A New N-Acyl Homoserine Lactone Synthase in an Uncultured Symbiont of the Red Sea Sponge Theonella swinhoei

Abstract

Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N-acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei. The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales, here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli, produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.

FIG 1

Mass spectrometry analysis. (A) Extracted ion chromatograms at m/z 228.1549 ± 0.0010 (C₈-HSL, denoted by 1), m/z 256.1907 ± 0.0010 (C₁₀-HSL, denoted by 2), and m/z 384.3108 ± 0.0010 (3OH-C₁₈-HSL, denoted by 3), derived from fractions of extract of T. swinhoei. (B) Extracted ion chromatograms at m/z 228.1594 ± 0.0010 (C₈-HSL, denoted by 1), m/z 256.1907 ± 0.0010 (C₁₀-HSL, denoted by 2), and m/z 284.2220 ± 0.0010 (C₁₂-HSL, denoted by 4), derived from fractions of extract of E. coli DH5α harboring tswI-pBBR1MCS. The characteristic product ion at m/z 102.05, corresponding to the deacylated homoserine lactone, was detected.

FIG 2

(A) tswI–tswR–tswR-t gene layout. (B) Alignment of LuxR-type transcriptional regulators. The alignment starts from position 60 of TswR (accession no.): Rhodobacterales bacterium TS309 (KP092522), Actibacterium mucosum KCTC 23349 (KAJ55423), Oceanicola granulosus (WP_007253988.1), Octadecabacter arcticus (WP_015494454.1), Loktanella vestfoldensis (WP_007205120.1), Roseobacter sp. CCS2 (WP_008235250.1), Pseudomonas (WP_003119559), Burkholderia xenovorans (WP_011492631), Aliivibrio fischeri (WP_005423460), Rhizobium etli (WP_004672081), Ruegeria sp. KLH11 (WP_008756899), Rhodobacter (WP_002720271), Ruegeria sp. KLH11 (WP_008757266), A. tumefaciens (WP_010892389), Rhodobacterales bacterium TS309 (KP092523), L. vestfoldensis (WP_026352205.1), Roseobacter sp. CCS2 (WP_008235255.1), and A. tumefaciens (AAC18654.1). The highlighted regions indicate high similarity between all the proteins presented. The nine highly conserved amino acids are indicated with stars. The red star indicates the missing amino acid in the truncated LuxR homologues.

FIG 3

Maximum-likelihood distance tree based on concatenated sequences for 6 conserved proteins. The numbers on the nodes indicate the bootstrap percentages out of 500. The bacteria published in this study are shown in bold.

FIG 4

(A) Phylogenetic distribution of EMIRGE 16S rRNA OTUs (cutoff, 97% similarity) at the phylum level; (B) phylogenetic distribution of EMIRGE 16S rRNA OTUs at the Proteobacteria class level.

FIG 5

Relative abundances of OTUs with 99% identity to the 16S rRNA of Rhodobacterales bacterium TS309 in the Sponge Microbiome Project. Presented in the figure are all sponge species with median values greater than 0 and three control habitats (blue, seawater and freshwater; brown, sediment). Sponge species with a median equal to zero are shown in Fig. S5 in the supplemental material. The vertical bars represent the medians, the boxes represent the first to third quartiles, and the whiskers show the lowest or highest datum within 1.5 times the interquartile range of the lowest and upper quartile, respectively. The data points beyond the ends of the whiskers are outliers. Information on samples (e.g., geographic location, sample identification [ID], etc.) is provided in Table S4 in the supplemental material.