Abstract
Dominant coral-associated Endozoicomonas bacteria species are hypothesized to play a role in the global sulfur cycle by metabolizing Dimethylsulfoniopropionate (DMSP) into Dimethylsulfide (DMS); a climate active-gas, which releases sulfur into the atmosphere; however, no sequenced genome to date harbors genes for this process. We assembled high-quality (>95% complete) genomes of two new strains (Acr-1 and Acr-5) of a recently added species Endozoicomonas acroporae isolated from the coral Acropora muricata. We identified the first DMSP lyase—a dddD gene homolog found in all E. acroporae, and functionally characterized bacteria being capable of metabolizing DMSP into DMS via the DddD cleavage pathway using RT-qPCR and Gas chromatography (GC). This study confirms the role of Endozoicomonas in the global sulfur cycle.
Introduction
The genus Acropora contains some of the most abundant reef-building corals in the Indo-Pacific [1], and these corals are also some of the most significant producers of dimethylsulphoniopropionate (DMSP) [2, 3]. DMSP is present in coral tissue, mucus and endosymbiont dinoflagellates (Symbiodiniaceae) [4, 5]. It is the central molecule in the marine sulfur cycle and precursor to dimethylsulphide (DMS), a climate-active gas [6, 7]. DMSP is hypothesized to be part of the coral holobiont antioxidant system [8] and it act as an osmoprotectant against salinity fluctuations [3]. DMSP also acts as a signal molecule that attracts specific bacterial groups, which can form coral holobionts and underpin coral health [9].
Coral-associated bacteria use DMSP produced by corals and their symbiotic algae as a reduced sulfur and carbon source [9, 10]; they can also metabolize it into DMS [6, 7]. DMSP degradation by marine organisms takes place via two pathways, the cleavage pathway and the demethylation pathway [10, 11]. A recent study reported that the majority of DMSP-degrading bacteria belong to class Gammaproteobacteria, which includes Alteromonas-, Arhodomonas-, Idiomarina-, Pseudomonas- and Spongiobacter-related organisms [12]. Of these, Arhodomonas-, Pseudomonas-, and Roseobacter-related species harbor a DMSP lyase—i.e. the dddD gene, first identified in Marinomonas sp. for degrading DMSP [13]. Endozoicomonas species, which are predominantly associated with keeping their coral host healthy [14], have been hypothesized to play role in the global sulfur cycle by effectively metabolizing DMSP into DMS [15, 16]. However, no previous study has confirmed the genus’ role. Here, we provide the conclusive evidence that one of Endozoicomonas species metabolize DMSP into DMS.
Material and Methods
We de-novo assembled high quality (>95% complete) genomes of two new strains (Acr-1 and Acr-5) of a recently added species Endozoicomonas acroporae isolated from the coral Acropora muricata and identified for the first time a dddD gene homolog capable of metabolizing DMSP into DMS via the DddD cleavage pathway in all the E. acroporae strains. Furthermore, we functionally characterized the expression of the dddD gene and quantified the amount of DMS released using RT-qPCR and Gas chromatography (GC). Comparative genomic analysis of genus Endozoicomonas was performed to ascertain its genomic characteristics and features. We also profiled the abundance of E. acroporae species from two previous studies on different species of corals in Penghu, Taiwan [17] and the Red Sea, Saudi Arabia [18] (for details see supplementary data: material and methods).
Results and Discussion
E. acroporae species are dominant coral-associated bacteria in the Red Sea, Saudi Arabia (Fig S2A, B) and Penghu, Taiwan (Fig S2C, D), depicting their wide distribution among different coral species. When comparing E. acroporae Acr-1, Acr-5 with previously assembled type strain E. acroporae Acr-14T [19, 20] (supplementary results Table S1, Fig S1), all three strains of E. acroporae have a dddD gene homolog that encodes a DMSP lyase. RT-qPCR analysis of the dddD gene from E. acroporae Acr-14T cultured in 1 mM DMSP resulted in 42.77, 56.52, and 91.37 times higher expression than samples cultured without DMSP after 16, 24 and 48hrs, respectively (Fig 1A). The amount of DMS released when the culture (E. acroporae Acr-14T) was incubated in a DMSP-rich environment was significantly higher (t-test, p-value <0.05) than controls (Fig 1B). The temporal increase in the concentration of released DMS confirms that E. acroporae can metabolize DMSP into DMS. The discovery of the dddD gene in Endozoicomonas provides new insights into the evolution of the DMSP cleavage pathway and further confirms the hypothesis that Endozoicomonas plays a role in the global sulfur cycle.
Comparative genomic analysis identified that only E. acroporae have the DMSP metabolism gene(s) so far (Table S2). Further, we report high genomic divergence using Amino-Acid Identity (AAI), Average Nucleotide Identity (ANI) and DNA-DNA Hybridization (DDH) (Fig 2A, B, and C) in the genus and also a reduced core genome (308 genes) (supplementary data: results, Fig S5). Genomes of Endozoicomonas species are large (5.43 ∼ 6.69 Mb) (Table S2) and encode genes for all essential amino-acids [21], giving clues about not predominant genome streamlining as identified in symbiotic bacteria [22] and other symbiotic life stages [21]. Moreover, E. acroporae species have the highest number of T3SS genes in Endozoicomonas (supplementary data: results, Table S3), suggesting an intricate relationship with their host. Besides, E. acroporae strains have different Insertion Sequence (IS) elements than E. montiporae, hinting that the two coral-isolates have different evolution histories [23] (supplementary data: results, Fig S3). Furthermore, diverse phage insertions in Endozoicomonas species genomes suggest different infection histories (supplementary data: results, Table S4). In addition, E. montiporae and E. acroporae do not share any branches, according to core-genome based phylogenetic analysis; instead, their strains cluster tightly within their clades (Fig 2D). These results indicate that host and Endozoicomonas species have a complex nature of co-diversification. All species in this genus have a high percentage of oxidative stress responsive genes, which might be attributed to resistance against low oxygen environment in the ocean as well as highlight the genus Endozoicomonas’ adaptation to marine environments (supplementary data: results, Fig S4).
Conclusion
Endozoicomonas is the most dominant coral-associated bacterial group. Here, we link their function in the global element cycle. In addition, comparative genomic analysis of the genus Endozoicomonas gives clues about high genomic divergence and genome plasticity. Although, current understanding of the interaction among coral-microbe-sulfur cycle is still not clear, the results from this study will be beneficial for investigating the global change in the reef ecosystem functioning with the changing environment.
Data Availability
E. acroporae Acr-1 and Acr-5 assembled draft genomes are submitted to GenBank under accession numbers SAUT00000000 and SAUU00000000, respectively.
Author Contributions
K.T and S.L.T conceived the idea of this study. K.T assembled the genomes, performed bioinformatics analysis and wrote the manuscript. P.W.C cultured the strains and performed RT-qPCR analysis. C.Y.L and Y.F.C performed GC experiments and analysis. S.H.Y and N.W helped write the manuscript. P.Y.C, H.Y.C, and M.S.C helped in GC experiments and provided the instruments for conducting the experiment. W.M.C provided the cultures. S.L.T supervised the overall study and modified the manuscript. All authors read and approved the manuscript.
Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported by funding from Academia Sinica. KT would like to acknowledge the Taiwan International Graduate Program (TIGP) for its fellowship towards his graduate studies. Authors would like to acknowledge support from Dr. Shu-Fen Chiou for GC experiment.