Potentially Mobile Denitrification Genes Identified in Azospirillum sp. Strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS6 family insertion sequences (ISAz581 and ISAz582). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers.IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD, located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification.

In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

BACKGROUND: The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. RESULTS: The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5T, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-ST, A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation. CONCLUSIONS: Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

The genetic diversity of culturable nitrogen-fixing bacteria in the rhizosphere of wheat.

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.

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.

[Extracellular proteolytic enzymes of Azospirillum brasilensis strain Sp7 and regulation of their activity by a homologous lectin].

It was found that Azospirillum brasilensis strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.

Azospirillum irakense pectate lyase displays a toroidal fold.

The three-dimensional structure of Azospirillum irakense pectate lyase (PelA) has been determined at a resolution of 2.65 A. The crystals are hexagonal, belonging to space group P6(5)22, with unit-cell parameters a = b = 85.37, c = 231.32 angstroms. Phase information was derived from a multiple-wavelength anomalous dispersion (MAD) experiment using a Hg derivative. Refinement of the model converged to Rcryst = 20.08% and Rfree = 25.87%. The overall structure of PelA does not adopt the characteristic parallel beta-helix fold displayed by pectate lyases from polysaccharide lyase (PL) families PL1, PL3 and PL9. Instead, it displays a predominantly alpha-helical structure with irregular coils and short beta-strands, similar to the recently reported structure of the catalytic module of the Cellvibrio japonicus pectate lyase Pel10Acm. The topologies of the two structures have been compared. They show two 'domains' with the interface between them being a wide-open central groove in which the active site is located. The active sites of the crystal structures are also compared and their similarities and differences are discussed.

Structural characterization of glutamine synthetase from Azospirillum brasilense.

CD spectroscopic study of the secondary structure of partly adenylylated glutamine synthetase (GS) of the bacterium Azospirillum brasilense showed both the native and cation-free (EDTA-treated) enzyme to be highly structured (58 and 49% as alpha-helices, 10 and 20% as beta-structure, respectively). Mg(2+), Mn(2+), or Co(2+), when added to the native GS, had little effect on its CD spectrum, whereas their effects on the cation-free GS were more pronounced. Emission ((57)Co) Mössbauer spectroscopic (EMS) study of (57)Co(2+)-doped cation-free GS in frozen solution and in the dried state gave similar spectra and Mössbauer parameters for the corresponding spectral components, reflecting the ability of the Co(2+)-enzyme complex to retain its properties upon drying. The EMS data show that (a) A. brasilense GS has 2 cation-binding sites per active center and (b) one site has a higher affinity to Co(2+) than the other, in line with the data on other bacterial GSs.

Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria.

In this work, we report the detection of aromatic amino acid aminotransferase (AAT) activity from cell-free crude extracts of nine strains of N(2)-fixing bacteria from three genera. Using tyrosine as substrate, AAT activity ranged in specific activity from 0.084 to 0.404 micromol min(-1)mg(-1). When analyzed under non-denaturating PAGE conditions; and using tryptophan, phenylalanine, tyrosine, and histidine as substrates Pseudomonas stutzeri A15 showed three isoforms with molecular mass of 46, 68 and 86 kDa, respectively; Azospirillum strains displayed two isoforms which molecular mass ranged from 44 to 66 kDa and Gluconacetobacter strains revealed one enzyme, which molecular mass was estimated to be much more higher than those of Azospirillum and P. stutzeri strains. After SDS-PAGE, some AAT activity was lost, indicating a differential stability of proteins. All the strains tested produced IAA, especially with tryptophan as precursor. Azospirillum strains produced the highest concentrations of IAA (16.5-38 microg IAA/mg protein), whereas Gluconacetobacter and P. stutzeri strains produced lower concentrations of IAA ranging from 1 to 2.9 microg/mg protein in culture medium supplemented with tryptophan. The IAA production may enable bacteria promote a growth-promoting effect in plants, in addition to their nitrogen fixing ability.

Catalytic properties of an endogenous beta-lactamase responsible for the resistance of Azospirillum lipoferum to beta-lactam antibiotics.

Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6-beta-Bromopenicillanic acid, an irreversible inhibitor of serine-beta-lactamases, completely abolished this resistance. A beta-lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31000 Da) and on isoelectric focussing (pI9.35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable k(cat) values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified beta-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum beta-lactamases inhibited by clavulanic acid (group 2b of the Bush-Jacoby-Medeiros scheme). The effect of pH on k(cat) and K(m) values for benzylpenicillin hydrolysis was studied. The dependence of k(cat) on pH suggests that the enzyme-substrate (ES) complex must be in at least three protonation states: two with k(cat) values equal to 2800 and 1450 s(-1) and a third inactive one [pK(1(ES)) 4.7 and pK(2(ES)) 7.9]. Similarly, the dependence of k(cat)/K(m) on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with k(cat)/K(m) values equal to 2.7 x 10(6) and 3.7 x 10(8) M(-1) s(-1) and a third one unable to productively bind substrate. Interestingly, the dependence of k(cat)/K(m) on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK(1(E)) 8.5 and pK(2(E)) 7.2].

Modeling growth and biochemical activities of Azospirillum spp.

An unstructured mathematical model was developed and used in the evaluation of biochemical activities of four Azospirillum spp. strains grown in batch cultures in a high C/N-ratio medium. The strains were evaluated for their ability to grow on fructose and produce exo-polysaccharide, and to sustain nitrogenase activity by using fructose or polysaccharides. Quantitative expression of the regulation of polysaccharide synthesis and nitrogenase (acetylene reduction) activity from the mineral nitrogen and sugar concentration in the culture medium was achieved. It was found that, during growth, Azospirillum spp. produced significant quantities of exocellular and capsular polysaccharide, whereas after depletion of the carbon source from the culture medium polysaccharides were consumed, especially in A. lipoferum strains. Significant nitrogenase activity was detected during polysaccharide degradation. Oxygen uptake was high during assimilation of fructose and low during polysaccharide degradation.

Crystallization and preliminary X-ray diffraction analysis of a novel pectate lyase from Azospirillum irakense.

The PelA gene from the N(2)-fixing plant-associated bacterium Azospirillum irakense encodes a pectate lyase. Analysis of the corresponding amino-acid sequence revealed no homology to other bacterial, plant and fungal pectinases of known published structure, resulting in the classification of the enzyme in a new pectate lyase family. The A. irakense PelA has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are hexagonal, with unit-cell parameters a = b = 85.55, c = 230.13 A, gamma = 120 degrees, and belong to space group P6(5)22 or P6(1)22, having one molecule per asymmetric unit. Diffraction data to a resolution of 1.97 A were collected at synchrotron facilities, as well as a three-wavelength MAD data set from an Hg-derivative crystal to a resolution of 2.6 A.

Evidence for the presence of DNA-binding proteins involved in regulation of the gene expression of indole-3-pyruvic acid decarboxylase, a key enzyme in indole-3-acetic acid biosynthesis in Azospirillum lipoferum FS.

We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS. Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.

Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum.

A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp. aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation. Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration. Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations. SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants. Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A. lipoferum and transformants.

The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides.

The CelA beta-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C(3), C(4), and C(5) oligosaccharides. The growth of a DeltacelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 beta-glucosidases appear to be functional alternatives for the GHF1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.

[Relationship between lectin, alpha-, beta-glucosidase, and beta-galactosidase activities of Azospirillum]. / Izuchenie vzaimosviazi lektinovoi, al'fa-, beta-gliukozidaznykh i beta-galaktozidaznoi aktivnostei azospirill.

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.

Loss of cytochrome c oxidase activity and acquisition of resistance to quinone analogs in a laccase-positive variant of Azospirillum lipoferum.

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.

Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease.

A novel psychrotrophic bacterium secreting a protease was isolated from a mountain soil in Korea. On the basis of a 16S rDNA sequence analysis and physiological properties, the isolate was identified as an Azospirillum sp. The protease purified from the culture supernatant was a monomer in its native form with an apparent molecular mass of 48.6 kDa on SDS-PAGE. The protease was active in a broad pH range around 8.5 and at temperatures up to 40 degrees C and stable at temperatures below 30 degrees C for 3 days. The proteolytic activity was inhibited by iodoacetamide and EDTA. The Mg2+ ion did not activate the enzyme much but reversed the inhibition by EDTA, suggesting that the protease belongs to a cysteine protease stabilized by the Mg2+ ion.

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process.

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.

Growth of Azospirillum irakense KBC1 on the aryl beta-glucoside salicin requires either salA or salB.

The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.