Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

Alterations in the primary structure of an 85-MDa plasmid affecting flagellation and motility of bacterium Azospirillum brasilense Sp245

Earlier such Azospirillum brasilense Sp245 mutants as flagellation-defective SK051, SK248 with immobilized flagella, and BK570 swimming and swarming faster than Sp245 were obtained. In SK051 and SK248 the self-killer vector pJFF350 integrated into the 18.3-kb XhoI fragment ofplasmid 85MDa (p85) while in BK570, it integrated into the 9.1-kb XhoI-fragment of p85. In the present work, analysis of the nucleotide sequence of fusion products of p85 and pJFF350 was performed. In p85, in addition to three IS elements (two of which caused cointegrate formation) and phage integrase gene, 22 open reading frames with coding sequence properties were identified. Possible participation of predicted translation products of several p85 genes in bacterial motility detection is discussed. Since differences in the primary structure of p85::pJFF350 cointegrates from SK051 and SK248 cells are localized within pJFF350 DNA, different effects of DNA-folding changes on expression of corresponding p85 genes are suggested.     

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.     

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.     

Effect of integrating the pJFF350 vector into the 85-MDa plasmid of Azospirillum brasilense Sp245 on bacterial flagellation and mobility

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.     

Identification and mapping of loci involved in motility, adsorption to wheat roots, colony morphology, and growth in minimal medium on the Azospirillum brasilense Sp7 90-MDa plasmid.

We have constructed a cosmid library of the Azospirillum brasilense Sp7 90-MDa plasmid (p90) and established the EcoRI restriction map of this plasmid. The central regions of cloned p90 DNA fragments from several recombinant cosmids were deleted by restriction endonuclease digestion and replaced by a DNA cassette encoding kanamycin resistance. Using these in vitro constructed deletions for marker exchange in Sp7, we made six different p90 deletion derivatives spanning all together 50% of the total length of p90. Comparison of the deletion derivatives with Sp7 for several properties revealed p90 loci involved in colony morphology, growth on minimal medium, motility, and adsorption to wheat roots. In analogy with the rhizobial symbiotic plasmids (pSym), we propose to denote the p90 plasmid as a rhizocoenotic plasmid (pRhico), carrying several genes involved in the A. brasilense-plant root interaction.     

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.     

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.     

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.     

Energy transduction efficiencies in nitrogenous oxide respirations of Azospirillum brasilense Sp7

For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H⁺-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth yields (Y _s ^max -values) were essentially the same with O₂ or N₂O but were 1/3 and 2/3 lower with NO ₂ ^- or NO ₃ ^- , respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N₂-fixing conditions drastically reduced the Y _s ^max -values in the N₂O and O₂-respiring cells. In the H⁺-translocation measurements, the /oxidant ratios were 5.6 for O₂→H₂O, 2.5–2.8 for NO ₃ ^- →NO ₂ ^- , 2.2 for NO ₂ ^- →N₂O and 3.1 for N₂O→N₂ respirations when the cells were preincubated with valinomycin and K⁺. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP⁺) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H⁺-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore, nitrate and nitrite respirations were accompanied by a H⁺-uptake . Any significant H⁺-translocation could not be detected in N₂O- and O₂-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal molar growth yield determinations and the absence of any H⁺-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of respiration. The low H⁺-extrusion values for N₂O-respiration compared to O₂-respiration in cells treated with valinomycin plus TPMP⁺ are, however, not in accord with such an interpretation.     

Electroporation of Azospirillum brasilense with plasmid DNA

Abstract The feasibility of electric field mediated transformation of the nitrogen fixing bacterium Azospirillum was studied. The broad host range plasmid pRK290 was used throughout this study. Transformants were obtained with all A. brasilense strains tested, although with strain dependent efficiency. No transformants were obtained with an A. lipoferum strain. Transfer of the pRK290 plasmid DNA in the A. brasilense strains was confirmed by DNA extraction of the transformants and gel electrophoresis. The effects of the physiological status of the cells and the electric field strength during electroporation were studied in detail for one particular A. brasilense strain.     

Effect of the Integration of Vector pJFF350 into Plasmid 85-MDa of Azospirillum brasilense Sp245 on Bacterial Flagellation and Motility

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.     

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.     

The nodPQ genes in Azospirillum brasilense Sp7 are involved in sulfation of lipopolysaccharides

Here we report on the presence of sulfated lipopolysaccharide molecules in Azospirillum brasilense, a plant growth-promoting rhizosphere bacterium. Chemical analysis provided structural data on the O-antigen composition and demonstrated the possible involvement of the nodPQ genes in O-antigen sulfation.     

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.     

Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.     

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