Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges

Abstract

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Figure 1. Synthetic and naturally occurring polyhalogenated molecules

Structural similarity between (A) synthetic and (B) naturally produced marine halogenated phenolic toxins and pollutants.

Figure 2. Host and symbiont phylogenies

(A) Left: Midpoint-rooted, maximum likelihood (ML) phylogeny based on sponge ribosomal ITS-2 sequences from the current study (in bold) along with representative literature examples. Identical sequences are listed at a single branch tip, with symbols denoting dominant natural product chemistry. Bootstrap values ≥70 are shown; scale bar indicates substitutions per site. Matching superscripts indicate literature ITS-2 and 16S rRNA sequences obtained from the same sponge specimen. Right: H. spongeliae ML tree of near-full length 16S rRNA gene sequences, including closest free-living relative Oscillatoria corallinae, and rooted on closest relative with a sequenced genome, Moorea producens 3L. One full-length 16S rRNA gene sequence (GUM098) was obtained from clone library (see Figure 3). Remaining Clade IV 16S rRNA sequences in this study, denoted in parentheses, are represented by 370 bp sequences obtained from high-throughput community data that are 100% identical to the GUM098 full-length sequence. (B) Chemical profiles, as monitored by absorbance at 280 nm by HPLC with in situ photographs of representative sponges for each clade.

Figure 3. Representative 16S rRNA gene diversity profiles

Genomic DNA for 16S amplification was isolated from cross sections of sponge tissues. Taxonomic groups are shown at the phylum level, with the exception of operational taxonomic units identified as H. spongeliae (dark green). For clarity, groups representing <0.1% of the data are not labeled.

Figure 4. Discovery of hs_bmp gene clusters

(A) Previously identified bmp gene loci in the marine bacterium Pseudoalteromonas luteoviolacea 2ta16. Genes bmp1–4, together with bmp8, participate in the biosynthesis of bromopyrroles, while bmp9–10, encoding ferredoxin and ferredoxin reductase respectively, are electron transporters for the activity of CYP450-Bmp7. The catalytic roles of bmp5 gene products (yellow), bmp6 (blue), and bmp7 (red) are color-coded. In addition to ortho-OH-BDEs such as 1, catalytically promiscuous CYP450-Bmp7 also generates para-OH-BDE and biphenyl products as shown. (B) All Clade Ia (Sample SP12) scaffolds greater than 5 kb in length are displayed in grey. The scaffold containing the hs_bmp operon is designated by a gold circle. Other colored points represent scaffolds classified by amino acid sequence similarity of multiple predicted proteins to known sequences from the GenBank non-redundant database. (C) The color scheme from panel A is preserved for hs_bmp genes. Nucleotide identity ≥90% between different regions of the hs_bmp gene loci from different sponge clades is shown in grey shading.

Figure 5. Functional characterization of Clade Ia hs_bmp genes

(A) UV-absorption chromatograms monitored at 280 nm for organic extracts of the following strains supplemented with 50 μM 10: (bottom) control strain Se7942-C, (middle) Se7942-hs_bmp7, and (top) Se7942-hs_bmp7–12. Production of 1 was observed in Se7942-hs_bmp7, while the production of 6 and 11 was observed for Se7942-hs_bmp7–12. Together with 6 and 11, the production of a third tribrominated molecule was observed, the MS1 signature for which corresponded to the tribrominated dioxin 7 as shown in panel B.