* 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
Correspondence: E-mail: maier{at}staff.uni-marburg.de.
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Abstract |
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Key Words: spheroid body Rhopalodia gibba nitrogen fixation endosymbiont
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Introduction |
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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.
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Methods |
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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.
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Results |
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Discussion |
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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, Söderbäck, 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.
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Acknowledgements |
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Footnotes |
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Laura A. Katz, Associate Editor
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Literature Cited |
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