Genome Erosion in a Nitrogen-Fixing Vertically Transmitted Endosymbiotic Multicellular Cyanobacterium

Background

An ancient cyanobacterial incorporation into a eukaryotic organism led to the evolution of plastids (chloroplasts) and subsequently to the origin of the plant kingdom. The underlying mechanism and the identities of the partners in this monophyletic event remain elusive.

Methodology/Principal Findings

To shed light on this evolutionary process, we sequenced the genome of a cyanobacterium residing extracellularly in an endosymbiosis with a plant, the water-fern Azolla filiculoides Lam. This symbiosis was selected as it has characters which make it unique among extant cyanobacterial plant symbioses: the cyanobacterium lacks autonomous growth and is vertically transmitted between plant generations. Our results reveal features of evolutionary significance. The genome is in an eroding state, evidenced by a large proportion of pseudogenes (31.2%) and a high frequency of transposable elements (∼600) scattered throughout the genome. Pseudogenization is found in genes such as the replication initiator dnaA and DNA repair genes, considered essential to free-living cyanobacteria. For some functional categories of genes pseudogenes are more prevalent than functional genes. Loss of function is apparent even within the ‘core’ gene categories of bacteria, such as genes involved in glycolysis and nutrient uptake. In contrast, serving as a critical source of nitrogen for the host, genes related to metabolic processes such as cell differentiation and nitrogen-fixation are well preserved.

Conclusions/Significance

This is the first finding of genome degradation in a plant symbiont and phenotypically complex cyanobacterium and one of only a few extracellular endosymbionts described showing signs of reductive genome evolution. Our findings suggest an ongoing selective streamlining of this cyanobacterial genome which has resulted in an organism devoted to nitrogen fixation and devoid of autonomous growth. The cyanobacterial symbiont of Azolla can thus be considered at the initial phase of a transition from free-living organism to a nitrogen-fixing plant entity, a transition process which may mimic what drove the evolution of chloroplasts from a cyanobacterial ancestor.     

Phylogenetic affinities of an enigmatic nannoplankton,Braarudosphaera bigelowii based on the SSU rDNA sequences

The phylogenetic position of an enigmatic calcareous nannoplankton Braarudosphaera bigelowii, whose taxonomic position has long been questioned, was investigated using molecular genetic methods. The SSU rDNA sequences of B. bigelowii were obtained using a single cell PCR technique independently from two single cells isolated from field samples. A BLAST search revealed that the SSU rDNA of B. bigelowii was most similar to those of the division Haptophyta. Subsequent phylogenetic analyses showed that B. bigelowii was included in a clade comprising members of the orders Isochrysidales and Coccolithales, the genus Chrysoculter and two unidentified haptophytes. Morphometric measurements of extant B. bigelowii populations were made to obtain precise information on their intraspecific size variation. The results showed that these populations could be subdivided into a small form (< 2.4 μm), an intermediate form A (4.0–5.3 μm), and an intermediate form B (5.3–7.2 μm), although the borders of the latter two forms overlapped. The SSU rDNA sequences showed the presence of 10-bp substitutions plus six insertions/deletions between specimens of intermediate forms A and B. The levels of DNA variation between them suggested that these two forms are genetically distinct from each other.     

Geosiphon pyriforme,an Endosymbiotic Consortium of a Fungus and a Cyanobacterium (Nostoc), Fixes Nitrogen

The bladders of Geosiphon pyriforme, an endosymbiotic consortium of a fungus and the cyanobacterium Nostoc punctiforme, show nitrogenase activity. This suggests that the organism is capable of nitrogen fixation.     

Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium Anabaena torulosa

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.     

Carotenoids of Prymnesiophyceae (Haptophyceae)

A quantitative study, including mass spectrometric identification, of the carotenoids isolated from some selected prymnesiophytes harvested in exponential growth phase has been carried out. Isochrysis galbana, Hymenomonas carterae, Prymnesium parvum, Pavlova (Monochrysis) lutheri and a Pavlova sp. all produce β,β-carotene, diatoxanthin, diadinoxanthin and fucoxanthin (major carotenoid) in various proportions, in addition to several minor carotenoids were found characteristic of each alga. 19'-Hexanoyloxyfucoxanthin, previously shown to be the major carotenoid in the prymnesiophyte Emeliana (Coccolithus) huxleyi, was not encountered in the Prymnesiophyceae studied here, and we conclude that this carotenoid should be critically looked for in other members of the Gephyrocapsaceae to which E. huxleyi belongs. We further conclude that the carotenoid complement of the Chrysophyceae (in the narrow sense) should be compared with that of the Prymnesiophyceae.     

Coccolithogenesis In Scyphosphaera apsteinii (Prymnesiophyceae)

Coccolithophores are the most significant producers of marine biogenic calcite, although the intracellular calcification process is poorly understood. In the case of Scyphosphaera apsteinii Lohmann 1902, flat ovoid muroliths and bulky, vase‐shaped lopadoliths with a range of intermediate morphologies may be produced by a single cell. This polymorphic species is within the Zygodiscales, a group that remains understudied with respect to ultrastructure and coccolith ontogeny. We therefore undertook an analysis of cell ultrastructure, morphology, and coccolithogenesis. The cell ultrastructure showed many typical haptophyte features, with calcification following a similar pattern to that described for other heterococcolith bearing species including Emiliania huxleyi. Of particular significance was the reticular body role in governing fine‐scale morphology, specifically the central pore formation of the coccolith. Our observations also highlighted the essential role of the inter‐ and intracrystalline organic matrix in growth and arrangement of the coccolith calcite. S. apsteinii secreted mature coccoliths that attached to the plasma membrane via fibrillar material. Time‐lapse light microscopy demonstrated secretion of lopadoliths occurred base first before being actively repositioned at the cell surface. Significantly, growth irradiance influenced the coccosphere composition with fewer lopadoliths being formed relative to muroliths at higher light intensities. Overall, our observations support dynamic metabolic (i.e., in response to growth irradiance), sensory and cytoskeletal control over the morphology and secretion of polymorphic heterococcoliths. With a basic understanding of calcification established, S. apsteinii could be a valuable model to further study coccolithophore calcification and cell physiological responses to ocean acidification.     

Extracellular calcification in Chrysotila lamellosa (Prymnesiophyceae)

Extracellular calcification has been observed in two strains of Chrysotila lamellosa using both light and electron microscopy and differences between the structure of the calcification in each strain noted. It is considered that the morphology of such structures is not a sufficiently reliable character to be used alone in distinguishing between species, but may be useful in conjunction with other information. A comparison is made with extracellular calcification in other microorganisms and the significance of such calcification to our understanding of the nature of certain Cretaceous nanofossils is discussed.     

Temperature-Induced Viral Resistance in Emiliania huxleyi (Prymnesiophyceae)

Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi’s susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Toxicity of coastal coccolithophores (Prymnesiophyceae, Haptophyta)

Over the last decade, certain coccolithophores have been thesubject of extensive multidisciplinary research. Several speciesof coccolithophore, belonging mainly to the families Pleurochrysidaceaeand Hymenomonadaceae, inhabit inshore coastal waters where theymay occasionally bloom and hence impact aquaculture resources.The toxicity to Artemia salina larvae of 11 species of coccolithophore(nine coastal and two oceanic members of the order Coccolithales)was tested. For the nine coastal species, tests were conductedwith rapidly growing and stationary phase cultures at a rangeof cellular concentrations and for two different exposure times(24 and 48 h). Five of the coastal species (four in the genusPleurochrysis as well as Jomonlithus littoralis) were foundto be toxic to A. salina nauplii. Allelopathic effects of acell-free filtrate of a culture of a toxic coccolithophore werealso tested on three flagellate microalgal species: Scrippsiellatrochoidea, Tetraselmis sp. and Isochrysis galbana. Negativeeffects of the filtrate on growth rates and motility of S. trochoideaand Tetraselmis sp. were recorded, suggesting that the toxinof the coccolithophore tested could be an exotoxin similar tothat produced by other non-calcifying members of the Prymnesiophyceae.The fact that certain coccolithophores were found to be toxicto invertebrates and were shown to exhibit allelopathic activitycould imply negative effects at different trophic levels incoastal areas.     

Cell division in Hymenomonas carterae (Braarud et Fagerland) Braarud (Prymnesiophyceae)

The cell division cycle of Hymenomonas carterae (Braarud et Fagerland) Braarud was investigated at the ultrastructural level. DNA synthesis and cytokinesis occurred during the 8-hour dark period. All organelles, including the flagellar bases were replicated prior to nuclear division. Prophase consisted of a clustering of the chromosomes into distinct groups and the disappearance of the nucleolus. During metaphase there was complete dissociation of the nuclear envelope resulting in the formation of an open spindle containing no major organelles. The metaphase plate formed at right angles to an imaginary line joining the two pairs of flagellar bases. Elongation of the cell and separation of the chromosomes occurred at anaphase. During early telophase the nuclear envelope veformed and was closely associated with the chromosome masses, resulting in the nuclear possessing convoluted profiles. Telophase was characterized by complete break down of spindle fibres, rounding off of the nuclear profiles, reappearance of the nucleolus, emergence of the flagella and the final separation of the two daughter cells.     

The vitamin B requirement of Phaeocystis globosa (Prymnesiophyceae)

In batch cultures of flagellates and non-flagellate cells ofPhaeocystis globosa, the biomass yield was significantly enhancedby the addition of a mixture of the vitamins thiamine (B1),cyanocobalamin (B12) and biotin (H). A bioassay with B1 andB12 using the non-flagellate cells of P.globosa showed thatthis prymnesiophyte is a B1 auxotroph. The bioassay also indicateda significant difference in growth rate between culture mediumwith 10 nmol l^–1 B1 (µ = 0.80 day^–1) and culturemedium with 10 nmol l^–1 B12 (µ = 0.52 day^–1).These findings are discussed in relation to the hypothesis thatcentric diatoms, through vitamin B1 excretion or B12 depletion,initiate Phaeocystis blooms. It is concluded, however, thatan alternative hypothesis, that diatoms provide a solid substratefor colony initiation, has more experimental support.     

Sexual reproductive ecology of Carex bigelowii an arctic-alpine sedge

Carex bigelowii an arctic-alpine sedge that shows an extensive clonal growth, and in many years also has a high degree of flowering. To see which factors are most important in determining the sexual reproductive success, the effects of self- and cross-pollination, pollen donor distance, amount of pollen, temperature, and time of flowering were studied. The study was conducted at Latnjajaure Field Station, northern Sweden and Thingvellir National Park, Iceland during three field seasons. Experimentally increased air temperature raised the fruit set and the fruit weight, in two of the three seasons. Pollen donor distance did not influence either fruit set or fruit weight, and there was no difference in fruit weight between selfing and cross-pollination. An increased amount of pollen raised the fruit set. but did not affect fruit weight. The time of flowering was also important; there was a negative relationship between flowering time and fruit set and fruit weight, i.e. late flowering ramets had a lower fruit set and fruit weight. The results suggest that Carex bigelowii is an outcrossed species where the reproductive success is mainly determined by the weather, and that there are differences between the two study sites.     

The ultrastructure of mitosis inIsochrysis galbana parke (Prymnesiophyceae)

Summary Mitosis and cytokinesis have been studied in the flagellate algaIsochrysis galbana Parke (Prymnesiophyceae). Nuclear division is preceded by replication of the flagella and haptonema, the Golgi body and the chloroplast; fission in the chloroplast occurs in the region of the pyrenoid. During prophase, spindle microtubules radiating from two ill-defined poles are formed. The nuclear envelope breaks down and the chromatin condenses. At metaphase the spindle is fully developed, some pole-to-pole microtubules passing through the well-defined chromatin plate, others terminating at it. No kinetochores or individual chromosomes were observed. By late metaphase, many Golgi-derived vesicles may be seen against the two poleward faces of the metaphase plate. During anaphase, the two daughter masses of chromatin move towards the poles. In early telophase, the nuclear envelope of each daughter nucleus is complete only on the side towards the adjacent chloroplast, remaining open on the interzonal side. However, during telophase each nucleus becomes reorientated so that it lies lateral to the long axis of the spindle and with its open side towards the chloroplasts. By late telophase, each new nuclear envelope is complete and confluence with the adjacent chloroplast ER established.Cytokinesis and subsequent segregation of the daughter cells are effected by the dilation of Golgi- and ER-derived vesicles in the interzonal region. No microtubular structures are involved. Comparisons with the results from other studies of mitosis in members of thePrymnesiophyceae show that they all have a number of features in common, but that there are differences in detail between species.     

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet-b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m⁻² ·d⁻¹ (BED_DNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.     

Nitrogen-Fixing (Acetylene Redution) Activity and Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland Rice

Nitrogen-fixing activity associated with different wetland rice varieties was measured at various growth stages by an in situ acetylene reduction method after the activities of blue-green algae (cyanobacteria) in the flood water and on the lower portion of the rice stem were eliminated. Nitrogen-fixing activities associated with rice varieties differed with plant growth stages. The activities increased with plant age, and the maximum was about at heading stage. The nitrogen fixed during the whole cropping period was estimated at 5.9 kg of N per ha for variety IR26 (7 days) and 4.8 kg of N per ha for variety IR36 (95 days). The population of aerobic heterotrophic N₂-fixing bacteria associated with rice roots and stems was determined by the most-probable-number method, using semisolid glucose-yeast extract and semisolid malate-yeast extract media. The addition of yeast extract to the glucose medium increased the number and activity of aerobic heterotrophic N₂-fixing bacteria. The glucose-yeast extract medium gave higher counts of aerobic N₂-fixing bacteria associated with rice roots than did the malate-yeast extract medium, on which Spirillum-like bacteria were usually observed. The lower portion of the rice stem was also inhabited by N₂-fixing bacteria and was an active site of N₂ fixation.