A Conserved Biosynthetic Gene Cluster Is Regulated by Quorum Sensing in a Shipworm Symbiont

Abstract

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites. We determined that 2052S produces the signal N-decanoyl-l-homoserine lactone (C₁₀-HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms. IMPORTANCE Bacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step toward linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

Keywords: metabolomics; quorum sensing; shipworm; symbiosis.

FIG 1

A quorum sensing system in the Teredinibacter sp. strain 2052S genome is adjacent to a conserved biosynthetic gene cluster. (A) Quorum sensing genes (tbaI and tbaR) neighbor a predicted hybrid trans-AT-PKS-NRPS biosynthetic gene cluster (3). Identified putative TbaR-binding sites are represented by dotted lines from their position in the cluster and list the number of base pairs they are located upstream of the start codon of the indicated gene. Numbers correspond to locus tags (K256DRAFT_XXXX) in the Joint Genome Institute Integrated Microbial Genomes (IMG) system (25). Genes are colored according to predicted function in antiSMASH 6.0 (26). (B) Comparison of known LuxR-type binding sites in the promoter sequences of Aliivibrio fischeri luxI, Ralstonia solanacearum solI, and Methylobacter tundripaludum mbaI with the putative TbaR-binding sites upstream of the acyl-HSL synthase gene tbaI and predicted PKS gene K256DRAFT_2890. (C) Response of E. coli reporter strains containing gfp fused to different promoter regions with putative TbaR-binding sites shown in B to 100 nM C₁₀-HSL or ethyl acetate (vehicle). Data are the mean ± standard deviation of three technical replicates and are representative of two independent experiments. RFU, relative fluorescence units; OD, optical density.

FIG 2

Teredinibacter sp. strain 2052S produces the quorum sensing signal C₁₀-HSL. (A) P_tbaI-gfp activity of HPLC-fractionated culture supernatant extracts from 2052S and the ΔtbaI mutant. The dashed line shows the methanol gradient. (B) Extracted ion chromatogram of supernatant extracts of 2052S and the ΔtbaI mutant compared with a commercial C₁₀-HSL signal for m/z 279.1812, corresponding to the sodiated adduct of C₁₀-HSL. Mass tolerance, <5 ppm. (C) Structure of C₁₀-HSL. RFU, relative fluorescence units; OD, optical density.

FIG 3

The conserved BGC is transcriptionally activated by the addition of C₁₀-HSL to the ΔtbaI mutant. (A) Pigmentation phenotype of 2052S (left) and ΔtbaI mutant (middle) cultures, and partial reversion to wild-type phenotype upon addition of QS signal to the ΔtbaI culture (right). (B) RT-qPCR results showing relative expression of GCF_3 genes K256DRAFT_2890, 2886, and 2881 upon addition of C₁₀-HSL to the ΔtbaI strain normalized to ΔtbaI expression in the absence of signal. The 16S rRNA gene was used as a reference gene. Data are the mean ± standard deviation of three technical replicates and are representative of two independent experiments.

FIG 4

Quorum sensing regulates the majority of extracellular metabolites produced by 2052S. (A) Molecular network of untargeted metabolomics data of supernatant extracts from cultures of 2052S, ΔtbaI, and ΔtbaI supplemented with C₁₀-HSL. Features found in samples where QS is on (2052S and ΔtbaI + C₁₀-HSL) are shown as cyan nodes; features found in the ΔtbaI culture, where QS is off, are shown as red nodes; and features found in both samples where QS is on and off are shown as split cyan and red nodes. Features identified as associated with GCF_3 (present in 2052S, ΔtbaI + C₁₀-HSL, and Δ2886 + pAWP275, but absent in ΔtbaI and Δ2886) are shown as square nodes, and C₁₀-HSL is shown as a hexagonal node. Edge width is scaled with cosine similarity score. (B) Venn diagram of features associated with the QS regulon. (C and D) Extracted ion chromatograms of m/z 464.2708 (C) and m/z 394.2985 (D) in culture supernatant extracts. Mass tolerance, <5 ppm.