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Herbaspirillum seropedicae is a diazotrophic β-Proteobacterium found endophytically associated with gramineae (Poaceae or graminaceous plants) such as rice, sorghum and sugar cane. In this work we show that nitrate-dependent growth in this organism is regulated by the master nitrogen regulatory two-component system NtrB/NtrC, and by NtrY/NtrX which functions to specifically regulate nitrate metabolism. NtrY is a histidine kinase sensor protein predicted to be associated with the membrane and NtrX is the response regulator partner. The ntrYntrX genes are widely distributed in Proteobacteria. In α-Proteobacteria they are frequently located downstream from ntrBC, whereas in β-Proteobacteria these genes are located downstream from genes encoding a RNA methyltransferase and a proline-rich protein with unknown function. The α-Proteobacteria NtrX protein has an AAA+ domain, absent in those from β-Proteobacteria. An ntrY mutant of H. seropedicae showed wild type fixing nitrogen phenotype, but the nitrate dependent growth was abolished. Gene fusion assays indicated that NtrY is involved in the expression of genes coding for the assimilatory nitrate reductase as well as the nitrate-responsive two-component system NarX/NarL (narK and narX promoters, respectively). The purified NtrX protein was capable of binding the narK and narX promoters, and the binding site at the narX promoter for the NtrX protein was determined by DNA footprinting. In silico analyses revealed similar sequences in other promoter regions of H. seropedicae that are related to nitrate assimilation, supporting the role of the NtrY/NtrX system in regulating nitrate metabolism in H. seropedicae. This article is protected by copyright. All rights reserved.

Rhizosphere engineering with beneficial plant growth promoting bacteria offers great promise for sustainable crop yield. Potato is an important food commodity that needs large inputs of nitrogen and phosphorus fertilizers. To overcome high fertilizer demand (especially nitrogen), five bacteria, i.e., Azospirillum sp. TN10, Agrobacterium sp. TN14, Pseudomonas sp. TN36, Enterobacter sp. TN38 and Rhizobium sp. TN42 were isolated from the potato rhizosphere on nitrogen-free malate medium and identified based on their 16S rRNA gene sequences. Three strains, i.e., TN10, TN38, and TN42 showed nitrogen fixation (92.67-134.54 nmol h(-1)mg(-1) protein), while all showed the production of indole-3-acetic acid (IAA), which was significantly increased by the addition of L-tryptophan. Azospirillum sp. TN10 produced the highest amount of IAA, as measured by spectrophotometry (312.14 mu g mL(-1)) and HPLC (18.3 mu g mL(-1)). Inoculation with these bacteria under axenic conditions resulted in differential growth responses of potato. Azospirillum sp. TN10 incited the highest increase in potato fresh and dry weight over control plants, along with increased N contents of shoot and roots. All strains were able to colonize and maintain their population densities in the potato rhizosphere for up to 60 days, with Azospirillum sp. and Rhizobium sp. showing the highest survival. Plant root colonization potential was analyzed by transmission electron microscopy of root sections inoculated with Azospirillum sp. TN10. Of the five test strains, Azospirillum sp. TN10 has the greatest potential to increase the growth and nitrogen uptake of potato. Hence, it is suggested as a good candidate for the production of potato biofertilizer for integrated nutrient management.

International audience; This study focuses on the chemotaxis, colonization and rice growth promoting ability of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing rhizobacteria Rhizobium leguminosarum bv. trifolii SN10, previously isolated from root nodules of Trifolium alexandrium L. We show here that R. leguminosarum bv. trifolii SN10 promote the growth of four different rice varieties grown in India in terms of biomass, root branching and N content. In addition, using scanning electron microscopy and viable cell counts, we provide evidence that the bacteria successfully colonize the root surface of the rice variety which showed maximum growth promotion upon inoculation. Not only this, R. leguminosarum bv. trifolii SN10 exhibit a strong chemotaxis response towards the rice seed and root exudates despite the presence of a bacteriostatic phenolic compound, 7-hydroxycoumarin (umbelliferone). Further, R. leguminosarum bv. trifolii SN10 secretion of phytohormones such as IAA and ACC deaminase suggest the potential of the plant growth promoting rhizobacteria to be used as biofertilizer to enhance rice crop production in the subcontinent.

Pseudomonas putida strain angstrom 313, a deleterious rhizosphere bacterium, reduced pea nitrogen content when inoculated alone or in combination with Rhizobium leguminosarum bv. viceae on plants in the presence of soil under greenhouse conditions. When plants were grown gnotobiotically in liquid media, mixed inocula of angstrom 313 and rhizobia gave a higher proportion of small evenly distributed nodules when compared with a single rhizobial inoculation. In addition, the rhizobial root establishment was reduced by angstrom 313 irrespective of inoculum, density, indicating that angstrom 313 has the capacity to interact with the early rhizobial infection process. When pea seedlings were simultaneously inoculated with angstrom 313 and rhizobia, angstrom 313 colonised the root hairs to the same extent as the rhizobia, according to analysis by immunofluorescence microscopy. This suggests that the root hair colonisation trait of P. putida interferes with the onset of the symbiotic process. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Despite more than 40 years of research on free-living and endophytic bacteria associated with cereals and grasses, conclusive examples of impacts of non-symbiotic N2-fixation in agriculture are lacking. All available methods for measurement of N2-fixation associated with cereals and grasses have been employed, and N2-fixation has been demonstrated to occur under controlled conditions, but this is insufficient evidence to prove a significant role for N2-fixation by heterotrophic bacteria in the field. Recently attention has focused on endophytic N2-fixation with claims of major inputs of N from the atmosphere, particularly in sugar cane for which the largest body of information exists, although some methodological concerns remain unanswered. Here we analyse the evidence for N2-fixation in the rhizosphere and within graminaceous plants. We provide guidelines suggesting the type of information required to allow a critical analysis of the amounts of non-symbiotic N2-fixation contributed in agriculture. Given the substantial availability of C as a substrate for N2-fixation in sugar cane it remains the most likely candidate plant to benefit from non-symbiotic N2-fixation. Measurements of elevated respiration in zones of high bacterial colonization may give an indication of whether these really are focal points of the rapid metabolic activity required to support significant N2-fixation activity. The only conclusive evidence for a major role of non-symbiotic N2-fixation in the field will come from long-term N balance studies in which ALL processes of potential gain and loss are measured. We conclude that evidence for a large contribution from heterotrophic, non-symbiotic N2-fixation in both natural and agricultural systems is weak. Although amounts of non-symbiotic N2-fixation in natural systems may be small (

In this study, the role of extracellular polymeric substances (EPS) in the initial adhesion of EPS-producing Pseudomonas aeruginosa SG91 and SG81R1, a non-EPS-producing strain, to substrata with different hydrophobicity was investigated. The release of EPS by SG81 was concurrent with a decrease in surface tension of a bacterial suspension from 70 to 45 mJ m(-2) that was absent for SG81R1. Both strains adhered faster and in higher numbers to a hydrophilic than to a hydrophobic substratum, but the initial deposition rates and numbers of adhering bacteria in a stationary-end point were highest for the non-EPS-producing strain SG81R1, regardless of substratum hydrophobicity. Both strains adhered less to substrata pre-coated with isolated EPS of strain SG81. Furthermore, it was investigated whether bacteria, detached by passing air-bubbles, had left behind 'footprints' with an influence on adhesion of newly redepositing bacteria. Redeposition on glass was highest for non-EPS-producing SG81R1 and decreased linearly with the number of times these cycles of detachment and deposition were repeated to become similar to the redeposition of SG81 after six cycles. This indicates that P. aeruginosa SG81 leaves the substratum surface nearly completely covered with EPS after detachment, while SG81R1 releases only minor amounts of surface active EPS, completely covering the substratum after repeated cycles of detachment and adhesion. Atomic force microscopy showed a thick and irregular EPS layer (up to 32 nm) after the first detachment cycle of EPS-producing strain SG81, whereas the putatively non-EPS-producing strain SG81R1 left a 9 nm thin layer after one cycle. X-ray photoelectron spectroscopy indicated that the bacterial footprints consisted of uronic acids, the prevalence of which increased with the number of detachment and deposition cycles.

In this study, the role of extracellular polymeric substances (EPS) in the initial adhesion of EPS-producing Pseudomonas aeruginosa SG91 and SG81R1, a non-EPS-producing strain, to substrata with different hydrophobicity was investigated. The release of EPS by SG81 was concurrent with a decrease in surface tension of a bacterial suspension from 70 to 45 mJ m(-2) that was absent for SG81R1. Both strains adhered faster and in higher numbers to a hydrophilic than to a hydrophobic substratum, but the initial deposition rates and numbers of adhering bacteria in a stationary-end point were highest for the non-EPS-producing strain SG81R1, regardless of substratum hydrophobicity. Both strains adhered less to substrata pre-coated with isolated EPS of strain SG81. Furthermore, it was investigated whether bacteria, detached by passing air-bubbles, had left behind 'footprints' with an influence on adhesion of newly redepositing bacteria. Redeposition on glass was highest for non-EPS-producing SG81R1 and decreased linearly with the number of times these cycles of detachment and deposition were repeated to become similar to the redeposition of SG81 after six cycles. This indicates that P. aeruginosa SG81 leaves the substratum surface nearly completely covered with EPS after detachment, while SG81R1 releases only minor amounts of surface active EPS, completely covering the substratum after repeated cycles of detachment and adhesion. Atomic force microscopy showed a thick and irregular EPS layer (up to 32 nm) after the first detachment cycle of EPS-producing strain SG81, whereas the putatively non-EPS-producing strain SG81R1 left a 9 nm thin layer after one cycle. X-ray photoelectron spectroscopy indicated that the bacterial footprints consisted of uronic acids, the prevalence of which increased with the number of detachment and deposition cycles.

International audience; A facile method of using optical trapping to measure cell adhesion forces is presented and applied to the adhesion of Saccharomyces cerevisiae on glass, in contact with solutions of different compositions. Trapping yeast cells with optical tweezers (OT) is not perturbed by cell wall deformation or cell deviation from a spherical shape. The trapping force calibration requires correction not only for the hydrodynamic effect of the neighboring wall but also for spherical aberrations affecting the focal volume and the trap stiffness. Yeast cells trapped for up to 5 h were still able to undergo budding but showed an increase of doubling time. The proportion of adhering cells showed the expected variation according to the solution composition. The detachment force varied in the same way. This observation and the fact that the detachment stress was exerted parallel to the substrate surface point to the role of interactions involving solvated macromolecules. Both the proportion of adhering cells and the removal force showed a distribution which, in our experimental conditions, must be attributed to a heterogeneity of surface properties at the cell level or at the subcellular scale. As compared with magnetic tweezers, atomic force microscopy, and more conventional ways of studying cell adhesion (shear-flow cells), OT present several advantages that are emphasized in this paper.

International audience; The three-dimensional structure of the hexameric (alpha beta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alpha beta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The(alpha beta)(6) hexamer exhibits a concentration-dependent equilibrium with alpha beta monomers and (alpha beta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alpha beta protomer only 3-fold by increasing the K-m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (alpha beta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.

Despite more than 40 years of research on free-living and endophytic bacteria associated with cereals and grasses, conclusive examples of impacts of non-symbiotic N2-fixation in agriculture are lacking. All available methods for measurement of N2-fixation associated with cereals and grasses have been employed, and N2-fixation has been demonstrated to occur under controlled conditions, but this is insufficient evidence to prove a significant role for N2-fixation by heterotrophic bacteria in the field. Recently attention has focused on endophytic N2-fixation with claims of major inputs of N from the atmosphere, particularly in sugar cane for which the largest body of information exists, although some methodological concerns remain unanswered. Here we analyse the evidence for N2-fixation in the rhizosphere and within graminaceous plants. We provide guidelines suggesting the type of information required to allow a critical analysis of the amounts of non-symbiotic N2-fixation contributed in agriculture. Given the substantial availability of C as a substrate for N2-fixation in sugar cane it remains the most likely candidate plant to benefit from non-symbiotic N2-fixation. Measurements of elevated respiration in zones of high bacterial colonization may give an indication of whether these really are focal points of the rapid metabolic activity required to support significant N2-fixation activity. The only conclusive evidence for a major role of non-symbiotic N2-fixation in the field will come from long-term N balance studies in which ALL processes of potential gain and loss are measured. We conclude that evidence for a large contribution from heterotrophic, non-symbiotic N2-fixation in both natural and agricultural systems is weak. Although amounts of non-symbiotic N2-fixation in natural systems may be small (

n this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

Some biological activities of Azotobacter chroococcum, strain Azcap 1, (spontaneous mutant, captan resistant up to 300 micrograms/ml) were assayed on RM medium with and without the presence of the fungicide. Comparisons were also carried out with Az. chroococcum sensitive strains Azwt, Azcan 10 and 14. The hydrolysis of captan, incorporated in agar plates of RM at 100 micrograms/ml, was rapid, since on 4-day plates, no effect was found on the strain Azwt, while on freshly prepared ones its growth was completely blocked. As for Azcap 1, grown on RM only, the behaviour was similar to that of sensitive strains, whereas when grown on captan the results of experiments showed: (i) a lag of approximately 12 h to reach the maximum nitrogen-fixing activity; (ii) delay of 12-24 h in the full consumption of glucose present in the medium, although the invertase activity did not present differences; (iii) high ATP culture content during the 50 h of the experiment; (iv) approximately 6-10-fold lower production of PHB (poly-B-hydroxybutyrate); (v) lack of typical encystment phase, for the tested 96 h and reduced viability in developing colonies on agar RM medium. In contrast, when captan was added to cultural medium at sublethal concentration, 50 micrograms/ml for sensitive strain Azwt and 200 micrograms/ml for Azcap 1, the amount of glutathione produced (to remove the fungicide toxicity) was several times higher for the former.