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PubMed2005

Nitric oxide is involved in the Azospirillum brasilense-induced lateral root formation in tomato.

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g-1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.

Azospirillum brasilense↗

PubMed2001

Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus.

A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C4-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.

Azospirillum↗

PubMed2000

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Anthranilate Synthase↗

PubMed2006

Root phospholipids in Azospirillum-inoculated wheat seedlings exposed to water stress.

Azospirillum-plant association is accompanied by biochemical changes in roots which, in turn, promote plant-growth and tolerance to water stress. To shed light on the possible factors underlying these effects, roots from Azospirillum brasilense Sp245-inoculated Triticum aestivum seedlings growing in darkness under osmotic stress were analyzed for phospholipid (PL) composition, fatty acid (FA) distribution profiles and degree of unsaturation of the major PL classes. Azospirillum inoculation diminished ion leakage and increased 2,3,5-tripheniltetrazolium reducing ability in roots of well irrigated and water-stressed wheat seedlings. Total root PL content remained unaltered in all treatments. Six PL classes were detected, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprising over 80% of the total. While water stress increased PC content and diminished that of PE, none of these changes were observed either under Azospirillum inoculation alone or when both treatments were combined. The major FAs found in both PC and PE were 16:0, 18:0, 18:1, 18:2, and 18:3. Higher PC and lower PE unsaturation than in well irrigated controls were observed in roots from Azospirillum-inoculated, water-stressed seedlings. Azospirillum inoculation could contribute to protect wheat seedlings from water stress through changes in the FA distribution profiles of PC and PE major root phospholipids.

Azospirillum↗

PubMed1996

Production and characterization of strain-specific monoclonal antibodies against outer membrane components of Azospirillum brasilense Sp245.

Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) directed against outer membrane components of the Gram-negative rhizosphere bacterium Azospirillum brasilense Sp245 have been established and characterized. Whole bacterial cells were used as immunogens. Among the clones obtained, 14 hybridoma cell lines were selected for further characterization. Eight MAbs were strain-specific and 6 MAbs showed cross-reactivity with a closely related strain Azospirillum brasilense Sp246. According to the biochemical characterization of the antigenic determinants, MAbs were classified into four groups. The corresponding antigens were lipopolysaccharides (class 1) and an outer membrane protein (class 4), which is common to Azospirillum brasilense Sp245 and Azospirillum brasilense Sp246 as well as two outer membrane proteins (class 2 and class 3) that are characteristic for Azospirillum brasilense Sp245. The number of antigens per cell varied from 4000 (class 1) to 100 (class 4). In each class high affinity MAbs were identified, which made a sensitive direct quantification of Azospirillum brasilense Sp245 possible.

Animals↗

PubMed2005

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea.

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.

Acetylene↗

PubMed2000

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots.

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1993

Chemotaxis of azospirillum species to aromatic compounds.

Chemotaxis of Azospirillum lipoferum Sp 59b and Azospirillum brasilense Sp 7 and Sp CD to malate and to the aromatic substrates benzoate, protocatechuate, 4-hydroxybenzoate, and catechol was assayed by the capillary method and direct cell counts. A. lipoferum required induction by growth on 4-hydroxybenzoate for positive chemotaxis to this compound. Chemotaxis of Azospirillum spp. to all other substrates did not require induction. Maximum chemotactic responses for most aromatic compounds occurred at concentrations of 1 to 10 mM for A. lipoferum and 100 muM to 1 mM for A. brasilense. Threshold levels of these chemoattractants ranged from nanomolar to micromolar, with A. brasilense Sp CD showing the lowest threshold levels for the substrates tested. Benzoate was the strongest chemoattractant tested, with threshold concentrations in the nanomolar to picomolar range for all strains. Azospirillum spp. clearly have more sensitive chemosensory mechanisms for certain aromatic substrates than previously reported in some other soil bacteria. This sensitivity allows Azospirillum spp. to detect and respond to aromatic substrates at concentrations relevant to the soil and rhizosphere environments. The ability to detect such low concentrations of aromatic compounds in soils may confer advantages in survival and colonization of the rhizosphere by Azospirillum species.

Journal Article↗

PubMed1985

Purification and properties of the nitrogenase of Azospirillum amazonense.

The nitrogenase of the free-living, microaerobic, N2-fixing bacterium Azospirillum amazonense (strain Y1) was purified by chromatography on DEAE-52 cellulose, by heat treatment, and by preparative polyacrylamide gel electrophoresis. The specific nitrogenase activities were 2,400 nmol of C2H4 formed per min per mg of protein for dinitrogenase (MoFe protein) and 1,800 nmol of C2H4 formed per min per mg of protein for dinitrogenase reductase (Fe protein). The MoFe protein was composed of a minimum of 1,852 amino acid residues, had an isoelectric point of 5.2, and contained 2 atoms of Mo, 24 atoms of Fe, and 28 atoms of acid-labile sulfide per molecule. The Fe protein had 624 amino acid residues and an isoelectric point of 4.6 and contained four atoms of Fe and six atoms of acid-labile sulfide per molecule. The purified MoFe protein showed two subunits with molecular weights of 55,000 and 50,000. The purified Fe protein revealed two polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weights of 35,000 and 31,000. The two Fe protein polypeptides were demonstrated with immunological techniques in the purified, highly active enzyme as well as in extracts. Also, Azotobacter vinelandii Fe protein showed two closely migrating polypeptides that migrated differently from the Fe protein polypeptides of Azospirillum brasilense or Rhodospirillum rubrum. The nitrogenase activity of Azospirillum amazonense Y1 was independent of Mn2+, and the addition of activating enzyme had no effect. No activating enzyme could be found in Azospirillum amazonense. Obviously, the nitrogenase system of Azospirillum amazonense Y1 is different from that of Azospirillum brasilense Sp7 and resembles the Azotobacter system.

Amino Acids↗

PubMed1984

[Differentiation among species of the genus Azospirillum].

To replace the Spirillum lipoferum denomination (2) for the nitrogen fixing group of soil spirilla, the Azospirillum genus has been created in recent years (12), which includes two species: A. lipoferum and A. brasilense. Both of them are nitrogen fixing bacteria able to induce a specific infection of grass roots (1, 6). Consequently it is important to differentiate correctly the species when plant-bacteria relationships must be studied or inoculation assays must be carried out. For this reason the response of 20 strains of Azospirillum to the classification tests will be described. Microorganisms were isolated from washed plant roots (Table 1) by similar methods to those described by Döbereiner et al. (5). NFb medium was used replacing the micronutrients with 0.05 g/l of yeast extract, which gave better results. For easily identification of Azospirillum colonies, Congo Red was added to the medium (11). Morphology, motility and type of colonies were typical of Azospirillum (1, 3, 4). Nitrogenase activity ranging from 7 to 57 nM C2H2/ml of culture medium/h, according to strain, was detected in all strains pure cultures, by a colorimetric ethylene determination (8). Differentiation tests were performed by Tarrand et al. method (12) (Table 2). Four strains were classified as A. lipoferum and 9 as A. brasilense. Five were A. brasilense-like organisms unable to grow in, a biotin-free medium. As these five strains have been isolated in a medium containing biotin (yeast extract), they might be A. brasilense mutants which have lost the biotin synthesizing capacity. The last two organisms could not be classified and a DNA study should be done to determine whether they belong to any of the Azospirillum species known (7, 10, 13). The results obtained show that to achieve a correct classification of Azospirillum strains (6, 9), all known tests should be performed.

Bacterial Proteins↗

PubMed2000

Surface characteristics of Azospirillum brasilense in relation to cell aggregation and attachment to plant roots.

The free-living bacteria of the genus Azospirillum live in close association with plant roots and represent one of the best-characterized plant growth promoting rhizobacteria (PGPR). The attachment of Azospirillum to the roots is essential for the establishment of an efficient association with the host plant. Azospirillum cells are able to aggregate under certain environmental conditions, leading to the formation of bacterial flocs. The bacterial surface plays an important role in the establishment of the bacteria-plant association as well as in the bacterial aggregation and data suggesting the involvement of extracellular polysaccharides and proteins in these phenomena have been published. This review summarizes the current knowledge on the involvement of surface components in the adhesion processes of Azospirillum. Emphasis is placed on A. brasilense, the species that has been the subject of most studies in the Azospirillum genus.

Azospirillum brasilense↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMed1996

The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis.

The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis.

Amino Acid Sequence↗

PubMed2004

Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression.

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

Antibiosis↗

PubMed1989

Membrane protein patterns of three Azospirillum species and Herbaspirillum seropedicae.

Membrane protein patterns in SDS-PAGE clearly characterized Azospirillum amazonense, A. brasilense, A. lipoferum, and Herbaspirillum seropedicae. Each species showed also a typical major band with approximate molecular weights (AMWs) of 44, 39.5, 35.5, and 37.5 Kdaltons for the four species respectively. Characteristic lighter bands with AMWs of 63.5 and 19 Kdaltons were shown by A. amazonense. Similar bands of 54, 24 and 24.5 Kdaltons were typical of A. brazilense, A. lipoferum and H. seropedicae, respectively. The three Azospirillum species showed four identical proteins in common, out of the six main proteins presented in SDS-PAGE; H. seropedicae had only two major proteins with one of them common to all three Azospirillum species. The data strongly favors the maintenance of Azospirillum and Herbaspirillum as valid generic taxa as well as supports the validity of the three Azospirillum species.

Azospirillum brasilense↗

PubMed1995

The development of Azospirillum as a commercial inoculant for improving crop yields.

Bacteria of the genus Azospirillum are nitrogen-fixing organisms that live in close association with plants in the rhizosphere. The Azospirillum-plant association leads to the enhanced development and yield of different host plants under appropriate growth conditions. This increase in yield is attributed mainly to an improvement in root development, an increase in the rate of water and mineral uptake by roots, and to a lesser extent, biological N(2) fixation. Worldwide data accumulated in the field over the past 20 years indicates that Azospirillum is capable of promoting the yield of agriculturally important crops in different soils and climatic regions. A.brasilense shows both chemotaxis and chemokinesis in response to temporal gradients of different chemoeffectors, thereby increasing the chance of root-bacterial interactions. Phytohormones synthesized by Azospirillum influence the host root respiration rate, metabolism and root proliferation and hence better the mineral and water uptake in inoculated plants. Positive effects of combined inoculation with Rhizobium have been reported for different legumes and were related to the favorable influence of Azospirillum on the nodule number, plant development, dry weight, and N(2) fixation. Additionally, A. brasilense produces the reserve material polyhydroxybutyrate comprising up to 70% of the cell dry weight This substance has received much attention recently as it can be extracted and formed into a biodegradable thermoplastic.

Journal Article↗

PubMed1985

Influence of azospirillum strains on the nodulation of clovers by Rhizobium strains.

Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cell preparations of Azospirillum strains were used for mixing experiments, no effect on nodulation was observed. A decrease in the effectiveness of normally Nod FixR. trifolii strains was observed when an Azospirillum strain caused an increase in nodule number.

Journal Article↗

PubMed1999

Degradation of desferrioxamines by Azospirillum irakense: assignment of metabolites by HPLC/electrospray mass spectrometry.

Based on a recent finding that an Azospirillum isolate ASP-1 possessing high 16S rDNA similarity to Azospirillum irakense was able to degrade desferrioxamine type siderophores (Winkelmann et al. BioMetals 9, 78-83, 1996), various members of the genus Azospirillum were analyzed for their ability to degrade desferrioxamines. While the desferrioxamine-degrading activity was absent or scarcely detectable in strains of A. lipoferum, A. brasilense, A. amazonense, degradation activity seemed to be confined to the species A. irakense (KBC-1, KA3). Also the identity of strain ASP-1 as A. irakense could be confirmed by species-specific oligonucleotide hybridization, Inter-LINE PCR fingerprinting and carbon source utilization pattern (BIOLOG) analysis. Products of desferrioxamine B degradation were analyzed by analytical HPLC and HPLC/electrospray mass spectrometry. Using whole cells and purified enzyme it was shown that the trihydroxamate desferrioxamine B (561 amu) is split at the N-terminal amide bond yielding a monohydroxamate (MH1, 219 amu) and a dihydroxamate (DH1, 361 amu) metabolite. A second monohydroxamate (MH2, 319 amu) resulted from DH1 after splitting the acetylhydroxamate bond. Minor amounts of a further dihydroxamate (DH2, 419 amu) originated from splitting the second amide bond in desferrioxamine B. In addition to desferrioxamine B, several other linear and cyclic desferrioxamines and derivatives were degraded, whereas desferricoprogen and desferri-ferrichrome were not degraded, indicating high substrate specificity of the desferrioxamine hydrolase in A. irakense species. A simple microtiter plate assay was developed which can be used to phenotypically discriminate and identify species of A. irakense from other Azospirillum species by their characteristic feature of desferrioxamine degradation.

Azospirillum↗