Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Effect of water-soluble vitamins on the production of indole-3-acetic acid by Azospirillum brasilense

The effects of six water-soluble vitamins on tryptophan-dependent synthesis of indole-3-acetic acid in Azospirillum brasilense were investigated. A multifactorial regression analysis was employed to produce models of indole-3-acetic acid synthesis versus concentrations of tryptophan and the vitamins added to the growth medium. Very low levels of the B-group vitamins added at 10 to 100 microg l(-1) affected production of indole-3-acetic acid in A. brasilense. The largest release of this phytohormone was observed after amendment with pyridoxine and nicotinic acid. Results of the study suggest a role these vitamins may fulfil in the regulation of indole-3-acetic acid synthesis in A. brasilense.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

The role of chemotaxis genes in establishing the associative relations between Azospirillum brasilense and wheat

The chemotactic properties of a number of Azospirillum brasilense natural strains have been studied. Azospirillum demonstrate the positive chemotactic reaction towards the organic acids salts but a poor reaction towards the presence of the attractants like hydrocarbons and aminoacids except for arabinose and glutamic acid. The series of Che- mutants deficient in general chemotaxis has been selected by introducing the transposon Tn5 into the cells of rifampicinresistant mutant strain Azospirillum brasilense 5T-2. The ability of the mutant cells to fast and solid adsorption on the roots of the sterile wheat sprouts is shown to be decreased 2-3 folds as compared with the one of the wild type strain. Chemotaxis is suggested to affect the adsorbtion ability of azospirillum and their associative properties.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Molecular Genetics and Genomics - Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid...     

Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245

Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.     

Potential roles for the glnB and ntrYX genes in Azospirillum brasilense

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.     

Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense

Azospirillum brasilense is a plant growth promoting rhizobacterium (PGPR) that is being increasingly used in agriculture in a commercial scale. Recent research has elucidated key properties of A.?brasilense that contribute to its ability to adapt to the rhizosphere habitat and to promote plant growth. They include synthesis of the auxin indole-3-acetic acid, nitric oxide, carotenoids, and a range of cell surface components as well as the ability to undergo phenotypic variation. Storage and utilization of polybetahydroxyalkanoate polymers are important for the shelf life of the bacteria in production of inoculants, products containing bacterial cells in a suitable carrier for agricultural use. Azospirillum brasilense is able to fix nitrogen, but despite some controversy, as judging from most systems evaluated so far, contribution of fixed nitrogen by this bacterium does not seem to play a major role in plant growth promotion. In this review, we focus on recent advances in the understanding of physiological properties of A.?brasilense that are important for rhizosphere performance and successful interactions with plant roots.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally present in this bacterium. In the presence of L-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid. Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plasmid of A. brasilense Sp245, gains the ability to produce anthranilic acid.     

Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.     

Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression

Rhizospheric bacterial strains are known to produce indole-3-acetic acid (IAA) through different pathways, and such IAA may be beneficial to plants at low concentrations. IAA biosynthesis by a natural isolate of Azospirillum brasilense SM was studied and observed to be tryptophan-inducible and -dependent in nature. While our work demonstrated the operation of the indole pyruvic acid pathway, the biochemical and molecular evidence for the genes of the indole acetamide (IAM) pathway were lacking in A. brasilense SM. This led us to use the IAM pathway genes as targets for metabolic engineering, with the aim of providing an additional pathway of IAA biosynthesis and improving IAA levels in A. brasilense SM. The introduction of the heterologous IAM pathway, consisting of the iaaM and iaaH genes, not only increased the IAA levels by threefold but also allowed constitutive expression of the same genes along with efficient utilization of IAM as a substrate. Such an engineered strain showed a superior effect on the lateral branching of sorghum roots as well as the dry weight of the plants when compared with the wild-type strain. Such an improved bioinoculant could be demonstrated to enhance root proliferation and biomass productivity of treated plants compared with the parental strain.     

Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense

The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant-bacteria associations (Rhizobium-legume, Pseudomonas-potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.