Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

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 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) 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.     

Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245

The expressed difference in the plasmid profile of A. brasilense Sp245 is registered as a result of Tn5-Mob-mutability. Integration of the vector pSUP5011 into one of the A. brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported. The properties of A. brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed. The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium. The results suppose 85Md plasmid participation in melaninogenesis.     

Effect of Azospirillum brasilense Sp245 lipopolysaccharide on the functional activity of wheat root meristematic cells

We studied changes in the physiological and biochemical parameters of wheat (Triticum aestivum L. ??Saratovskaya 29??) seedlings treated with lipopolysaccharide isolated from the outer membrane of the associative bacterium Azospirillum brasilense Sp245. The obtained data were compared with (i) the results of plant inoculation with whole Sp245 cells and (ii) the effects exerted by the lipopolysaccharide and whole cells of the enterobacterium Escherichia coli K12 and the specific legume symbiont Rhizobium leguminosarum 249. The functional activity of meristematic cells was judged by their mitotic index and by the results of immunochemical determination of the proliferative antigen of initials, a molecular marker for wheat meristem cells. Treatment of the seedling root system with 10 ??g mL^?1 of Sp245 lipopolysaccharide increased the mitotic index (1.8-fold) and the antigen content (approximately 1.4-fold). These increases were comparable to the effects produced by whole cell inoculation (2- and 1.4-fold, respectively). Our findings give grounds to consider lipopolysaccharide as an active component of the Azospirillum cell surface that not only determines bacterial contact interactions with wheat roots but also participates in the induction of plant responses to these interactions. We finally discuss the linkage between the proliferative antigen of initials and the transduction of a hormonal signal to the cell, as well as the informational value of this antigen as an indicator of effectiveness of plant?Cbacterial interactions.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.     

Identification and sequencing of pyrG, the CTP synthetase gene of Azospirillum brasilense Sp7

Abstract An 18.5-kb DNA fragment carrying the trpGDC cluster of Azospirillum brasilense Sp7 was previously cloned, yielding cosmid pAB1005. Attempts to identify trpA in the vicinity of trpGDC failed but led to the detection of a locus strongly homologous to pyrG , the structural gene for the CTP synthetase. The function of the A. brasilense pyrG gene was verified by complementation of the cytidine-requiring PyrG-deficient mutant JF646 of Escherichia coli . A second open reading frame was identified downstream of pyrG . The deduced amino acid sequence showed homology to dienelactone hydrolases of Pseudomonas and Alcaligenes , enzymes involved in utilization of halogenated aromatic compounds.     

Survival of Azospirillum brasilense in the Bulk Soil and Rhizosphere of 23 Soil Types

The survival of Azospirillum brasilense Cd and Sp-245 in the rhizosphere of wheat and tomato plants and in 23 types of plant-free sterilized soils obtained from a wide range of environments in Israel and Mexico was evaluated. Large numbers of A. brasilense cells were detected in all the rhizospheres tested, regardless of soil type, bacterial strain, the origin of the soil, or the amount of rainfall each soil type received prior to sampling. Survival of A. brasilense in soils without plants differed from that in the rhizosphere and was mainly related to the geographical origin of the soil. In Israeli soils from arid, semiarid, or mountain regions, viability of A. brasilense rapidly declined or populations completely disappeared below detectable levels within 35 days after inoculation. In contrast, populations in the arid soils of Baja California Sur, Mexico, remained stable or even increased during the 45-day period after inoculation. In soils from Central Mexico, viability slowly decreased with time. In all soils, percentages of clay, nitrogen, organic matter, and water-holding capacity were positively correlated with bacterial viability. High percentages of CaCO(inf3) and fine or rough sand had a highly negative effect on viability. The percentage of silt, pH, the percentage of phosphorus or potassium, electrical conductivity, and C/N ratio had no apparent effect on bacterial viability in the soil. Fifteen days after removal of inoculated plants, the remaining bacterial population in the three soil types tested began to decline sharply, reaching undetectable levels 90 days after inoculation. After plant removal, percolating the soils with water almost eliminated the A. brasilense population. Viability of A. brasilense in two artificial soils containing the same major soil components as the natural soils from Israel did was almost identical to that in the natural soils. We conclude that A. brasilense is a rhizosphere colonizer which survives poorly in most soils for prolonged periods of time; that outside the rhizosphere, seven abiotic parameters control the survival of this bacterium in the soil; and that disturbance of the soil (percolation with water or plant removal) directly and rapidly affects the population levels.     

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.     

小麦根际联合固氮菌(固氮螺菌,Azospirillum brasilense)田间接种效果的研究

据黑龙江省1990年7个中试点试验证明,小麦根际联合固氮菌田间接种效果在5.2—20.6％,平均为9.8％;每田可增产小麦19.1公斤。小麦根际联合固氮菌能促进小麦本长发育,在生育期间可使株高、鲜重增加,叶色变缘。秋收考种证明,对产量构成因子的平方米穗数、穗粒数和千粒重均有增加。     

Improving micropropagation: effect of Azospirillum brasilense Sp245 on acclimatization of rootstocks of fruit tree

Abstract

The effect of Azospirillum brasilense Sp245 on the micropropagation of three fruit rootstocks: Mr.S 2/5 plum (Prunus cerasifera×P. spinosa), GF 677 hybrid (Prunus persica×P. amigdalus), and MM 106 apple (Northen Spy×M1) was assessed. Rooted shoots were treated with 3×10⁷ of Sp245 cells during transplantation from in vitro cultures to the acclimatization phase. After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of Azospirillum can significantly contribute to optimize plant performance during the phase of adaptation of plants to post-vitrum conditions.     

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: Involvement of nifA, glnA and glnB gene products

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.     

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Abstract The effect of certain nitrogen compounds on nitrogenase activity was studied in cells of Azospirillum brasilense strain Sp6, grown under microaerophilic conditions with nitrogenase fully derepressed. 0.5 mM NH₄Cl, 0.5 mM glutamine, 1.0 mM KNO₃ and 0.1 mM KNO₂ completely blocked nitrogenase activity. 1.0 mM asparagine, 1.0 mM aspartate, 1.0 mM histidine and 1.0 mM adenine did not caused no inhibition of nitrogenase; indeed asparagine, aspartate and histidine showed a slight stimulatory effect on N₂ fixation. The addition of 10 mM dl -methionine- dl -sulphoximine prevented the inhibitory effect of NH₄Cl and glutamine but did not counteract the effect of KNO₂. Rifampicin and chloramphenicol did not prevent the inhibition of nitrogenase by NH₄Cl.     

Inhibition of Biosynthesis and Activity of Nitrogenase in Azospirillum brasilense Sp7 Under Salinity Stress

Azospirillum brasilense is a microaerophilic, plant growth-promoting bacterium, whose nitrogenase activity has been shown to be sensitive to salinity stress. Growth of A. brasilense in semi-solid medium showed that diazotrophic growth in N-free medium was relatively less sensitive to high NaCl concentrations (200–400 mM) than that in presence of NH₄ ⁺. Increase in salinity stress to diazotrophic A. brasilense in the semi-solid medium led to the migration of the pellicle to deeper anaerobic zones. Assays of acetylene reduction and nifH-lacZ and nifA-lacZ fusions indicated that salinity stress inhibited nitrogenase biosynthesis more strongly than nitrogenase activity. Under salt stress, the amount of dinitrogenase reductase inactivated by ADP-ribosylation was strongly reduced, indicating that the dinitrogenase reductase ADP ribosyl transferase (DRAT) activity was also inhibited by increased NaCl concentrations. Movement of the pellicle to the anaerobic zone and inhibition of DRAT might be adaptive responses of A. brasilense to salinity stress under diazotrophic conditions. Supplementation of glycine betaine, which alleviates salt stress, partially reversed both responses. Received: 2 August 2001 / Accepted: 28 August 2001     

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.     

Aerobic nitric oxide production by Azospirillum brasilense Sp245 and its influence on root architecture in tomato

The major feature of the plant-growth-promoting bacteria Azospirillum brasilense is its ability to modify plant root architecture. In plants, nitric oxide (NO) mediates indole-3-acetic acid (IAA)-signaling pathways leading to both lateral (LR) and adventitious (AR) root formation. Here, we analyzed aerobic NO production by A. brasilense Sp245 wild type (wt) and its mutants Faj009 (IAA-attenuated) and Faj164 (periplasmic nitrate reductase negative), and its correlation with tomato root-growth-promoting effects. The wt and Faj009 strains produced 120 nmol NO per gram of bacteria in aerated nitrate-containing medium. In contrast, Faj164 produced 5.6 nmol NO per gram of bacteria, indicating that aerobic denitrification could be considered an important source of NO. Inoculation of tomato (Solanum lycopersicum Mill.) seedlings with both wt and Faj009 induced LR and AR development. In contrast, Faj164 mutant was not able to promote LR or AR when seedlings grew in nitrate. When NO was removed with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), both LR and AR formation were inhibited, providing evidence that NO mediated Azospirillum-induced root branching. These results show that aerobic NO synthesis in A. brasilense could be achieved by different pathways and give evidence for an NO-dependent promoting activity on tomato root branching regardless of bacterial capacity for IAA synthesis.     

Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance

The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.     

Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.     

Estimation by PLFA of Microbial Community Structure Associated with the Rhizosphere of Lygeum spartum and Piptatherum miliaceum Growing in Semiarid Mine Tailings

The objective of this study was to compare the microbial community composition and biomass associated with the rhizosphere of a perennial gramineous species (Lygeum spartum L.) with that of an annual (Piptatherum miliaceum L.), both growing in semiarid mine tailings. We also established their relationship with the contents of potentially toxic metals as well as with indicators of soil quality. The total phospholipid fatty acid (PLFA) amount was significantly higher in the rhizosphere soil of the annual species than in the rhizosphere soil of the perennial species. The fungal/bacterial PLFA ratio was significantly greater in the perennial species compared to the annual species. The fatty acid 16:1ω5c, the fungal/bacterial PLFA ratio and monounsaturated/saturated PLFA ratio were correlated negatively with the soluble contents of toxic metals. The cyc/prec (cy17:0 + cy19:0/16:1ω7 + 18:1ω7) ratio was correlated positively with the soluble contents of Pb, Zn, Al, Ni, Cd, and Cu. The results of the PLFA analysis for profiling microbial communities and their stress status of both the plant species indicate that perennial and annual gramineous species appear equally suitable for use in programmes of revegetation of semiarid mine tailings.     

The Use of Fragments of the 85- and 120-MDa Plasmids of Azospirillum brasilense Sp245 to Study the Plasmid Rearrangement in This Bacterium and to Search for Homologous Sequences in Plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

The use of fragments of the 85- and 120-MDa plasmids of Azospirillum brasilense Sp245 to study the plasmid rearrangement in this bacterium and to search for homologous sequences in plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of the Azospirillum brasilense Sp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilense Sp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.