Sulfur, sterol and trehalose metabolism in the deep-sea hydrocarbon seep tubeworm Lamellibrachia luymesi

Abstract

Background: Lamellibrachia luymesi dominates cold sulfide-hydrocarbon seeps and is known for its ability to consume bacteria for energy. The symbiotic relationship between tubeworms and bacteria with particular adaptations to chemosynthetic environments has received attention. However, metabolic studies have primarily focused on the mechanisms and pathways of the bacterial symbionts, while studies on the animal hosts are limited.

Results: Here, we sequenced the transcriptome of L. luymesi and generated a transcriptomic database containing 79,464 transcript sequences. Based on GO and KEGG annotations, we identified transcripts related to sulfur metabolism, sterol biosynthesis, trehalose synthesis, and hydrolysis. Our in-depth analysis identified sulfation pathways in L. luymesi, and sulfate activation might be an important detoxification pathway for promoting sulfur cycling, reducing byproducts of sulfide metabolism, and converting sulfur compounds to sulfur-containing organics, which are essential for symbiotic survival. Moreover, sulfide can serve directly as a sulfur source for cysteine synthesis in L. luymesi. The existence of two pathways for cysteine synthesis might ensure its participation in the formation of proteins, heavy metal detoxification, and the sulfide-binding function of haemoglobin. Furthermore, our data suggested that cold-seep tubeworm is capable of de novo sterol biosynthesis, as well as incorporation and transformation of cycloartenol and lanosterol into unconventional sterols, and the critical enzyme involved in this process might have properties similar to those in the enzymes from plants or fungi. Finally, trehalose synthesis in L. luymesi occurs via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. The TPP gene has not been identified, whereas the TPS gene encodes a protein harbouring conserved TPS/OtsA and TPP/OtsB domains. The presence of multiple trehalases that catalyse trehalose hydrolysis could indicate the different roles of trehalase in cold-seep tubeworms.

Conclusions: We elucidated several molecular pathways of sulfate activation, cysteine and cholesterol synthesis, and trehalose metabolism. Contrary to the previous analysis, two pathways for cysteine synthesis and the cycloartenol-C-24-methyltransferase gene were identified in animals for the first time. The present study provides new insights into particular adaptations to chemosynthetic environments in L. luymesi and can serve as the basis for future molecular studies on host-symbiont interactions and biological evolution.

Keywords: Adaptation to deep-sea chemosynthetic environment; Cholesterol; Cold seep; Cycloartenol-C-24-methyltransferase; Cysteine; Fungi; Sulfate; Transcriptome; Trehalase.

Fig. 1

A summary of the functional annotation of the L. luymesi transcriptome. Functional classification of transcripts annotated by A KOG; B KEGG; C GO. D Species distribution of annotated transcripts

Fig. 2

A Scheme of L. luymesi sulfation pathways and biosynthetic pathways for cysteine. TST, thiosulfate sulfurtransferase; tauD, alpha-ketoglutarate-dependent taurine dioxygenase; SLC26A2, sulfate transporter; SLC26A11, sodium-independent sulfate anion transporter; PAPSS, bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase; PAPST1/2, adenosine 3'-phospho 5'-phosphosulfate transporter 1/2; CYS2, probable serine-O-acetyltransferase cys2; CYS3, putative cystathionine gamma-lyase 2; CYS4, cystathionine beta-synthase. B Heatmap of FPKM expression values for the annotated genes in L. luymesi sulfation pathway from three individual samples (TW1, TW2 and TW3). C Heatmap of FPKM expression values for the annotated genes in L. luymesi biosynthetic pathways for cysteine. FPKM values were listed in Table S4

Fig. 3

PAPS synthesis in L. luymesi. A PAPS synthesis pathway. B The domain formation of L. luymesi PAPSS, a fused gene encoding a protein with two conserved domains. APS, adenosine 5'-phosphosulfate; PAPS, bifunctional 3'-phosphoadenosine-5'-phosphosulfate; PAPSS, PAPS synthase; APSK, APS kinase; ATPS, ATP sulfurylase

Fig. 4

Phylogenetic tree of CS sequences. L. luymesi CS is highlighted in bold

Fig. 5

A Overview of cholesterol synthesis pathway in L. luymesi. Circles represent the following enzymes: 1, acetyl-CoA acetyltransferase; 2, hydroxymethylglutaryl-CoA synthase; 3,3-hydroxy-3-methylglutaryl-coenzyme A reductase; 4, mevalonate kinase; 5, promyelvalonate kinase; 6, diphosphomevalonate decarboxylase; 7, isopentenyl-diphosphate delta-isomerase; 8, farnesyl pyrophosphate synthase; 9, geranylgeranyl pyrophosphate synthase; 10, squalene synthase; 11, squalene monooxygenase; 12, lanosterol synthase; 13, lanosterol 14-alpha demethylase; 14, delta(14)-sterol reductase; 15, lamin-B receptor; 16, sterol-4-alpha-carboxylate 3-dehydrogenase; 17, 3-keto-steroid reductase; 18, lathosterol oxidase; 19, 7-dehydrocholesterol reductase. C. elegans lacks the branch inside the dashed box. B Heatmap of FPKM expression values for the annotated genes in L. luymesi cholesterol synthesis pathway from three individual samples (TW1, TW2 and TW3). FPKM values were listed in Table S4

Fig. 6

Sequence alignment of 42 CYP51 family members from different biological kingdoms [bacteria (1–4), plants (5–10), fungi (11, 13–31) and animals (12, 32–42)] in CYP51 substrate recognition sites (SRS) 1 and 4; 100% and more than 95% conserved residues are shaded in black and grey, respectively. Phyla-specific residues are highlighted in red or shaded in yellow. The accession number of sequences can be found in Supplemental data file 1

Fig. 7

Phylogenetic tree of 42 CYP51 family members with 1000 bootstrap replications. L. luymesi CYP51A1 is highlighted in bold. The accession number of sequences can be found in Supplemental data file 1

Fig. 8

A Trehalose metabolic pathway. TPS, trehalose 6-phosphate synthase; TPP, trehalose 6-phosphate phosphatase; TREH, trehalase. B Heatmap of FPKM expression values for the annotated genes in L. luymesi trehalose metabolic pathway from three individual samples (TW1, TW2 and TW3). FPKM values were listed in Table S4

Fig. 9

Phylogenetic tree of trehalase genes and protein isoform diversity in L. luymesi and other taxa

Fig. 10

Partial sequence alignment of L. luymesi ATHs and human PGGHG. HsPGGHG, Homo sapiens PGGHG (NCBI accession no. NP_079368.3); LlATH1/2, L. luymesi acid trehalase 1/2. Identical amino acids are marked by an asterisk and shaded in grey. More than 50% of the conserved residues are shaded in blue. The amino acids in red indicate that three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) are essential for catalytic activity