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Discovery of Symbiotic Nitrogen-Fixing Cyanobacteria in Corals

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Science  13 Aug 2004:
Vol. 305, Issue 5686, pp. 997-1000
DOI: 10.1126/science.1099128

Abstract

Colonies of the Caribbean coral Montastraea cavernosa exhibit a solar-stimulated orange-red fluorescence that is spectrally similar to a variety of fluorescent proteins expressed by corals. The source of this fluorescence is phycoerythrin in unicellular, nonheterocystis, symbiotic cyanobacteria within the host cells of the coral. The cyanobacteria coexist with the symbiotic dinoflagellates (zooxanthellae) of the coral and express the nitrogen-fixing enzyme nitrogenase. The presence of this prokaryotic symbiont in a nitrogen-limited zooxanthellate coral suggests that nitrogen fixation may be an important source of this limiting element for the symbiotic association.

The success of scleractinian corals since the Triassic (1) has been attributed to the establishment of a mutualistic symbiosis between the cnidarian host and a diverse group of endosymbiotic dinoflagellates (zooxanthellae). Zooxanthellae, which are localized within gastrodermal cells of the cnidarian host, can provide more than 100% of the carbon requirements of the animal partner, primarily in the form of carbohydrates and low–molecular-weight lipids. Experimental manipulations of zooxanthellate corals suggest that inorganic nitrogen limits the growth and abundance of zooxanthellae in the coral; indeed, this limitation has been suggested to be essential for the stability of the symbiotic association (2, 3).

In addition to zooxanthellae, a variety of bacteria appear to be associated with scleractinian corals (46), and although these associations also appear to be widely distributed, stable, and nonpathogenic, the function of these bacteria remains largely unknown. However, the presence of cyanobacteria is associated with photosynthesis-dependent nitrogen fixation on coral reefs (7) and is suggested to be responsible for nitrogen fixation in living coral tissue (8). In this paper, we show that large numbers of endosymbiotic cyanobacteria capable of fixing nitrogen occur in a common scleractinian coral, Montastraea cavernosa.

Scleractinian corals, including M. cavernosa (911), express a variety of fluorescent proteins, but colonies of M. cavernosa have also been observed to fluoresce orange during the daytime (Fig. 1A). This fluorescence is not due to a fluorescent protein but to phycoerythrin. In vivo excitation/emission spectra of these corals showed an emission peak at 580 nm and a shoulder at 630 nm, with excitation bands at 505 and 571 nm (Fig. 1B). Although this spectral signature is similar to those reported for red fluorescent proteins of corals (12, 13), the two excitation peaks also corresponded to absorption by phycoerythrin in marine cyanobacteria that contain both the phycourobilin and phycoerythrobilin chromphores (14, 15). Immunoblots (16) of coral homogenates challenged with a polyclonal antibody against phycoerythrin revealed a positive cross-reaction with the 18- to 20-kD β-polypeptide of phycoerythrin (Fig. 1C). These results clearly suggest that intracellular cyanobacteria are associated with the coral.

Fig. 1.

(A) Underwater photograph of M. cavernosa exhibiting orange daytime fluorescence throughout the colony. The colony is approximately 0.6 m high. (B) Measured fluorescence excitation/emission spectrum of an orange-fluorescing M. cavernosa. Orange-fluorescing corals have an emission peak at 580 nm and excitation peaks at 505, 533, and 571 nm. (C) Immunoblot of zooxanthellae-free tissues of M. cavernosa, showing the presence of positive staining for the 18- to 20-kD β-polypeptide of phycoerythrin. (D) Fluorescence lifetime analysis for the orange fluorescent chromophore from M. cavernosa single decay at 580 nm. The results produce a single lifetime component at 3.93 ns.

Fluorescence lifetime analyses (16) indicate that the 580-nm excited state is dominated by a single component with a 3.93-ns time constant (Fig. 1D). The slow single-exponential decay of the orange pigment is longer than described for fluorescent proteins (2.6 to 3.7 ns) (11, 17, 18) and suggests energetic isolation and the absence of excitation energy transfer out of the chromophore. In contrast, phycoerythrin fluorescence normally observed in cyanobacteria exhibits faster kinetics in vivo owing to efficient energy transfer within the phycobilisomes. The lifetime data and the daytime fluorescence indicate that the energy coupling of these pigments to primary photochemistry in the symbiotic cyanobacteria is weak, leading to the relatively high quantum yield of fluorescence for this pigment.

Epifluorescence microscopy (16) of host tissue homogenates revealed zooxanthellae exhibiting red chlorophyll fluorescence, as well as many smaller orange-fluorescent cells resembling cyanobacteria (Fig. 2A). An analysis of tissue homogenates using flow cytometry (16) showed a distinct phycoerythrin signature and a size range for these cells of 1.0 to 3.0 μm in diameter. The number of phycoerythrin-positive cells from fluorescent samples of M. cavernosa, normalized to surface area, ranged from 1.14 × 107 to 2.55 × 107 cells cm–2, whereas nonfluorescent colonies have « 102 phycoerythrin-positive cells per square centimeter. Transmission electron micrographs (16) of the coral tissue revealed that the cyanobacteria-like cells are located in the epithelial cells of the animal host and are surrounded by host membrane (Fig. 2B). The cyanobacteria-like cells exhibit an unusual arrangement of their thylakoid membranes that cross randomly throughout the cell and occasionally appear both expanded and appressed. These cells also appear to have fewer electron-dense phycobilisomes associated with the thylakoid membranes than has been reported for other cyanobacteria. Immunogold probing of thin sections (Fig. 2C), using the antibody to phycoerythrin, showed that the antibody binds significantly [analysis of variance (ANOVA), P < 0.001] to the cyanobacterial cells [134.9 particles per cell ± 19.4 (SE)] when compared to controls [6.5 particles per cell ± 2.1 (SE)] without the primary antibody. These cells also bind positively to a cyanobacteria-specific (CYA762) 16S ribosomal RNA–targeted oligonucleotide probe (Fig. 2D) which cross-reacts with a large number of cyanobacteria (16, 19). Additionally, 16S ribosomal DNA sequencing using cyanobacteria-specific primers (16, 20) yielded a 556-bp sequence (GenBank accession number AY580333) from genomic DNA preparations that match cyanobacterial sequences (n = 100) related to Synechococcus sp., Prochlorococcus sp., or uncultured cyanobacteria in the Order Chroococcales, a paraphyletic group (21), with 93 to 97% sequence homology.

Fig. 2.

(A) Epifluorescent micrograph (magnification, ×1000) of coral homogenate showing red-fluorescing (685-nm fluorescence from chlorophyll) zooxanthellae and 1- to 3-μm orange-fluorescing cyanobacteria (arrows). (B) Electron micrograph of epithelial tissues of M. cavernosa. Scale bar, 1.0 μm. C, cyanobacterium; N, nematocyst; T, thylakoid; HM, host membrane; SV, secretory vesicle. (C) Immunogold labeling (20-nm gold particles, arrows) of thin sections for phycoerythrin (magnification, ×50,000). (D) Fluorescent in situ hybridization micrograph (magnification, ×1000) of M. cavernosa epithelial tissues showing positive binding of cyanobacterial-specific probe (arrows).

To assess the potential for nitrogen fixation in this coral, we challenged protein extracts with a polyclonal antibody to the 32-kD Fe protein subunit of nitrogenase (Fig. 3A). The results yielded a single positive cross-reaction, strongly indicating expression of the gene in the coral. Again, using the same antibody to nitrogenase, we used immunogold probing of thin sections (Fig. 3B) and found that the antibody binds significantly (ANOVA, P = 0.006) to the cyanobacterial cells [8.8 particles per cell ± 1.3 (SE)] when compared to controls [4.5 particles per cell ± 0.5 (SE)] without the primary antibody. Many members of the Order Chroococcales are capable of fixing nitrogen. Our results clearly suggest that endosymbiotic cyanobacteria capable of fixing nitrogen are present in M. cavernosa and form a stable long-term association within host cells. This symbiont could potentially be a source of the limiting element nitrogen for the symbiosis through the release of fixed nitrogen products to the coral host.

Fig. 3.

(A) Positive immunoblot for the 32-kD Fe protein of nitrogenase in zooxanthellae-free tissues of M. cavernosa. (B) Immunogold labeling (20-nm gold particles, arrow) of thin sections for nitrogenase (magnification, ×50,000).

Like the symbiotic cyanobacteria of M. cavernosa, free-living cyanobacteria and prochlorophytes that contain phycoerythrin often exhibit strong fluorescence under certain conditions, with a maximum emission between 570 to 580 nm (22, 23). Uncoupled phycoerythrin has been proposed to serve as a storage pool of nitrogen in phycobilin-containing cyanobacteria (22) but not in prochlorophytes (23). Phycoerythrin detachment from the photosynthetic apparatus in cyanobacteria and prochlorophytes can be caused by exposure to glycerol and results in strong fluorescence by eliminating the quenching associated with energy transfer from phycoerythrin to the reaction centers (22, 23). The cyanobacterial symbionts of M. cavernosa are exposed to high concentrations of glycerol in the coral, because it is the major carbon compound translocated from the symbiotic zooxanthellae to the host tissues (24), and this may explain both the unusual ultrastructure and the characteristic orange fluorescence emission of the symbiotic cyanobacteria. Because little or no energy is transferred from phycoerythrin to primary photochemistry, glycerol supplied from the zooxanthellae may serve as an energy source for the cyanobacteria operating heterotrophically and provide a steady supply of reductant and adenosine triphosphate for nitrogen fixation in the symbionts. Nitrogenase is also sensitive to molecular and reactive species of oxygen that accumulate during photosynthesis (25, 26), and the symbiotic cyanobacteria, operating heterotrophically, could quench molecular oxygen via respiration and/or by the Mehler reaction (27). Additionally, the coral environment is well suited for the temporal separation of photosynthesis and nitrogen fixation, because coral tissues experience extreme hypoxia at night (25). The presence of an additional symbiont in a zooxanthellate coral that is nitrogen-limited (2) suggests that nitrogen fixation may be an important supplemental source of the limiting element for the symbiotic association, and it highlights the potential significance of microbial consortia composed of photosynthetic eukaryotes and prokaryotes (28). Cyanobacteria are involved in many diverse mutualistic symbioses in both terrestrial and marine environments, and they provide critical ecological services, including important contributions to the global nitrogen cycle (29).

Supporting Online Material

www.sciencemag.org/cgi/content/full/305/5686/997/DC1

Materials and Methods

Fig. S1

References and Notes

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