Pleiotropic physiological effects in the plant growth-promoting bacterium Azospirillum brasilense following chromosomal labeling in the clpX gene (original) (raw)
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Regulation of IAA Biosynthesis in Azospirillum brasilense Under Environmental Stress Conditions
Current microbiology, 2018
Indole-3-acetic acid (IAA) is one of the most important molecules produced by Azospirillum sp., given that it affects plant growth and development. Azospirillum brasilense strains Sp245 and Az39 (pFAJ64) were pre-incubated in MMAB medium plus 100 mg/mL L-tryptophan and treated with or exposed to the following (a) abiotic and (b) biotic stress effectors: (a) 100 mM NaCl or NaSO 4.0% (w/v) PEG 0.5 mM HO 0.1 mM abscisic acid, 0.1 mM 1-aminocyclopropane 1-carboxylic acid, 45 °C or daylight, and (b) 4.0% (v/v) filtered supernatant of Pseudomonas savastanoi (Ps) or Fusarium oxysporum (Fo), 0.1 mM salicylic acid (SA), 0.1 mM methyl jasmonic acid (MeJA), and 0.01% (w/v) chitosan (CH). After 30 and 120 min of incubation, biomass production, cell viability, IAA concentration (µg/mL), and ipdC gene expression were measured. Our results show that IAA production increases with daylight or in the presence of PEG, ABA, SA, CH, and Fo. On the contrary, exposure to 45 °C or treatment with HO NaCl, N...
Environmental Microbiology, 2005
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 gfp mut3 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 colo-nizing the rhizoplane documented for the first time an in situ expression of ipdC .
Genetics and molecular biology of Azospirillum
Biology and fertility of Soils, 1999
Genetic manipulation of Azospirillum spp. has facilitated a better understanding of the mode of action of this plant-growth promoting bacterium and should help to improve its ability to stimulate plant growth and development. This review considers and discusses Agospirillum plasmids, promoter sequences, the isolation of Azospirillum mutants, the genetic transformation of Azospirillum, the transfer of foreign genes into Azospirillum by conjugation and the Azospirillum genes that have been isolated and characterized. The Azospirillum genes that are discussed include genes involved in nitrogen fixation, plant root attachment, phytohormone biosynthesis, tryptophan biosynthesis, carbon metabolism and a few other less well characterized processes.
Potential roles for the glnB and ntrYX genes in Azospirillum brasilense
Fems Microbiology Letters, 2001
Three Azospirillum brasilense mutants constitutive for nitrogen fixation (NifC) 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.
2000
Azospirillum brasilense strains, CDJA and A40, capable of growing at sub-optimal temperature were tagged with stable chromogenic marker Tn5-lacZ. Mutants were screened for plant growth promoting activities at 20, 22, 25, 30 and 37°C. Mutants MC48 and MA3 were found to ®x nitrogen upto 85% and produced indole acetic acid (IAA) and siderophore in isogenic manner to their respective wild type strains, CDJA and A40, at sub-optimal temperatures. Co-inoculation of mutants with their respective parent (1:1 ratio) to the wheat revealed that colonization potential of the mutants was aected greatly. Tn5-lacZ tagged mutants MC48 and MA3 were found isogenic to their respective wild type Azospirillum strain, with regards to plant growth promoting activities and root colonization ability.
Molecular and General Genetics, 1986
We report the successful mutagenesis of Azospirillum brasilense 29710 Rif Sm with transposon Tn5. The narrow host-range plasmid pGS9 (p15A replicon), which possesses broad host-range N-type transfer genes, was used as the suicide vehicle to deliver Tn5 in Azospirillum. Out of 900 colonies tested, 0.8% proved to be auxotrophic. One mutant altered in indoleacetic acid (auxin) biosynthesis was isolated and, in addition, three mutants completely defective in nitrogen fixation (nif) were obtained. All the mutants tested contained a single copy of Tn5 integrated randomly in the genome. The Tn5-mutagenized EcoRI fragments were cloned from the three Nif- mutants. Physical analysis of cloned DNA showed that Tn5 was present on a different EcoRI fragment in each case, ranging in size from 15–17 kb. The nitrogenase structural genes (nifHDK) in A. brasilense 29710 Rif Sm were localized on a 6.7 kb EcoRI fragment. We found that Tn5 is not inserted in the nifHDK genes in the Nif- mutants reported here. Site-directed mutagenesis using the cloned, Tn5-containing DNA from mutant Nif27(pMS188), produced a large number of Nif- transconjugants of the A. brasilense 29710 Rif wild-type strain, showing the linkage between Tn5 insertion and the Nif- phenotype. This is the first time that transposon-mutagenized auxotrophic, Nif- and other mutants have been available for genetic analysis in Azospirillum. This should greatly facilitate the cloning and mapping of genes involved in nitrogen fixation as well as in many other phenotypic characteristics of Azospirillum.
FEMS Microbiology Letters, 2005
Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38°C in a standard minimal medium (MMAB) containing 2.5 g l À1 L-malate and 50 lg ml À1 tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense. (J. Vanderleyden). www.fems-microbiology.org FEMS Microbiology Letters 246 (2005) 125-132
Regulation of Azospirillum brasilense nifA gene expression by ammonium and oxygen
FEMS Microbiology Letters, 1999
The structure and activity of the nifA promoter of Azospirillum brasilense was studied using deletion analysis. An essential region for nifA promoter activity was identified between nucleotides 367 and 347 from the identified transcription start site. A sequence resembling a c 70 recognition site occurs in this region and may constitute the nifA gene promoter. The regulation of the nifA gene was studied in plasmid and chromosomal nifA: :lacZ fusions. Full expression was obtained under low oxygen levels and in the absence of ammonium ions. Repression of nifA expression involves a synergistic effect between oxygen and ammonium. ß