Intracellular Spheroid Bodies of Rhopalodia gibba Have Nitrogen-Fixing Apparatus of Cyanobacterial Origin
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分子生物学进展 2004年第8期
* Cell Biology, Philipps-University Marburg, Marburg, Germany
Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand
E-mail: maier@staff.uni-marburg.de.
Abstract
Nitrogen fixation is not regarded as a eukaryotic invention. The process has only been reported as being carried out by bacteria. These prokaryotes typically interact with their eukaryotic hosts as extracellular and temporary nonobligate nitrogen-fixing symbionts. However, intracellular permanent "spheroid bodies" have been reported within the fresh-water diatom Rhopalodia gibba, and these, too, have been speculated as being able to provide nitrogen to their host diatom. These spheroid bodies have gram-negative characteristics with thylakoids. We demonstrate that they fix nitrogen under light conditions. We also show that phylogenetic analyses of their 16rRNA and nif D genes predict that their genome is closely related to that of Cyanothece sp. ATCC 51.142, a free-living diazotrophic cyanobacterium. We suggest that the intracellular spheroid bodies of Rhopalodia gibba may represent a vertically transmitted, permanent endosymbiotic stage in the transition from a free-living diazotrophic cyanobacterium to a nitrogen-fixing eukaryotic organelle.
Key Words: spheroid body ? Rhopalodia gibba ? nitrogen fixation ? endosymbiont
Introduction
Cyanobacteria occur not only as free-living bacteria. Some species exist in extra or intracellular associations with eukaryotic hosts (Rai, S?derb?ck, and Bergmann 2000). In plants, these associations are of significant scientific and agricultural interest and, thus, have been the subject of much research (Bergmann and Osborne 2002). In protists by contrast, where cyanobacterial symbionts and endosymbionts with the potential to fix nitrogen have been known for decades, scientific description is much less advanced (Rai, S?derb?ck, and Bergmann 2000; Carpenter 2002; Stoebe and Maier 2002). Nevertheless, it is clear that symbiotic interactions with protists range from those that involve temporary extracellular cyanobacteria to those that involve more permanent intracellular endosymbionts (Janson et al. 1999; Rai, S?derb?ck, and Bergmann 2000). Furthermore, kleptoplastidic interactions between heterotrophic euglenids and cyanobacteria are also known (Schnepf, Schlegel, and Hepperle 2002).
Diatoms are widespread protists with high ecological impact. Some genera have symbiotic associations with extracellular or intracellular cyanobacteria, and it has been suggested that the hosts benefit from the nitrogen fixation capacity of their symbionts (Janson et al. 1999; Carpenter and Janson 2000). Intracellular inclusions in the form of "spheroid bodies" have been identified in the diatom genera Epithemia and Rhopalodia (Geitler 1977). Ultrastructural characterizations show a double membrane surrounding the spheroid body and invaginations of the inner membrane. These observations have been interpreted to suggest that the spheroid bodies may be either unique organelles or obligate endosymbiotic intracellular organisms (Drum and Pankratz 1965).
Floener and Bothe (1980) reported that one diatom strain, Rhopalodia gibba, has the unusual capacity to fix nitrogen. This observation led these authors to speculate that the spheroid bodies may contain the enzymatic machinery for the nitrogen-fixation reaction (Floener and Bothe 1980). Here we describe observations consistent with the permanent nature of the interaction between spheroid bodies and R. gibba. We demonstrate that the spheroid bodies fix nitrogen in light, and through analysis of 16SrRNA and nifD genes, we identify their closest phylogenetic relatives.
Methods
Culture
Rhopalodia gibba was isolated from the Botanical Garden in Marburg, Germany and grown according to Floener and Bothe (1980).
Nitrogen Fixation Assay
Nitrogen fixation was estimated by the C2H2-reduction assay (Stewart, Fitzgerald, and Burris 1967). The experiments were carried out at 20°C in 450 ml Fernbach flasks. Rhopalodia gibba was grown for 3 to 4 days in 100 ml medium containing no nitrogen source and with an O2 concentration of approximately 20% in the gas phase. To analyze the reduction activity, the fixation assay was accomplished with cultures incubated under a light/dark regime for 16/8 h and by growing cultures in the dark. After addition of 10% acetylene, the reduction to ethylene was measured for 72 h in a Varian model 3380 gas chromatograph fitted with a flame ionization detector and a Chromopak column. As a control, intrinsic ethylene production of the diatom was measured. The protein content of cultures was determined with the Bradford assay after complete destruction of the cells with glass beads.
Isolation of Spheroid Bodies and Genomic DNA
R. gibba cells were harvested at 3,500 x g for 5 min and washed several times in culture medium. The cell pellet was resuspended in isolation buffer (330 mM sorbitol 20 mM MOPS, 13 mM Tris-base, 3 mM MgCl2, 0.1% [w/v] BSA) and the cells disrupted at 4°C by homogenization. Large cell debris and intact cells were afterwards removed by centrifugation at 100 x g for 3 min. A crude spheroid fraction, which was obtained by centrifugation of the supernatant at 3,000 x g for 5 min was loaded on a discontinuous Percoll gradient in 1 x g HMS (50 mM HEPES/KOH, 3 mM MgCl2, 330 mM sorbitol, pH 7.6). Intact spheroid bodies were detected at the 70%/60% Percoll boundary. This fraction was collected and washed in 1x HMS. Isolation of genomic DNA from the spheroid body fraction was carried out using standard protocols.
PCR Reactions
The primer pair 5'-AGA GTT TGA TCA TGG CTC AG-3' and 5'-AAG GAG GTG ATC CAA CCG CA-3' was used for the amplification of the 16S rDNA. The nifD fragment was obtained with the primers 5'-CAC CAC ATT GCT AAC GA-3' and 5'-AAG AGT GCA TTT GAC GG-3'.
In Situ Hybridization
Cell were fixed with 4% glutaraldehyde, dehydrated and embedded in LRgold. Sections were prehybridized for 2 to 3 h in hybridization buffer (McFadden 1991). Hybridizations were carried out overnight and used the biotinylated primer 5'-GCA CGG CTT GGG TCG ATA CAA-3'. After washing the sections with 4xSSC, 2xSSC, and 1xSSC, respectively, cross reactivity was tested using an anti-biotin IgG and a secondary anti-mouse IgG Cy2. Images were obtained with a Leica confocal laser microscope.
In Situ Localizations
These procedures were performed according to the protocols in Fraunholz, Moerschel, and Maier (1998).
Phylogenetic Analyses
For rDNA sequences, a minimum-evolution tree was built using PAUP* (Swofford 2001). Sites containing insertions or deletions were excluded from the RDP secondary structure alignment for 16S rRNA sequences (http://rdp.cme.msu.edu/html). Objective distances were estimated for 1,013 sequence positions. These estimates, made using a general time reversible (GTR) model assumed a proportion of invariable sites and gamma distributed rates. For nifD protein sequences, the most conservative regions (230 residues) of a ClustalX version 1.8 (Thompson et al. 1997) alignment were used. These regions were ungapped and bounded by columns of residues from positively scoring groups in Gonnet log odds matrices. Objective distances were estimated assuming a JTT model of substitution and a gamma distribution of rates. The alpha shape parameter was estimated using PAML (Yang 1997), and objective distances and minimum-evolution trees were calculated using PHYLIP version 3.6a3 (Felsenstein 2002). For both rRNA and nifD sequences, nonparamateric bootstrapping used 1,000 replicates. The sequences generated from this study have been deposited in the DDBJ/EMBL/GenBank under the accession numbers AJ582391 and AJ582390.
Results
Rhopalodia gibba is a pennate diatom of the family Epithemiaceae (Geitler 1977). There are reports indicating different numbers of spheroid bodies per cell, depending on the culture conditions (DeYoe, Lowe, and Marks 1992). However, under our laboratory environment, we always detected four permanent spheroid bodies per cell. Ultrastructural investigations showed that the spheroid bodies are intracellular and separated from the cytoplasm of the host cell by one membrane (fig. 1a). The endosymbionts are surrounded by a double membrane with no cell wall and show internal membranes reminiscent of thylakoids. Such a phenotype is similar to that of nonfilamentous, unicellular cyanobacteria. By mechanical treatment of the cells and the use of Percoll gradients, we were able to separate the spheroid bodies from other cell compartments and organelles, leading to a spheroid body fraction as shown in figure 1b. Apart from the diatom cell wall debris, no other contaminating cell components were detected in this preparation. Consistent with a permanent nature of the endosymbiosis, the spheroid bodies were found to be unable to grow in a variety of different media outside the diatom host, including media that has been used to successfully culture Cyanothece sp. ATCC 51.142.
FIG. 1. Spheroid bodies are shown. (a) Ultrastruture of a spheroid body. (b) Spheroid body fraction from Percoll gradients, showing that spheroid bodies can be isolated without substantial cellular contamination. SM, symbiontophoric membrane; SBM1, inner membrane of the spheroid body; SBM2, outer membrane of the spheroid body
DNA was extracted from the spheroid body fraction, and a 1,479-bp fragment was amplified using standard 16S-specific primers (Wuyts et al. 2002) and sequenced. Blast searches identified homology between this sequence and cyanobacterial 16S rDNA genes. To identify a cyanobacterial-specific stretch for the synthesis of a spheroid body–specific primer, the sequence was aligned with 16S rDNA genes from a variety of eubacteria, including sequences from rhizobia. Southern hybridizations with DNA fractions from the spheroid bodies and control DNAs were used to demonstrate that this primer was specific for a cyanobacterium-like 16S rDNA originating from the spheroid body genome (supporting figure 1). In situ hybridizations with this 16S rDNA–specific primer specifically label the spheroid bodies (fig. 2b), thereby demonstrating that the isolated 16S rDNA originated from the spheroid bodies.
FIG. 2. (a) In situ hybridization of spheroid-specific 16S rDNA within the spheroid bodies. Phase contrast image of a spheroid body within the diatom host cell. (b) In situ hybridization of a primer spanning 21 bp of the spheroid body–specific rDNA. One spheroid body shows specific hybridization
The spheroid-DNA deduced 16S sequence was integrated into an RDP database secondary structure alignment (1,013 ungapped positions) of 175 taxa containing a representative sequence from eight major groups of nonphotosynthetic eubacteria, nine diverse plastids, and 158 cyanobacteria. Included in this alignment were cyanobacterial sequences not present in the RDP database, but which had very high alignment scores with the spheroid body sequence when this sequence was Blasted against GenBank. Preliminary analyses with neighbor joining (Jukes-Cantor substitution model) and nonparametric bootstrapping showed that the spheroid body sequence grouped strongly within the cyanobacterial clade (figure not shown). Minimum evolution (using optimal GTR- distances: pinv = 0.34093, = 0.52797) was then used to test more precisely for the closest relative to the spheroid body sequence among a diverse selection of plastid and cyanobacterial sequences. The spheroid sequence was found to be most closely related to two strains of the nitrogen-fixing cyanobacterium Cyanothece, Cyanothece sp. ATCC51.142 and Cyanthece sp. PCC8801 and with the cyanobacterial symbiont of the diatom Climacodium frauenfeldianum (fig. 3), an intracellular symbiont speculated as having the potential for nitrogen fixation (Carpenter and Janson 2000).
FIG. 3. Minimum-evolution tree for 16S rDNA sequences. Branch lengths and nonparametric bootstrap values for important internal branches are shown
Floener and Bothe (1980) investigated the diatom R. gibba for its capacity to fix molecular nitrogen. They were able to demonstrate that the eukaryotic diatom cell can reduce acetylene to ethylene. Thus, they speculated that the spheroid bodies may provide fixed nitrogen to its endosymbiotic host. We reinvestigated R. gibba for its nitrogen fixation capacity and have confirmed the findings of Floener and Bothe (1980). As shown in figure 4, acetylene is converted into ethylene (0.367 nmol ethylene per h per 2.5x106 cells) in daylight. This is an observation consistent with Nif-dependent, nitrogen-fixing machinery being located in this cell. These results prompted us to investigate the spheroid body DNA for genes encoding Nif proteins. By aligning GenBank database entries for nifD genes, we designed a cyanobacterium-specific primer pair. These primers were used to amplify a fragment of 751 bp, encoding part of the nifD gene. A minimum-evolution phylogenetic tree (using opimal JTT- distance: = 0.57200) was constructed, showing that the amplified sequence from the spheroid body was of cyanobacterial ancestry (fig. 5), with highest similarity to the Cyanothece sp. ATCC 51.142 sequence. To test whether nitrogenase could be detected at the protein level, an antibody specific for the subunit of nitrogenase MoFe protein was used for in situ localization. In these studies, a negative control without the antinitrogenase antibody, detected only unspecific binding of the secondary gold-coupled antibody to the cell wall of the diatom (data not shown). In contrast, a specific signal was detected inside the spheroid bodies by the use of the antinitrogenase antibody, thereby showing that the nitrogenase was expressed inside the endosymbiont (supporting figure 2).
FIG. 4. "Nitrogen fixation" of Rhopalodia. Solid lines show the conversion of acetylene into ethylene under light conditions. Dashede lines indicate activity in the dark
FIG. 5. Minimum-evolution tree for NifD protein sequences. Branch lengths and bootstrap values for internal branches receiving greater than 80% support are shown
Discussion
Our finding that spheroid bodies are intracellular nitrogen-fixing endosymbionts of cyanobacterial origin extends current understanding of plant biodiversity. To our knowledge, the spheroid body of R. gibba is the first demonstrated example of an intracellular, nonfilamentous cyanobacterial symbiont that has the potential to provide fixed nitrogen to its eukaryotic host. This we have shown by a combination of gene and protein detection, as well as by enzymatic assay. The spheroid bodies of R. gibba are closely related to the intracellular cyanobacterium detected in Climacodium frauenfeldianum, for which the potential of nitrogen fixation has been speculated (Carpenter and Janson 2000). Moreover, DNA sequences from the spheroid bodies group with free-living cyanobacteria of the genus Cyanothece in phylogenetic reconstructions. The latter genus is an important component of phytoplankton in tropical environments (Falcón et al. 1999).
Spheroid bodies seem not to be photosynthetically active. This implies that the energy-consuming nitrogen fixation process is powered by import of energy-rich molecules from the host, which requires modification of host and cyanobacterial physiology. Moreover, the physiology of the endosymbiont is additionally modified in its photoperiodicity, because the free-living Cyanothece fixes nitrogen at night (Reddy et al. 1993), whereas the spheroid body is active in nitrogen fixation when the diatom chloroplast is converting light energy. Unlike the intracellular symbiosis known to occur in Gunnera, where Nostoc invades preexisting stem glands and forms nitrogen-fixing heterocysts, spheroid bodies in diatoms persist from generation to generation (Rai, S?derb?ck, and Bergmann 2000). These are all observations that support a hypothesis that spheroid bodies are in a transitional stage of becoming an intracellular organelle or at the very least an obligate permanent, vertically inherited endosymbiont.
Phylogenetic and functional genomic comparisons of Cyanothece strains and diatom spheroid bodies offer a unique situation to develop understanding of endosymbiosis. Although endosymbiotic events have led to the evolution of cyanelles, plastids, and mitochondria, these organelles are all anciently diverged from their nearest eubacterial relatives. This makes studying the transition from free-living prokaryote to permanent and obligate endosymbiont difficult. The very close phylogenetic relationship between intracellular spheroid bodies and extant cyanobacteria is likely to make for a much better model system.
An important question is whether spheroid bodies have lost genes in the course of their intracellular association and whether genes have been transferred into the cell nucleus of the diatom. If the latter scenario, including a reimport of the gene product, is demonstrated, the spheroid bodies would need to be regarded as a new and obligate, DNA-containing organelle, equal to plastids and mitochondria.
Acknowledgements
We gratefully acknowledge the electron microscopical work done by Marinanne Johannsen and help in the nitrogen-fixing assay by Professor Erhard M?rschel, and we thank Dr. Karsten Niehaus (University of Bielefeld, Germany) for the anti–alpha subunit MoFe-protein antibody. Our work is supported by the Deutsche Forschungsgemeinschaft (SFB 395, TP B9), the Alexander von Humboldt Foundation, and New Zealand Marsden Fund.
Literature Cited
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Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand
E-mail: maier@staff.uni-marburg.de.
Abstract
Nitrogen fixation is not regarded as a eukaryotic invention. The process has only been reported as being carried out by bacteria. These prokaryotes typically interact with their eukaryotic hosts as extracellular and temporary nonobligate nitrogen-fixing symbionts. However, intracellular permanent "spheroid bodies" have been reported within the fresh-water diatom Rhopalodia gibba, and these, too, have been speculated as being able to provide nitrogen to their host diatom. These spheroid bodies have gram-negative characteristics with thylakoids. We demonstrate that they fix nitrogen under light conditions. We also show that phylogenetic analyses of their 16rRNA and nif D genes predict that their genome is closely related to that of Cyanothece sp. ATCC 51.142, a free-living diazotrophic cyanobacterium. We suggest that the intracellular spheroid bodies of Rhopalodia gibba may represent a vertically transmitted, permanent endosymbiotic stage in the transition from a free-living diazotrophic cyanobacterium to a nitrogen-fixing eukaryotic organelle.
Key Words: spheroid body ? Rhopalodia gibba ? nitrogen fixation ? endosymbiont
Introduction
Cyanobacteria occur not only as free-living bacteria. Some species exist in extra or intracellular associations with eukaryotic hosts (Rai, S?derb?ck, and Bergmann 2000). In plants, these associations are of significant scientific and agricultural interest and, thus, have been the subject of much research (Bergmann and Osborne 2002). In protists by contrast, where cyanobacterial symbionts and endosymbionts with the potential to fix nitrogen have been known for decades, scientific description is much less advanced (Rai, S?derb?ck, and Bergmann 2000; Carpenter 2002; Stoebe and Maier 2002). Nevertheless, it is clear that symbiotic interactions with protists range from those that involve temporary extracellular cyanobacteria to those that involve more permanent intracellular endosymbionts (Janson et al. 1999; Rai, S?derb?ck, and Bergmann 2000). Furthermore, kleptoplastidic interactions between heterotrophic euglenids and cyanobacteria are also known (Schnepf, Schlegel, and Hepperle 2002).
Diatoms are widespread protists with high ecological impact. Some genera have symbiotic associations with extracellular or intracellular cyanobacteria, and it has been suggested that the hosts benefit from the nitrogen fixation capacity of their symbionts (Janson et al. 1999; Carpenter and Janson 2000). Intracellular inclusions in the form of "spheroid bodies" have been identified in the diatom genera Epithemia and Rhopalodia (Geitler 1977). Ultrastructural characterizations show a double membrane surrounding the spheroid body and invaginations of the inner membrane. These observations have been interpreted to suggest that the spheroid bodies may be either unique organelles or obligate endosymbiotic intracellular organisms (Drum and Pankratz 1965).
Floener and Bothe (1980) reported that one diatom strain, Rhopalodia gibba, has the unusual capacity to fix nitrogen. This observation led these authors to speculate that the spheroid bodies may contain the enzymatic machinery for the nitrogen-fixation reaction (Floener and Bothe 1980). Here we describe observations consistent with the permanent nature of the interaction between spheroid bodies and R. gibba. We demonstrate that the spheroid bodies fix nitrogen in light, and through analysis of 16SrRNA and nifD genes, we identify their closest phylogenetic relatives.
Methods
Culture
Rhopalodia gibba was isolated from the Botanical Garden in Marburg, Germany and grown according to Floener and Bothe (1980).
Nitrogen Fixation Assay
Nitrogen fixation was estimated by the C2H2-reduction assay (Stewart, Fitzgerald, and Burris 1967). The experiments were carried out at 20°C in 450 ml Fernbach flasks. Rhopalodia gibba was grown for 3 to 4 days in 100 ml medium containing no nitrogen source and with an O2 concentration of approximately 20% in the gas phase. To analyze the reduction activity, the fixation assay was accomplished with cultures incubated under a light/dark regime for 16/8 h and by growing cultures in the dark. After addition of 10% acetylene, the reduction to ethylene was measured for 72 h in a Varian model 3380 gas chromatograph fitted with a flame ionization detector and a Chromopak column. As a control, intrinsic ethylene production of the diatom was measured. The protein content of cultures was determined with the Bradford assay after complete destruction of the cells with glass beads.
Isolation of Spheroid Bodies and Genomic DNA
R. gibba cells were harvested at 3,500 x g for 5 min and washed several times in culture medium. The cell pellet was resuspended in isolation buffer (330 mM sorbitol 20 mM MOPS, 13 mM Tris-base, 3 mM MgCl2, 0.1% [w/v] BSA) and the cells disrupted at 4°C by homogenization. Large cell debris and intact cells were afterwards removed by centrifugation at 100 x g for 3 min. A crude spheroid fraction, which was obtained by centrifugation of the supernatant at 3,000 x g for 5 min was loaded on a discontinuous Percoll gradient in 1 x g HMS (50 mM HEPES/KOH, 3 mM MgCl2, 330 mM sorbitol, pH 7.6). Intact spheroid bodies were detected at the 70%/60% Percoll boundary. This fraction was collected and washed in 1x HMS. Isolation of genomic DNA from the spheroid body fraction was carried out using standard protocols.
PCR Reactions
The primer pair 5'-AGA GTT TGA TCA TGG CTC AG-3' and 5'-AAG GAG GTG ATC CAA CCG CA-3' was used for the amplification of the 16S rDNA. The nifD fragment was obtained with the primers 5'-CAC CAC ATT GCT AAC GA-3' and 5'-AAG AGT GCA TTT GAC GG-3'.
In Situ Hybridization
Cell were fixed with 4% glutaraldehyde, dehydrated and embedded in LRgold. Sections were prehybridized for 2 to 3 h in hybridization buffer (McFadden 1991). Hybridizations were carried out overnight and used the biotinylated primer 5'-GCA CGG CTT GGG TCG ATA CAA-3'. After washing the sections with 4xSSC, 2xSSC, and 1xSSC, respectively, cross reactivity was tested using an anti-biotin IgG and a secondary anti-mouse IgG Cy2. Images were obtained with a Leica confocal laser microscope.
In Situ Localizations
These procedures were performed according to the protocols in Fraunholz, Moerschel, and Maier (1998).
Phylogenetic Analyses
For rDNA sequences, a minimum-evolution tree was built using PAUP* (Swofford 2001). Sites containing insertions or deletions were excluded from the RDP secondary structure alignment for 16S rRNA sequences (http://rdp.cme.msu.edu/html). Objective distances were estimated for 1,013 sequence positions. These estimates, made using a general time reversible (GTR) model assumed a proportion of invariable sites and gamma distributed rates. For nifD protein sequences, the most conservative regions (230 residues) of a ClustalX version 1.8 (Thompson et al. 1997) alignment were used. These regions were ungapped and bounded by columns of residues from positively scoring groups in Gonnet log odds matrices. Objective distances were estimated assuming a JTT model of substitution and a gamma distribution of rates. The alpha shape parameter was estimated using PAML (Yang 1997), and objective distances and minimum-evolution trees were calculated using PHYLIP version 3.6a3 (Felsenstein 2002). For both rRNA and nifD sequences, nonparamateric bootstrapping used 1,000 replicates. The sequences generated from this study have been deposited in the DDBJ/EMBL/GenBank under the accession numbers AJ582391 and AJ582390.
Results
Rhopalodia gibba is a pennate diatom of the family Epithemiaceae (Geitler 1977). There are reports indicating different numbers of spheroid bodies per cell, depending on the culture conditions (DeYoe, Lowe, and Marks 1992). However, under our laboratory environment, we always detected four permanent spheroid bodies per cell. Ultrastructural investigations showed that the spheroid bodies are intracellular and separated from the cytoplasm of the host cell by one membrane (fig. 1a). The endosymbionts are surrounded by a double membrane with no cell wall and show internal membranes reminiscent of thylakoids. Such a phenotype is similar to that of nonfilamentous, unicellular cyanobacteria. By mechanical treatment of the cells and the use of Percoll gradients, we were able to separate the spheroid bodies from other cell compartments and organelles, leading to a spheroid body fraction as shown in figure 1b. Apart from the diatom cell wall debris, no other contaminating cell components were detected in this preparation. Consistent with a permanent nature of the endosymbiosis, the spheroid bodies were found to be unable to grow in a variety of different media outside the diatom host, including media that has been used to successfully culture Cyanothece sp. ATCC 51.142.
FIG. 1. Spheroid bodies are shown. (a) Ultrastruture of a spheroid body. (b) Spheroid body fraction from Percoll gradients, showing that spheroid bodies can be isolated without substantial cellular contamination. SM, symbiontophoric membrane; SBM1, inner membrane of the spheroid body; SBM2, outer membrane of the spheroid body
DNA was extracted from the spheroid body fraction, and a 1,479-bp fragment was amplified using standard 16S-specific primers (Wuyts et al. 2002) and sequenced. Blast searches identified homology between this sequence and cyanobacterial 16S rDNA genes. To identify a cyanobacterial-specific stretch for the synthesis of a spheroid body–specific primer, the sequence was aligned with 16S rDNA genes from a variety of eubacteria, including sequences from rhizobia. Southern hybridizations with DNA fractions from the spheroid bodies and control DNAs were used to demonstrate that this primer was specific for a cyanobacterium-like 16S rDNA originating from the spheroid body genome (supporting figure 1). In situ hybridizations with this 16S rDNA–specific primer specifically label the spheroid bodies (fig. 2b), thereby demonstrating that the isolated 16S rDNA originated from the spheroid bodies.
FIG. 2. (a) In situ hybridization of spheroid-specific 16S rDNA within the spheroid bodies. Phase contrast image of a spheroid body within the diatom host cell. (b) In situ hybridization of a primer spanning 21 bp of the spheroid body–specific rDNA. One spheroid body shows specific hybridization
The spheroid-DNA deduced 16S sequence was integrated into an RDP database secondary structure alignment (1,013 ungapped positions) of 175 taxa containing a representative sequence from eight major groups of nonphotosynthetic eubacteria, nine diverse plastids, and 158 cyanobacteria. Included in this alignment were cyanobacterial sequences not present in the RDP database, but which had very high alignment scores with the spheroid body sequence when this sequence was Blasted against GenBank. Preliminary analyses with neighbor joining (Jukes-Cantor substitution model) and nonparametric bootstrapping showed that the spheroid body sequence grouped strongly within the cyanobacterial clade (figure not shown). Minimum evolution (using optimal GTR- distances: pinv = 0.34093, = 0.52797) was then used to test more precisely for the closest relative to the spheroid body sequence among a diverse selection of plastid and cyanobacterial sequences. The spheroid sequence was found to be most closely related to two strains of the nitrogen-fixing cyanobacterium Cyanothece, Cyanothece sp. ATCC51.142 and Cyanthece sp. PCC8801 and with the cyanobacterial symbiont of the diatom Climacodium frauenfeldianum (fig. 3), an intracellular symbiont speculated as having the potential for nitrogen fixation (Carpenter and Janson 2000).
FIG. 3. Minimum-evolution tree for 16S rDNA sequences. Branch lengths and nonparametric bootstrap values for important internal branches are shown
Floener and Bothe (1980) investigated the diatom R. gibba for its capacity to fix molecular nitrogen. They were able to demonstrate that the eukaryotic diatom cell can reduce acetylene to ethylene. Thus, they speculated that the spheroid bodies may provide fixed nitrogen to its endosymbiotic host. We reinvestigated R. gibba for its nitrogen fixation capacity and have confirmed the findings of Floener and Bothe (1980). As shown in figure 4, acetylene is converted into ethylene (0.367 nmol ethylene per h per 2.5x106 cells) in daylight. This is an observation consistent with Nif-dependent, nitrogen-fixing machinery being located in this cell. These results prompted us to investigate the spheroid body DNA for genes encoding Nif proteins. By aligning GenBank database entries for nifD genes, we designed a cyanobacterium-specific primer pair. These primers were used to amplify a fragment of 751 bp, encoding part of the nifD gene. A minimum-evolution phylogenetic tree (using opimal JTT- distance: = 0.57200) was constructed, showing that the amplified sequence from the spheroid body was of cyanobacterial ancestry (fig. 5), with highest similarity to the Cyanothece sp. ATCC 51.142 sequence. To test whether nitrogenase could be detected at the protein level, an antibody specific for the subunit of nitrogenase MoFe protein was used for in situ localization. In these studies, a negative control without the antinitrogenase antibody, detected only unspecific binding of the secondary gold-coupled antibody to the cell wall of the diatom (data not shown). In contrast, a specific signal was detected inside the spheroid bodies by the use of the antinitrogenase antibody, thereby showing that the nitrogenase was expressed inside the endosymbiont (supporting figure 2).
FIG. 4. "Nitrogen fixation" of Rhopalodia. Solid lines show the conversion of acetylene into ethylene under light conditions. Dashede lines indicate activity in the dark
FIG. 5. Minimum-evolution tree for NifD protein sequences. Branch lengths and bootstrap values for internal branches receiving greater than 80% support are shown
Discussion
Our finding that spheroid bodies are intracellular nitrogen-fixing endosymbionts of cyanobacterial origin extends current understanding of plant biodiversity. To our knowledge, the spheroid body of R. gibba is the first demonstrated example of an intracellular, nonfilamentous cyanobacterial symbiont that has the potential to provide fixed nitrogen to its eukaryotic host. This we have shown by a combination of gene and protein detection, as well as by enzymatic assay. The spheroid bodies of R. gibba are closely related to the intracellular cyanobacterium detected in Climacodium frauenfeldianum, for which the potential of nitrogen fixation has been speculated (Carpenter and Janson 2000). Moreover, DNA sequences from the spheroid bodies group with free-living cyanobacteria of the genus Cyanothece in phylogenetic reconstructions. The latter genus is an important component of phytoplankton in tropical environments (Falcón et al. 1999).
Spheroid bodies seem not to be photosynthetically active. This implies that the energy-consuming nitrogen fixation process is powered by import of energy-rich molecules from the host, which requires modification of host and cyanobacterial physiology. Moreover, the physiology of the endosymbiont is additionally modified in its photoperiodicity, because the free-living Cyanothece fixes nitrogen at night (Reddy et al. 1993), whereas the spheroid body is active in nitrogen fixation when the diatom chloroplast is converting light energy. Unlike the intracellular symbiosis known to occur in Gunnera, where Nostoc invades preexisting stem glands and forms nitrogen-fixing heterocysts, spheroid bodies in diatoms persist from generation to generation (Rai, S?derb?ck, and Bergmann 2000). These are all observations that support a hypothesis that spheroid bodies are in a transitional stage of becoming an intracellular organelle or at the very least an obligate permanent, vertically inherited endosymbiont.
Phylogenetic and functional genomic comparisons of Cyanothece strains and diatom spheroid bodies offer a unique situation to develop understanding of endosymbiosis. Although endosymbiotic events have led to the evolution of cyanelles, plastids, and mitochondria, these organelles are all anciently diverged from their nearest eubacterial relatives. This makes studying the transition from free-living prokaryote to permanent and obligate endosymbiont difficult. The very close phylogenetic relationship between intracellular spheroid bodies and extant cyanobacteria is likely to make for a much better model system.
An important question is whether spheroid bodies have lost genes in the course of their intracellular association and whether genes have been transferred into the cell nucleus of the diatom. If the latter scenario, including a reimport of the gene product, is demonstrated, the spheroid bodies would need to be regarded as a new and obligate, DNA-containing organelle, equal to plastids and mitochondria.
Acknowledgements
We gratefully acknowledge the electron microscopical work done by Marinanne Johannsen and help in the nitrogen-fixing assay by Professor Erhard M?rschel, and we thank Dr. Karsten Niehaus (University of Bielefeld, Germany) for the anti–alpha subunit MoFe-protein antibody. Our work is supported by the Deutsche Forschungsgemeinschaft (SFB 395, TP B9), the Alexander von Humboldt Foundation, and New Zealand Marsden Fund.
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