Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Cacarbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra-and extracellular Ca-carbonate biomineralization by cyanobacteria.calcification | amorphous calcium carbonate | polyphosphate

Abstract:The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM). Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

Soybean, Glycine max (L.) Merrill, is one of the most important food crops in the world. High soybean yields require large amounts of N fertilizers, which are expensive and can cause environmental problems. The industrial fixation of nitrogen accounts for about 50% of fossil fuel usage in agriculture. In contrast, biological fixation of N 2 is a low-cost source of N for soybean cropping through the symbiotic association between the plant and soil bacteria belonging to the genera Bradyrhizobium and Sinorhizobium, which are collectively called "soybean rhizobia". In general, symbiotic nitrogen fixation in crop legumes not only reduces fertilizer costs but also improves soil fertility through crop rotation and intercropping. Biological nitrogen fixation is due to symbioses between leguminous plants and species of Rhizobium bacteria. Replacing this natural N source by synthetic N fertilizers would cost around 10 billion dollars annually. Moreover, legume seed and foliage have a higher protein content than that of non-legumes, and this makes them desirable protein crops. There is a wide knowledge of the industrial elaboration and use of commercial soybean inoculants based on bradyrhizobia strains. At present, the technology to prepare different types of inoculants, either solid or liquid, is sufficiently developed to meet market requirements, although further research and investments are still required to improve the symbiotic efficacy of rhizobial inoculants. Inoculation of soybeans under field conditions has been successful in the USA, Brazil and Argentina, which are the world leaders in soybean cultivation in terms of acreage and grain yields. There are, however, limitations to a wider use of rhizobial inoculants: the size of indigenous soil rhizobial populations can prevent the successful use of inoculants in some particular areas. For example, many Chinese soils contain more than 10 5 soybean rhizobia per gram of soil, which imposes a serious barrier for nodule occupancy by the soybean rhizobia used as an inoculant. The use of inoculants based on soil bacteria other than rhizobia has also increased in the last decades. An example is the genus Azospirillum, which can be used for its capacity to increase plant growth and seed yields through different mechanisms, such as the production of plant hormones and the increase in phosphate uptake by roots. In addition, co-inoculation with Azospirillum and rhizobia enhances nodulation and nitrogen fixation. Although less developed, it is expected that inoculants based on mycorrhizal fungi will also play a relevant role in sustainable agriculture and forestry. In spite of any possible limitations, the use of inoculants appears compulsory in a frame of sustainable agriculture, which seeks to increase crop yields and nutrient-use efficiency while reducing the environmental costs associated with agriculture intensification. This review also summarizes some of the most relevant genetic aspects of soybean rhizobia in relation to their symbiosis with soybeans. They can be listed a...

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.