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      Discovery of an Endosymbiotic Nitrogen-Fixing Cyanobacterium UCYN-A in Braarudosphaera bigelowii (Prymnesiophyceae)

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          Abstract

          Braarudosphaera bigelowii (Prymnesiophyceae) is a coastal coccolithophore with a long fossil record, extending back to the late Cretaceous (ca. 100 Ma). A recent study revealed close phylogenetic relationships between B. bigelowii, Chrysochromulina parkeae (Prymnesiophyceae), and a prymnesiophyte that forms a symbiotic association with the nitrogen-fixing cyanobacterium UCYN-A. In order to further examine these relationships, we conducted transmission electron microscopic and molecular phylogenetic studies of B. bigelowii. TEM studies showed that, in addition to organelles, such as the nucleus, chloroplasts and mitochondria, B. bigelowii contains one or two spheroid bodies with internal lamellae. In the 18S rDNA tree of the Prymnesiophyceae, C. parkeae fell within the B. bigelowii clade, and was close to B. bigelowii Genotype III (99.89% similarity). Plastid 16S rDNA sequences obtained from B. bigelowii were close to the unidentified sequences from the oligotrophic SE Pacific Ocean (e.g. HM133411) (99.86% similarity). Bacterial16S rDNA sequences obtained from B. bigelowii were identical to the UCYN-A sequence AY621693 from Arabian Sea, and fell in the UCYN-A clade. From these results, we suggest that; 1) C. parkeae is the alternate life cycle stage of B. bigelowii sensu stricto or that of a sibling species of B. bigelowii, and 2) the spheroid body of B. bigelowii originated from endosymbiosis of the nitrogen-fixing cyanobacterium UCYN-A.

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          Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga.

          Symbioses between nitrogen (N)(2)-fixing prokaryotes and photosynthetic eukaryotes are important for nitrogen acquisition in N-limited environments. Recently, a widely distributed planktonic uncultured nitrogen-fixing cyanobacterium (UCYN-A) was found to have unprecedented genome reduction, including the lack of oxygen-evolving photosystem II and the tricarboxylic acid cycle, which suggested partnership in a symbiosis. We showed that UCYN-A has a symbiotic association with a unicellular prymnesiophyte, closely related to calcifying taxa present in the fossil record. The partnership is mutualistic, because the prymnesiophyte receives fixed N in exchange for transferring fixed carbon to UCYN-A. This unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N(2) fixation.
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            Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

            Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.
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              Globally distributed uncultivated oceanic N2-fixing cyanobacteria lack oxygenic photosystem II.

              Biological nitrogen (N2) fixation is important in controlling biological productivity and carbon flux in the oceans. Unicellular N2-fixing cyanobacteria have only recently been discovered and are widely distributed in tropical and subtropical seas. Metagenomic analysis of flow cytometry-sorted cells shows that unicellular N2-fixing cyanobacteria in "group A" (UCYN-A) lack genes for the oxygen-evolving photosystem II and for carbon fixation, which has implications for oceanic carbon and nitrogen cycling and raises questions regarding the evolution of photosynthesis and N2 fixation on Earth.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                4 December 2013
                : 8
                : 12
                : e81749
                Affiliations
                [1 ]Institute for Study of the Earth’s Interior, Okayama University, Yamada, Misasa, Tottori, Japan
                [2 ]Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan
                [3 ]National Institute for Environmental Studies, Onogawa, Tsukuba-City, Ibaraki, Japan
                [4 ]Department of Natural History Sciences, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Japan
                Université Claude Bernard - Lyon 1, France
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KH TH. Performed the experiments: KH RO TH. Analyzed the data: KH MK TH. Contributed reagents/materials/analysis tools: KH TH. Wrote the paper: KH TH.

                Article
                PONE-D-13-16157
                10.1371/journal.pone.0081749
                3852252
                24324722
                4fe46214-73f0-41a8-9f98-4924fc2784d3
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 April 2013
                : 16 October 2013
                Page count
                Pages: 11
                Funding
                This research was supported by Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Sciences (No. 20740296), Grant-in Aid for Environmental Studies from Tottori Prefecture, Japan, and by Grand-in Aid for Biodiversity Research Program (No. 1115AA051) from the National Institute for Environmental Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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