A bacterial symbiont in the gill of the marine scallop Argopecten irradians irradians metabolizes dimethylsulfoniopropionate

Abstract

Microbial lysis of dimethylsulfoniopropionate (DMSP) is a key step in marine organic sulfur cycling and has been recently demonstrated to play an important role in mediating interactions between bacteria, algae, and zooplankton. To date, microbes that have been found to lyse DMSP are largely confined to free-living and surface-attached bacteria. In this study, we report for the first time that a symbiont (termed "Rhodobiaceae bacterium HWgs001") in the gill of the marine scallop Argopecten irradians irradians can lyse and metabolize DMSP. Analysis of 16S rRNA gene sequences suggested that HWgs001 accounted for up to 93% of the gill microbiota. Microscopic observations suggested that HWgs001 lived within the gill tissue. Unlike symbionts of other bivalves, HWgs001 belongs to Alphaproteobacteria rather than Gammaproteobacteria, and no genes for carbon fixation were identified in its small genome. Moreover, HWgs001 was found to possess a dddP gene, responsible for the lysis of DMSP to acrylate. The enzymatic activity of dddP was confirmed using the heterologous expression, and in situ transcription of the gene in scallop gill tissues was demonstrated using reverse-transcription PCR. Together, these results revealed a taxonomically and functionally unique symbiont, which represents the first-documented DMSP-metabolizing symbiont likely to play significant roles in coastal marine ecosystems.

Keywords: Alphaproteobacteria; DMSP lyases; dddP; scallop; symbiont.

Figure 1

Fluorescence in situ hybridization (FISH) images of the gill filament at 20× and 40× magnification. (A) Phase‐difference micrograph of the gill filament. (B) Gill cells stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). (C) FISH image using an oligo‐DNA probe specifically targeting the 16S rRNA of the OTU_322591 (red); (D) The merged image of the OTU_322591 signals (red) and the DAPI‐stained gill cells (blue). The white boxes represent accumulated bacterial cells in the gill tissues which are shown by yellow arrows. Scale bars, 20 µm.

Figure 2

Phylogenetic relationships between the scallop symbiont HWgs001 and symbiont bacteria of other bivalves. Three alphaproteobacterial symbionts from the sponge Mycale laxissima, the sea urchin Heliocidaris, and the ciliate Cyclidium porcatum are also included in the analysis. The tree was built based on 16S rRNA gene sequences in a maximum‐likelihood mode. Bootstrap values were generated through 1000 replicated calculations and are shown on the branches. Triangles indicate symbionts whose sequences were used for subsequent functional genomic comparisons. The respective hosts are shown in the rightmost column.

Figure 3

Functional genomic comparison between the scallop symbiont HWgs001 and seven other symbionts. 200920, Candidatus Hydrogenosomobacter endosymbioticus 200920 from the ciliate Scuticociliate; H1, Rickettsiaceae bacterium H1 from the sea urchin Heliocidaris; KLH11, Rhodobacteraceae bacterium KLH11 from the sponge Mycale laxissima; MAGL205, Candidatus Thiodiazotropha taylori MAGL205 from the bivalve Loripes orbiculatus; MAGdivaljamaic004, Candidatus Thiodiazotropha endolucinida MAGdivaljamaic004 from the bivalve Loripes orbiculatus; MAGKOTO1, Candidatus Thiodiazotropha lotti MAGKOTO1 from the bivalve Loripes orbiculatus; WH, Gammaproteobacterium symbiont of the bivalve Solemya velum, strain WH; the phylogenetic relationships between these bacteria and their hosts are shown in Figure 2. The colored box indicates the existence of the corresponding pathway.

Figure 4

A schematic model showing the major carbon metabolic pathways in HWgs001. Dimethylsulfoniopropionate (DMSP) is cleaved to produce dimethylsulfide (DMS) and acrylate. Acrylate is transformed into acryloyl‐CoA, and finally, succinyl‐CoA enters the TCA cycle. FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NUO, NADH dehydrogenase complex.

Figure 5

Distribution pattern of the dddP gene in the genome of HWgs001 and its enzyme activity. (A) The dddP gene is adjacent to genes involved in energy metabolism. The gene cluster was drawn using a Python script. (B) The dddP gene was expressed in Escherichia coli BL21, and the dimethylsulfoniopropionate (DMSP) lysis activity was detected by measuring the dimethylsulfide (DMS) production using gas chromatography. The peaks indicated by arrows correspond to DMS.