorescent universal bacterial probe, indicating large numbers of bacterial symbionts in the bacteriocytes of the gill and paucity of bacteria in the cecum; green, background fluorescence. (Bottom) Collection locations of specimens included in this study. See Table S1 for details.

FIG 2

Cultivated bacterial isolates represent the major shipworm gill symbionts. (A) Isolated bacteria analyzed in this study are shown in abstracted schematic of a 16S rRNA phylogenetic tree. The complete tree with accurate branch lengths and bootstrap numbers is shown in Fig. S1. T. turnerae comprised 11 sequenced strains; for other groups, individual strains are shown. Each color indicates different bacteria appearing in the metagenomes in panel B. (B) Species composition of shipworm gill symbiont community based on shotgun metagenome sequence analysis. The y-axis data indicate the percentages of reads mapping to each bacterial species, while the x-axis data indicate the individual shipworm specimens used in the study. Colors indicate the origin of bacterial reads; gray represents minor, sporadic, unidentified strains.

FIG 3

Heat map of relationships between symbiont isolate genomes and gill metagenome bins. The scale bar is shaded according to identity on the basis of calculated values (AF × gANI). Color bars in the phylogenetic tree indicate bacterial species identity, either in the metagenomes or in the genome, and they are identical to the codes shown in Fig. 2. This figure indicates the high degree of certainty that the cultivated isolates are the same species as the major bacteria present in the gill.

FIG 4

Most BGCs found in the metagenomes and in the bacterial isolate genomes are shared. A total of 401 BGCs from metagenome sequences were compared to the bacterial isolate genomes, 305 of which were found in isolates. Conversely, 148 of 168 BGCs from sequenced bacterial isolates were found in the metagenomes. The shared numbers likely differ because the contigs assembled from the metagenome sequences were shorter on average such that several metagenome fragments can map to a single BGC in an isolate.

FIG 5

GCFs found in (A) bacterial genomes and (B) gill metagenomes. (A) A list of strains of cultivated bacterial genomes is provided in the x axis, while the number of total GCFs in different sequenced strains is shown in the y axis. Colors indicate bacteria as described for Fig. 2A. Because there were 11 isolates of T. turnerae, the levels of GCFs in this group (dark blue bars) are comparatively overrepresented in the diagram. (B) GCFs (x axis) found in each metagenome (y axis) are shown. The inset expands a region containing the most common GCFs found in our specimens. Colors indicate shipworm host species. See Table S4 for a complete list of GCFs used in this figure.

FIG 6

A possible tabtoxin pathway is found in the D. mannii metagenome. Tabtoxin is a phytotoxin β-lactam that was initially discovered in Pseudomonas spp. (top). Strain 2719K contained a tabtoxin-like cluster that was pseudogenized (shown as an insertion in tabB; middle). A nonpseudogenized tabtoxin-like cluster was found in the D. mannii metagenome gill (bottom), supporting the observation that multiple variants of each symbiont genome are represented in each metagenome.

FIG 7

GCF distribution across shipworm species. Shown is a similarity network diagram, in which circles indicate individual BGCs from sequenced isolates (gray) and gill metagenomes (colors indicate species of origin; see legend). Lines indicate the MultiGeneBlast scores from comparisons between identified BGCs, with thinner lines indicating a lower degree of similarity. For example, the cluster labeled “GCF_8” encodes the pathway for the siderophore turnerbactin, the structure of which is shown at the right. The main cluster, circled by a light blue box, includes BGCs that are very similar to the originally described turnerbactin gene cluster. More distantly related BGCs, with fewer lines connecting them to the majority nodes in GCF_8, might represent other siderophores. The GCF_11 data likely all represent tartrolon D/E, a boronated polyketide shown at the right. For detailed alignments of BGCs, see Fig. S4.

FIG 8

Integration of tBLASTn and networking analyses reveals the pattern of occurrence of GCFs in isolates and metagenomes. Here, we show only the most commonly occurring GCFs. The values in each box indicate the number of BGC occurrences per specimen for each GCF (see Fig. S5 for details). When the number equals 1, then the BGC is found in all specimens of that species. When the number is less than 1, it then indicates the fraction of specimens in which the pathway is found. A number greater than 1 is specific to GCF_3, for which two different types are possible (see Fig. 8). In that case, there were two different classes of GCF_3 in two D. mannii specimens and one N. reynei specimen and only one class in the other specimens.

FIG 9

Three types of GCF_3 gene clusters were found to be distributed in all cellulolytic shipworms in this study. tBLASTx was used to compare the clusters, demonstrating the presence of three closely related GCF_3 gene families in all cellulolytic shipworm gills.