Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae

Highlights

• A novel GS-like cDNA sequence, TSSGS1, encodes a protein with a GS and NDK domain.

• A GLT-like cDNA sequence, TSSGLT, encodes a plastid-localized TSSGOGAT.

• The proteins TSSGS1 and TSSGOGAT originated from the zooxanthellae of T. squamosa.

• The TSSGS1-TSSGOGAT cycle is present in the zooxanthellae of T. squamosa.

• The zooxanthellae in T. squamosa can assimilate endogenous ammonia and prevent ammonia excretion.

Abstract

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Introduction

Giant clams are bivalve molluscs belonging to family Cardiidae and subfamily Tridacninae. They live on shallow coral reefs in the tropical waters of Indo-Pacific, and harbor multi-clades (A, C and D) of symbiotic zooxanthellae belonging to genus Symbiodinium under the class Dinophyceae (Fitt et al., 1993a, Fitt et al., 1993b; Hernawan, 2008). The larval clam acquired Symbiodinium from the surrounding waters by ingestion (Norton et al., 1992). A single opening from one of the digestive diverticular ducts in the stomach of the host clam extends to form a primary tube that branches into secondary and tertiary tubules. The tertiary tubules pervade the host's body, particularly the mantle (Norton et al., 1992). The mantle encapsulates the organs of the giant clam and is differentiated into the siphonal and the lateral mantle. The lateral mantle comprises two distinct regions as demarcated by the pallial line on the inner surface of the shell-valve. They are the outer mantle, which is fleshy and colorful, and the inner mantle, which is thin and whitish. The inner mantle is located adjacent to the extrapallial fluid, contains only a small quantity of symbionts, and takes part in shell formation. The outer mantle, which is unique to giant clams, is extensible and retractable. It contains host pigments and iridophores, and harbors the majority of the symbiotic zooxanthellae extracellularly in a fluid inside the tertiary tubules (Holt et al., 2014). As the symbiotic zooxanthellae are positioned close to the upper surface of the outer mantle, they receive adequate sunlight for photosynthesis during the day (Norton et al., 1992). The hemolymph of the host clam pervades the tissues/organs through an open circulatory system (Yellowlees et al., 2008). Hence, tertiary tubules in the outer mantle are surrounded by the hemolymph, through which the host clam can supply nutrients, such as inorganic carbon and nitrogen, to the symbiotic zooxanthellae residing therein. Likewise, photosynthate produced by the zooxanthellae can be translocated to the host via the hemolymph (Hernawan, 2008).

Nitrogen is an essential element in organisms, as it is a fundamental component of amino acids, proteins and nucleic acids (Boutilier, 2012). The breakdown of nitrogen-containing compounds, like amino acids, releases toxic ammonia, which must be excreted or detoxified (Campbell, 1991; Ip and Chew, 2010). However, instead of excreting ammonia, many marine invertebrate-algae associations, including giant clams, absorb and assimilate exogenous ammonia from the ambient seawater during insolation (Muscatine et al., 1979; Wilkerson and Muscatine, 1984; Wilkerson and Trench, 1986; Miller and Yellowlees, 1989). The assimilated nitrogenous compound is then supplied to the symbiotic zooxanthellae, which are nitrogen-deficient. While symbiotic zooxanthellae can fix CO₂ into carbohydrates through photosynthesis, they need a supply of nitrogen from the host to augment the synthesis of amino acids, which are subsequently shared with the host. Zooxanthellae isolated from the host can absorb ammonia and nitrate from the external medium in vitro (Wilkerson and Trench, 1986), implying that they can probably do the same in vivo inside the host clam. For intact giant clam-zooxanthellae associations, the addition of inorganic nitrogen to the ambient seawater can augment photosynthesis in the symbionts (Summons et al., 1986) and enhance the hosts' growth rate (Hastie et al., 1988; Onate and Naguit, 1989; Hastie et al., 1992). In seawater containing elevated concentrations of ammonia, juvenile giant clams grow faster (Hastie et al., 1992; Belda et al., 1993; Fitt et al., 1993a) and harbor more zooxanthellae with higher division rate (Belda et al., 1993; Fitt et al., 1993a).

Based predominantly on information from symbiotic cnidarians, the depletion-diffusion model (D'Elia et al., 1983) and the host assimilation model (Miller and Yellowlees, 1989) have been proposed to explain light-enhanced ammonia absorption and assimilation in alga-invertebrate associations. In the depletion-diffusion model, the symbiotic zooxanthellae are allegedly responsible for keeping low concentrations of ammonia in the host tissues to facilitate the continual inward diffusion of exogenous ammonia into the host. Zooxanthellae absorb the ammonia and convert it into glutamine through a reaction catalyzed by the zooxanthellal Glutamine Synthetase (GS; EC 6.3.1.2): NH₄⁺ + glutamate + ATP → glutamine + ADP + Pi, (Anderson, 1986; Anderson and Burris, 1987; Rahav et al., 1989; Yellowlees et al., 1994). Subsequently, one mole of glutamine can react with one mole of 2-oxoglutarate to form two moles of glutamate: glutamine +2-oxoglutarate + NADPH + H⁺ → 2 glutamate + NADP⁺ (Rahav et al., 1989; McAuley and Cook, 1994; Roberts et al., 1999). This reaction is catalyzed by glutamine 2-oxoglutarate amidotransferase (GOGAT; or glutamate synthase; EC 1.4.1.13), which is peculiar to plants and plant-like organisms (Falkowski et al., 1985). Alternatively, the host assimilation model (Miller and Yellowlees, 1989) depicts that the host clam is purportedly responsible for the assimilation of ammonia into glutamine (Rees, 1986; Miller and Yellowlees, 1989), as the host has higher GS activity than the symbiont (Yellowlees et al., 1994). Nevertheless, the host still has to depend on a supply of carbon skeletons (glutamate) from the symbionts to support ammonia assimilation. Hence, both models attribute light-enhanced assimilation of exogenous ammonia to the symbiotic zooxanthellae and their photosynthetic activity.

Recently, Hiong et al. (2017) cloned and sequenced a host GS from the ctenidium (or gill) of the fluted giant clam, Tridacna squamosa. They demonstrated that the gene and protein expression levels of this host GS in the ctenidium could be up-regulated by light exposure. Their results implicate the host GS as a novel light-enhanced mechanism of increased ammonia assimilation, which lend support to the host assimilation model (Miller and Yellowlees, 1989). They also concluded that the host clam would supply exogenous nitrogen in the form of glutamine to the symbiotic zooxanthellae. Hence, it is logical to hypothesize that symbiotic zooxanthellae would possess a homolog of GOGAT to metabolize the glutamine supplied by the host, but there is currently no information available in this regard. As giant clams normally do not excrete ammonia except when exposed to continuous darkness (Cates and McLaughlin, 1976; Muscatine and D'Elia, 1978; Wilkerson and Muscatine, 1984; Wilkerson and Trench, 1986; Szmant and Gassman, 1990), endogenous ammonia produced through nitrogen metabolism in the host clam must be salvaged effectively by the nitrogen-deficient zooxanthellae, particularly during insolation. It is therefore also logical to hypothesize that symbiotic zooxanthellae would express an algal GS homolog with high affinity to NH₄⁺. Of note, enzyme assays would not be able to separate the activities of enzymes deriving from the symbiotic zooxanthellae and the host clam, but it is possible to elucidate their origins through molecular characterization of their amino acid sequences. Therefore, this study was performed to clone the complete cDNA coding sequences of GS and GLT (GLT is the gene acronym of the GOGAT protein) from the outer mantle, which harbors the majority of the symbiotic zooxanthellae, of T. squamosa. Sequence similarity and phylogenetic analyses of their deduced amino acid sequences were performed to verify that the GS and GOGAT sequences obtained had indeed a zooxanthellal origin. A molecular characterization of the putative GS amino acid sequence obtained from T. squamosa indicated that it was novel, because it displayed unique features not found in other GSs. Furthermore, custom-made anti-GS and anti-GOGAT antibodies were produced to confirm the co-expression of GS and GOGAT in the symbiotic zooxanthellae inside the outer mantle of T. squamosa through immunofluorescence microscopy. It was expected that results obtained would shed light on the mechanism of ammonia assimilation and recycling in the symbiotic zooxanthellae and why the host clam would not excrete ammonia under normal circumstance.