Integrated metabolism in sponge-microbe symbiosis revealed by genome-centered metatranscriptomics

Abstract

Despite an increased understanding of functions in sponge microbiomes, the interactions among the symbionts and between symbionts and host are not well characterized. Here we reconstructed the metabolic interactions within the sponge Cymbastela concentrica microbiome in the context of functional features of symbiotic diatoms and the host. Three genome bins (CcPhy, CcNi and CcThau) were recovered from metagenomic data of C. concentrica, belonging to the proteobacterial family Phyllobacteriaceae, the Nitrospira genus and the thaumarchaeal order Nitrosopumilales. Gene expression was estimated by mapping C. concentrica metatranscriptomic reads. Our analyses indicated that CcPhy is heterotrophic, while CcNi and CcThau are chemolithoautotrophs. CcPhy expressed many transporters for the acquisition of dissolved organic compounds, likely available through the sponge's filtration activity and symbiotic carbon fixation. Coupled nitrification by CcThau and CcNi was reconstructed, supported by the observed close proximity of the cells in fluorescence in situ hybridization. CcPhy facultative anaerobic respiration and assimilation by diatoms may consume the resulting nitrate. Transcriptional analysis of diatom and sponge functions indicated that these organisms are likely sources of organic compounds, for example, creatine/creatinine and dissolved organic carbon, for other members of the symbiosis. Our results suggest that organic nitrogen compounds, for example, creatine, creatinine, urea and cyanate, fuel the nitrogen cycle within the sponge. This study provides an unprecedented view of the metabolic interactions within sponge-microbe symbiosis, bridging the gap between cell- and community-level knowledge.

Figure 1

Predicted model of integrated metabolic processes within Cymbastela concentrica. Selected pathways and gene functions are shown. Transporters with at least partial genomic evidence and expression detected were included. Inferred nutrient fluxes (A–O) are discussed in the section ‘Model of integrated metabolism of the microbial community members in C. concentrica’. Expressed genes are listed in Supplementary Tables S2.

Figure 2

Most expressed CcPhy genes related to membrane transport. (a) CDS assigned to TCDB were grouped by families and super families. Groups were ordered according to maximum TPM_av values (x axis), and only the top 15 are shown. For reference, CDS not classified by TCDB are shown (not in TCDB). The median of all expressed genes is represented by the dashed line. (b) Most expressed transport-related genes. Gray scale refer to gene family or superfamily. Genes are ordered according to TPM_av values (x axis). Whiskers represent s.d. of biological replicates (n=3).

Figure 3

Expression of selected functional genes of CcNi. TPM_av values are shown for genes of selected functions. ‘Others’ stands for genes not grouped in the specific functions shown. The median of all expressed genes is represented by the dashed line. Highlighted genes encode for the nitrite oxidoreductase alpha (nxrA), beta (nxrB), putative C (nxrC) subunits (Supplementary Information), cyanate lyase (cya) and pyruvate ferredoxin oxidoreductase (PFOR).

Figure 4

Expression of selected functional genes of CcThau. TPM_av values are shown for genes of selected functions. ‘Others’ stands for genes not grouped in the specific functions shown. The median of all expressed genes is represented by the dashed line. Highlighted genes encode for the ammonia monooxygenase subunits alpha (amoA), beta (amoB) and C (amoC) subunits as well as ammonium transporter (Amt).

Figure 5

Expression of diatom and sponge transcripts in Cymbastela concentrica. (a) Transcripts taxonomically classified as diatoms (Bacillariophyta) or sponge (Porifera or Metazoan with best BLAST hit to sponge sequences) were sorted from other eukaryotic transcripts recovered from C. concentrica. (b) Overall expression of diatom and sponge is indicated by the sum of transcripts’ TPM. (c) Deduced peptide sequences from transcripts’ coding sequences were used for KEGG functional classification. Expression of KEGG functions was inferred from the sum of TPM values across coding sequences.

Figure 6

Expressed metabolic functions in diatoms and the sponge. Sums of TPM are shown for KEGG metabolic pathways found in (a) diatoms (Bacillariophyta) and (b) sponge (Porifera and Metazoan or Metazoan with best BLAST hit to sponge sequences). Legends list the top expressed pathways.

Figure 7

Visualization of microorganisms associated with Cymbastela concentrica by fluorescence in situ hybridization. (a) Rod-shaped cells (white arrows) were observed from hybridization with a Pacific Blue-labeled CcPhy-specific probe. Signals from (b) fluorescein-isothiocyanate-labeled CcNi-specific and (c) Cy3-labeled CcThau-specific probes are shown. (d) Red autofluorescence signals from diatoms were captured (arrows). (e) Clusters of CcNi-specific (green) and CcThau-specific (magenta) probes in the sponge mesohyl are highlighted in the merged color image by circles. CcPhy cells (cyan) did not specifically co-localize with CcNi or CcThau cells. Autofluorescence of the sponge tissue (light blue/cyan) served as a reference for the structure of the sponge mesohyl. Scale bars, 10 μm.