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PubMed2002

Diversity of azospirillum strains isolated from rice plants grown in saline and nonsaline sites of coastal agricultural ecosystem.

The diversity of indigenous Azospirillum spp. associated with rice cultivated along the coastline of Tamil Nadu was analyzed. Twelve sites with varying soil characteristics such as salinity, texture, and the host variety were chosen. Of the 402 strains isolated using NFB media, 302 were confirmed to be Azospirillum spp. and subjected to DNA polymorphism analysis using PCR-RFLP of 16S rDNA. They were also screened for their salt tolerance and microaerobic N2-fixing-dependent growth. On species identification, all the strains were found to be A. brasilense, A. lipoferum, or unidentified. On comparing the influence of the previously noted variability on the indigenous population, soil salinity was found to play a dominant role. This was revealed by PCR-RFLP studies and salt tolerance studies. A high association between soil salinity and the distribution of Azospirillum genotypes reveals that soil salinity should be taken into consideration while developing biofertilizers specifically for the coastal agricultural ecosystem.

Agriculture↗

PubMed1998

Cell-surface lectins of Azospirillum spp.

Cell-surface lectins were screened in seven strains of Azospirillum brasilense and A. lipoferum. The presence of lectins was determined by particle agglutination assays employing latex beads coated with neoglycoproteins and by Western blot with neoglycoproteins labeled with horseradish peroxidase as a probe. Seven strains were agglutinated with the assayed sugar residues. The highest agglutination was with fucose and glucose and to a lesser extent with mannose residues. Cell-wall proteins extracted from two Azospirillum spp. strains exhibit lectin-like activities. We believe that lectins are present in the cell-wall of Azospirillum spp.

Agglutination↗

PubMed2003

Plant growth regulators and amino acids released by Azospirillum sp in chemically defined media.

AIMS: To investigate the ability of Azospirillum sp., a facultative endophitic diazotrophic bacterium, to release plant growth regulators (PGR) such as polyamines, ethylene, indoleacetic acid and amino acids in both combined-N and N-free cultures. METHODS AND RESULTS: The presence of those substances was analysed by HPLC. Azospirillum sp. is capable of releasing PGR and amino acids into the culture medium. CONCLUSIONS: The type and quantity of the released substances varied, depending on the presence of combined-N in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of PGR produced by Azospirillum sp. has been gained.

Amino Acids↗

PubMed1991

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense.

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.

Ammonia↗

PubMed2006

N-acyl-homoserine lactone-mediated quorum-sensing in Azospirillum: an exception rather than a rule.

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

4-Butyrolactone↗

PubMed2006

Identification, cloning and characterization of cysK, the gene encoding O-acetylserine (thiol)-lyase from Azospirillum brasilense, which is involved in tellurite resistance.

O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.

Azospirillum brasilense↗

PubMed1995

Oligonucleotide probes based on 16S rRNA sequences for the identification of four Azospirillum species.

Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.

Azospirillum↗

PubMed2004

Influence of substrate composition and flow rate on growth of Azospirillum brasilense Cd in a co-culture with 3 sorghum rhizobacteria.

The ability of Azospirillum brasilense Cd to colonize the niche occupied by 3 bacterial strains previously isolated from sorghum rhizosphere was studied by means of the Biolog system. The isolates were identified by different methods as strains belonging to Pseudomonas putida, Stenotrophomonas maltophilia, and Klebsiella terrigena species. Several C sources, also chosen among the constituents of sorghum root exudates, were used to evaluate the metabolic profiles of Azospirillum and the sorghum rhizobacteria. Azospirillum brasilense Cd exploited the same class of C compounds as the sorghum rhizobacteria and overlapped in their niche requirements. Since structure and functioning of a microbial community are largely affected by the flow rate of nutrient supply, the competitive behavior of A. brasilense Cd was studied in a chemostat mixed culture under C-limited conditions using disodium succinate as C source. Only at high growth rates, i.e., when the C source was highly supplied, A. brasilense Cd appeared to be a good competitor and it became the dominant species, whereas at low growth rates, it was outnumbered by the other species. However, the coexistence of all the strains was always maintained, thus suggesting that interactions other than competition or a potential cross-feeding might occur within the mixed culture.

Amino Acids↗

PubMed2005

The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings.

Carnation cuttings treated with non-transformed and 1-aminocyclopropane (ACC) deaminase-containing Azospirillum brasilense Cd1843 produced significantly more roots than untreated controls and fewer roots than cuttings treated with 0.1% indolebutyric acid (IBA). The roots produced by cuttings treated with ACC deaminase-containing Azospirillum brasilense Cd1843 were the longest roots resulting from any of the treatments, followed by non-transformed Azospirillum brasilense Cd1843, 0.1% IBA, and treatment with water. The results are interpreted in terms of a previously proposed model of bacterial promotion of plant growth by ACC deaminase and indoleacetic acid, and may have implications for the use of plant growth-promoting bacteria in the flower industry.

Azospirillum brasilense↗

PubMedDate not supplied

Effect of Azospirillum brasilense inoculation on urease activity in soil and gamma-sterilized soil.

Azospirillum spp. is considered a PGPR (plant growth promoting rhyzobacteria) bacterium, besides this interest, there is little information about its effects on other functional microbial groups or on soil enzymes. In this paper, the impact that A. brasilense 7001 inoculation has on urease activity expression in a Typic Argiudoll was studied. Evolution of urease activity of soil and of gamma-irradiation (25 KGy) sterilized soil, and the inoculated strain survival were tested. The relation between soil urease activity and soil NH4(+)-N was also determined. In gamma-sterilized soil, urease activity of inoculated soil increased with time, showing significant differences with regard to the control soil without inoculum at day 15. In non-sterile soil, urease activity decreased during the studied period in all treatments; in inoculated soil, it showed higher or lower values than the control depending on sampling time. Azospirillum survival was important and different according to soil conditions. The negative relation between NH4(+)-N concentration and soil urease activity (r2 = 0.62) was not observed in inoculated soil. The role of the addition of autoclaved inoculum in the urease activity expression is discussed. The research proves that in both studied situations Azospirillum modified soil urease activity, and that the competition with native microorganisms and soil NH4(+)-N may affect this bacterium capacity.

Azospirillum brasilense↗

PubMed1989

[Formation of pAS8-1213 cointegrate with one of the plasmids of Azospirillum brasilense Sp245].

Inheritance of the plasmid vector pAS8-1213 in Azospirillum brasilense Sp245 cells has been studied. The plasmid pAS8-1213 is shown to be uncapable of autonomous replication in the new host but able to integrate into the genetic structures of Azospirillum with high frequency. 90-95% of KmR-transconjugants of A. brasilense harbor pAS8-1213 cointegrated with the smaller host plasmid pAbSP245c(85Md). The formed cointegrate can be transferred into Azospirillum spp. 75 and RecA- strains of E. coli (HB101 and DH1) and stably maintained in these cells. The IS21 element inherent of the plasmid pAS8-1213 is supposed to participate in pAS8-1213::pAbSP245c cointegrate formation.

Azospirillum brasilense↗

PubMed1993

RAPD fingerprinting is useful for identification of Azospirillum strains.

In vitro amplification of genomic DNA fragments with single primers of arbitrary sequence was used as a rapid and sensitive method to obtain fingerprints of ten strains belonging to three of the Azospirillum species: brasilense, lipoferum and amazonense. Each strain showed a distinctive pattern of bands that permitted its unequivocal identification. Closely related strains produced almost identical fingerprints. Pairwise comparison and cluster analysis of the amplification patterns allowed grouping of the strains. The resulting dendrograms are similar to previous dendrograms based on the restriction endonuclease analysis (REA) of total DNA and on the restriction fragment length polymorphism (RFLP). Our results indicate that the random amplified polymorphic DNA (RAPD) technique is a simple, fast and useful tool for the determination of genetic relationships among Azospirillum isolates and to evaluate the genomic stability of the Azospirillum strains released in the environment.

Azospirillum↗

PubMed1989

Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis.

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.

DNA Transposable Elements↗

PubMedDate not supplied

Nucleotide sequence of the Azospirillum brasilense Sp7 glutamine synthetase structural gene.

The complete nucleotide sequence of the glnA gene, encoding the glutamine synthetase subunit of Azospirillum brasilense Sp7, was established. This is the first Azospirillum gene sequenced. The gene encodes a 468 residue polypeptide of MW 51,917. The similarity coefficient (SAB) between the polypeptidic sequence of Azospirillum and Anabaena 7120, which is the only other glnA sequence available, is 58%. No significant homology with E. coli canonical and ntr promoters, or with the promoter region of the Anabaena glnA gene was found. When fused to an E. coli promoter, the gene could be translated in E. coli, despite a very biased codon usage and an atypical Shine-Dalgarno sequence.

Amino Acid Sequence↗

PubMed1980

Increase in Dry Weight and Total Nitrogen Content in Zea mays and Setaria italica Associated with Nitrogen-fixing Azospirillum spp.

The association between nitrogen-fixing bacteria from the genus Azospirillum and the grasses Zea mays and Setaria italica was investigated in sterilized Leonard-jar assemblies. Nitrogen-fixing bacteria isolated from Cynodon dactylon roots in Israel and Azospirillum brasilense (Sp-7, Sp-80, and Cd) were examined. C(2)H(2) reduction activity was detected in systems containing 0.0 to 0.08 but not in those containing 0.16 gram per liter NH(4)NO(3). The organisms tested significantly increased plant dry weight (50-100%), total N content of leaves (50-100%) and C(2)H(4) production (300-1000 nanomoles C(2)H(4) per plant per hour). Highest C(2)H(2) reduction activities were obtained above 30 C and with high light intensities. Significant increases in S. italica dry weight (DW) and nitrogen (N) content were observed in sand (DW = 80%, N = 150%), sandy loam soil (DW = 80%, N = 75%) and loess (DW = 37%, N = 25%). The results obtained in this work clearly demonstrate the potential benefit of inoculating grasses with Azospirillum.

Journal Article↗

PubMed1985

Interaction of azospirillum and Rhizobium strains leading to inhibition of nodulation.

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.

Journal Article↗

PubMed1986

Comparison of Two Cellulomonas Strains and Their Interaction with Azospirillum brasilense in Degradation of Wheat Straw and Associated Nitrogen Fixation.

A mutant strain of Cellulomonas sp. CS1-17 was compared with Cellulomonas gelida 2480 as the cellulolytic component of a mixed culture which was responsible for the breakdown of wheat straw to support asymbiotic nitrogen fixation by Azospirillum brasilense Sp7 (ATCC 29145). Cellulomonas sp. strain CSI-17 was more efficient than was C. gelida in cellulose breakdown at lower oxygen concentrations and, in mixed culture with A. brasilense, it supported higher nitrogenase activity (C(2)H(2) reduction) and nitrogen fixation with straw as the carbon source. Based on gravimetric determinations of straw breakdown and total N determinations, the efficiency of nitrogen fixation was 72 and 63 mg of N per g of straw utilized for the mixtures containing Cellulomonas sp. and C. gelida, respectively. Both Cellulomonas spp. and Azospirillum spp. exhibited a wide range of pH tolerance. When introduced into sterilized soil, the Cellulomonas sp.-Azospirillum brasilense association was more effective in nitrogen fixation at a pH of 7.0 than at the native soil pH (5.6). This was also true of the indigenous diazotrophic microflora of this soil. The potential implications of this work to the field situation are discussed.

Journal Article↗

PubMed1987

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass.

The effect of oxygen on N(2)-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 muM O(2), some of them being carbon limited. All strains needed a minimum amount of oxygen for N(2)-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O(2) concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N(2) fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N(2) fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O(2) concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.

Journal Article↗