A microbial factory for defensive kahalalides in a tripartite marine symbiosis

Abstract

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont ("Candidatus Endobryopsis kahalalidefaciens") uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. "Ca E. kahalalidefaciens" has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.

Fig. 1.. Bacterial composition and chemical defense of the Bryopsis–Elysia symbiotic system.

(A) The natural Bryopsis sp. bloom studied in this work. (B) E. rufescens feeding on Bryopsis sp. in the laboratory. (C) Composition of Bryopsis-2015–associated bacterial communities across four different replicates collected on two consecutive days (A and B: 29 March 2015; C and D: 30 March 2015) and grouped at the class level. Rare taxa (<1% of total sequences) are labeled as “Others.” The inset bar graph represents the percentage of a single 16S rDNA sequence (hereafter designated “cEK”) that dominates the class Flavobacteriia. (D) Molecular structure of kahalalide F (KF), the main defensive chemical in both Bryopsis sp. and E. rufescens. Structural features commonly associated with microbial biosynthesis are depicted in red, blue, and pink.

Fig. 2.. The large biosynthetic capacity of “Ca. E. kahalalidefaciens.”

(A) Circular map of the “Ca. E. kahalalidefaciens” chromosome. Tracks (from outermost to innermost) represent genes on the forward frame, genes on the reverse frame, RNAs, GC content, and GC skew. Genes are color coded according to the Cluster of Orthologous Groups (COG) categories in the Integrated Microbial Genome platform (21) (A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, and chromosome partitioning, E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures; X, mobilome, prophages, and transposons; Y, nuclear structure; Z, cytoskeleton; and NA, not assigned). Note that 99.7% of the aggregate length of genes classified in the COG category Q in the “Ca. E. kahalalidefaciens” chromosome (red) correspond to NRPS pathways and occupy 20% of the genome coding capacity. (B) Genetic organization of the 20 NRPS pathways in “Ca. E. kahalalidefaciens” (ordered by size), where each arrow indicates a single gene.

Fig. 3.. Structural and biosynthetic diversity of kahalalides from Bryopsis sp.

(A) Molecular structures of nine kahalalides that can be bioinformatically linked to specific NRPS pathways in “Ca. E. kahalalidefaciens” and chemically detected in Bryopsis-2015. (B) Extracted ion chromatograms (EICs) for the mass/charge ratios (m/z) corresponding to the (M + H)⁺ ions of the molecules in (A), as detected in the chemical extract of Bryopsis-2015. An asterisk indicates the peak corresponding to the molecule of interest. (C) Amino acid composition of the nine kahalalides shown in (A), indicating the position and stereochemistry of each amino acid on a linear scale. Kahalalide names are represented with two-letter abbreviations (e.g., kahalalide D = KD), and amino acids are denoted by their canonical one-letter codes (A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr) except for ornithine (O) and dehydrobutyrine (DB). FA, fatty acid. (D) Domain organization of “Ca. E. kahalalidefaciens”–encoded NRPS pathways that were bioinformatically linked to corresponding kahalalides in C (Cs, starting condensation domain; A, adenylation; T, thiolation; E, epimerization; C, condensation; and TE, thioesterase). The number of modules in a given NRPS and the number and position of epimerization domains encoded in it (black) match exactly what is observed in its linked product (see also substrate specificity analyses in figs. S3 and S6 and detailed HR-MS/MS analyses in table S1 and figs. S1 and S7).

Fig. 4.. Localization of “Ca. E. kahalalidefaciens” in Bryopsis sp. using FISH.

Epifluorescent micrographs of a Bryopsis sp. section hybridized with (A) universal eubacterial probes EUB338 I to III (labeled with Cy3, red) and (B) “Ca. E. kahalalidefaciens”–specific probe JZP2 (labeled with 6-FAM, green). (C) Algal cell wall of the same section viewed under a 4′,6-diamidino-2-phenylindole (DAPI) channel using calcofluor counterstaining. (D) Composite of images (A) to (C), showing the colocalization of the red and green signals for “Ca. E. kahalalidefaciens” but not for other bacteria. Arrows indicate bacteria: cEK, “Ca. E. kahalalidefaciens”; OB, other bacteria.

Fig. 5.. Intensive genetic exchange in “Ca. E. kahalalidefaciens” NRPS pathways.

(A) Pairs of sequence fragments sharing more than 60% identity across the whole “Ca. E. kahalalidefaciens” genome, shown on a linear scale on both the x and y axes. Diagonal matches were removed, and color indicates percent identity. Positions of the 20 NRPS pathways are indicated on the axes, matching the color code below. (B) Examples of aligned sequences between NRPS pathways. Note the abrupt rise and fall in sequence identity. Colors in pathways indicate different domains, matching the color code below. Encoded amino acids are indicated by their single-letter abbreviations, and underlined letters indicate D-amino acids. (C) Circular representation of “Ca. E. kahalalidefaciens” genome recombination events (coordinate one of the genome is indicated by a solid black line), in which NRPS pathways are shown at their respective positions around the genome and connecting colored lines indicate identified pairwise recombination events. Lines follow a yellow-to-blue color code that represents the percent identity between pairs of aligned sequences, and their thickness positively correlates with the length of the sequence. Sequences shared with NRPS-8 and NRPS-9 have a lower percent identity than ones shared with other pathways. Black lines indicate the three identified duplication events. (D) Phylogenetic trees for three groups of aligned sequences (red, green, and blue) that together cover the entire NRPS-15. Although the three aligned sequences are found in NRPS-15, they do not follow the same phylogenetic lineage and are likely a result of three independent recombination events. NRPS-15 is shown as a black rectangle, and NRPS pathways that share one or more of the three groups of aligned sequences with it are shown as gray rectangles. The scale bar at top right indicates 10% sequence divergence.

Fig. 6.. Metatranscriptomic analysis of “Ca. E. kahalalidefaciens.”

(A) Genome-wide transcriptional activity of “Ca. E. kahalalidefaciens” using metatranscriptomic data collected from Bryopsis-2015. Black bars indicate total counts for each mapped position and averaged per 5 kbps of the genome. The inset bar graph indicates the ranked contribution of each NRPS pathway as a percentage of the total genome transcriptional activity. (B) “Ca. E. kahalalidefaciens” genes (x axis, shown in percentage of the total number of genes in the genome) plotted against their cumulative expression (y axis, shown in percentage of the total expression of all genes in the genome). Note the trimodal distribution of genes according to their expression and that genes from the NRPS pathways appear in all three modes.