Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates.
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The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa(+) phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri(+) phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants' responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri(+) mutant BK759.G gave rise to stable Swa(++) derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa(++) derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates. (+info)
Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense.
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Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43). (+info)
Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245.
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Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1-3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant. (+info)
Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage.
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The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (phiAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the phiAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of phiAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd. (+info)
Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein.
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Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 microM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense. (+info)
Aerobic nitric oxide production by Azospirillum brasilense Sp245 and its influence on root architecture in tomato.
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