Iron distribution through the developmental stages of Medicago truncatula nodules.

Paramount to symbiotic nitrogen fixation (SNF) is the synthesis of a number of metalloenzymes that use iron as a critical component of their catalytical core. Since this process is carried out by endosymbiotic rhizobia living in legume root nodules, the mechanisms involved in iron delivery to the rhizobia-containing cells are critical for SNF. In order to gain insight into iron transport to the nodule, we have used synchrotron-based X-ray fluorescence to determine the spatio-temporal distribution of this metal in nodules of the legume Medicago truncatula with hitherto unattained sensitivity and resolution. The data support a model in which iron is released from the vasculature into the apoplast of the infection/differentiation zone of the nodule (zone II). The infected cell subsequently takes up this apoplastic iron and delivers it to the symbiosome and the secretory system to synthesize ferroproteins. Upon senescence, iron is relocated to the vasculature to be reused by the shoot. These observations highlight the important role of yet to be discovered metal transporters in iron compartmentalization in the nodule and in the recovery of an essential and scarce nutrient for flowering and seed production.

MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.

Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron provided by the host legume. Using synchrotron-based X-ray fluorescence, we have determined that this iron is released from the vasculature into the apoplast of zone II of M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen fixation is indicated by the 60% reduction of nitrogenase activity observed in knock-down lines, most likely due to deficient incorporation of this essential metal cofactor at the necessary levels.

Functional characterization of Rhizobium leguminosarum bv. viciae DmeRF, a cation diffusion facilitator system involved in nickel and cobalt resistance

In prokaryotes, nickel is an essential element participating in the structure of enzymes involved in multiple cellular processes. Nickel transport is a challenge for microorganisms since, although essential, high levels of this metal inside the cell are toxic. For this reason, bacteria have developed high-affinity nickel transporters as well as nickel-specific detoxification systems. Ultramafic soils, and soils contaminated with heavy metals are excellent sources of nickel resistant bacteria. Molecular analysis of strains isolated in the habitats has revealed novel genetic systems involved in adaptation to such hostile conditions.

Identification and analysis of a nickel-inducible cation diffusion facilitator-NiCoT combined system in Rhizobium leguminosarum bv. viciae

Nickel, like other transition metals, can be toxic to cells even at moderate concentration (low microM range) by displacing essential metals from their native binding sites or by generating reactive oxygen species that cause oxidative DNA damage. For this reason, cells have evolved mechanisms to deal with excess nickel. Efflux systems include members of the Resistance-Nodulation-cell Division (RND) protein family, P-type ATPases, cation diffusion facilitators (CDF) and other resistance factors. Nickel-specific exporters have been characterized in Cupravidus metallidurans, Helicobacter pylori, Achromobacter xylosoxidans, Serratia marcenses and Escherichia coli.

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.

A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.

Rizobactérias nativas da Caatinga com potencial para redução dos efeitos da seca em soja (Glycine max L.)

No semiárido brasileiro, a vegetação predominante é a Caatinga, bioma ainda pouco explorado, que apresenta plantas e micro-organismos com alta resistência aos períodos de seca imposto pelo clima. Os micro-organismos associados às plantas deste bioma, são capazes de desenvolver mecanismos de proteção celular contra o estresse hídrico, assim como proteção vegetal contra a dessecação. O presente estudo buscou compreender as rizobactérias associadas a Mimosa artemisiana a fim de selecionar bactérias tolerantes à seca com características de promover o crescimento de plantas sob condições de estresse hídrico, diminuindo assim, os efeitos adversos impostos pela seca. As amostras de solo rizosférico foram coletadas ao longo da Caatinga, englobando os estados da BA e PE, totalizando quatro pontos de coleta. Com o uso de metodologias dependentes de cultivo, foi isolado bactérias com algumas características de promoção de crescimento de plantas diretos e/ou indiretos, como produção de AIA e fixação de nitrogênio. Além disso, linhagens capazes de crescer em meio com reduzida atividade de água e com mecanismos de proteção contra a dessecação, como, produção de EPS, biofilme, produção da ACC deaminase e indução de resistência sistêmica através das enzimas peroxidase e polifenoloxidase. Uma linhagem de Paenibacillus sp. e outra de Bacillus sp. foram capazes de promover o crescimento de soja sob condições de estresse hídrico, aumentando alguns parâmetros vegetais como, parte aérea e sistema radicular analisados.

Culturable aerobic and facultative bacteria from the gut of the polyphagic dung beetle Thorectes lusitanicus Jeckel

Unlike other dung beetles, the Iberian geotrupid Thorectes lusitanicus exhibits polyphagous behavior; for example, it is able to eat acorns, fungi, fruits, and carrion in addition to the dung of different mammals. This adaptation to digest a wider diet has physiological and developmental advantages and requires key changes in the composition and diversity of the beetle's gut microbiota. In this study, we isolated aerobic, facultative anaerobic, and aerotolerant microbiota amenable to grow in culture from the gut contents of T. lusitanicus and resolved isolate identity to the species level by sequencing 16S rRNA gene fragments. Using BLAST similarity searches and maximum likelihood phylogenetic analyses, we were able to reveal that the analyzed fraction (culturable, aerobic, facultative anaerobic, and aerotolerant) of beetle gut microbiota is dominated by the phyla Proteobacteria, Firmicutes and Actinobacteria. Among Proteobacteria, members of the order Enterobacteriales (Gammaproteobacteria) were the most abundant. The main functions associated with the bacteria found in the gut of T. lusitanicus would likely include nitrogen fixation, denitrification, detoxification, and diverse defensive roles against pathogens.

Die verwertung des luftstickstoffs mit hilfe des elektrischen flammenbogens. Vortrag gehalten am 23. september 1910 auf der 82. versammlung Deutscher naturforscher und ärzte in Königsberg i. Pr., von Prof. Dr. J. Zenneck ...

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Laboratory culture studies of Trichodesmium isolated from the great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6 years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR similar to 40-50 mumol quanta m(-2) s(-1)). N-2 fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2-0.3 day(-1) and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR similar to 5-120 mumol quanta m(-2) s(-1)) with the maximum growth occurring at - 40-50 mumol quanta m(-2) s(-1). These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37 psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45 nM. No active growth was observed with the 4.5 nM Fe addition.

Phosphate uptake and growth kinetics of Trichodesmium (Cyanobacteria) isolates from the North Atlantic Ocean and the Great Barrier Reef, Australia

We compared inorganic phosphate (P-i) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate-limited cultures had up to six times higher maximum P-i uptake rates than P-replete cultures in both strains. For strain GBRTRLI101, cell-specific P-i uptake rates were nearly twice as high, due to larger cell size, but P-specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 muM for P-i uptake and 0.1 and 0.2 muM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing P-i up to 1.0 muM. The C:P ratios were 340-390 and N:P ratios were 40-45 for both strains under severely P-limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 muM P-i. The North Atlantic strain IMS101 is better adapted to growing on P-i at low concentrations than is GBRTRLI101 from the more P-i-enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) P-i concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.