| 1. |
Braker G,
Tiedje JM,
( 2003 ) Nitric oxide reductase (norB) genes from pure cultures and environmental samples. PMID : 12788753 : DOI : 10.1128/aem.69.6.3476-3483.2003 PMC : PMC161466 Abstract >>
A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.
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2. |
Liang YY,
de Zamaroczy M,
Arsène F,
Paquelin A,
Elmerich C,
( 1992 ) Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products. PMID : 1362170 : DOI : 10.1111/j.1574-6968.1992.tb14028.x Abstract >>
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.
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3. |
Kadouri D,
Jurkevitch E,
Okon Y,
( 2003 ) Poly beta-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant. PMID : 12898135 : DOI : 10.1007/s00203-003-0590-z Abstract >>
Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.
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4. |
Kadouri D,
Burdman S,
Jurkevitch E,
Okon Y,
( 2002 ) Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant. PMID : 12039753 : DOI : 10.1128/aem.68.6.2943-2949.2002 PMC : PMC123958 Abstract >>
Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.
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5. |
Hauwaerts D,
Alexandre G,
Das SK,
Vanderleyden J,
Zhulin IB,
( 2002 ) A major chemotaxis gene cluster in Azospirillum brasilense and relationships between chemotaxis operons in alpha-proteobacteria. PMID : 11934495 : DOI : 10.1111/j.1574-6968.2002.tb11061.x Abstract >>
Azospirillum brasilense shows chemotaxis to a variety of nutrients and oxygen. Genes encoding the central signal transduction pathway in chemotaxis were identified by phenotypic complementation of generally non-chemotactic mutants. Sequencing of a DNA fragment, which complemented two different mutants, revealed a region of five open reading frames translated in one direction and encoding homologs of known genes comprising excitation and adaptation pathways for chemotaxis in other bacterial species. The major chemotaxis gene cluster appears to be essential for all known behavioral responses that direct swimming motility in A. brasilense. Phylogenetic and genomic analysis revealed three groups of chemotaxis operons in alpha-proteobacterial species and assigned the A. brasilense operon to one of them. Interestingly, operons that are shown to be major regulators of behavior in several alpha-proteobacterial species are not orthologous.
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6. |
Steenhoudt O,
Keijers V,
Okon Y,
Vanderleyden J,
( 2001 ) Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245. PMID : 11409544 : Abstract >>
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.
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7. |
Wu Y,
Wang J,
Ma L,
( 1999 ) Site-directed mutagenesis analysis of draTG genes and their downstream region from Azospirillum brasilense Yu62. PMID : 10935160 : Abstract >>
draT and draG genes are involved in posttranslational regulation of nitrogenase activity of Azospirillum brasilense Yu62. Both genes and their downstream region were mutagenized by Kmr cassette insertions. Analysis of mutations introduced into the draTG region on the A. brasilense Yu62 chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Analysis of mutations introduced into the draTG downstream region (the mutagenized site is about 2 kb downstream from draG) showed that the mutant had higher nitrogenase activity than the wild strain while growing in nitrogen-free medium and medium with 2 mmol/L ammonium. These results reveal that there is no gene required for nitrogen fixation in this mutagenized region, but it is possible that there are genes which play a role in regulating nitrogen fixation. The results of monitoring the expression of transcriptional nifH-lacZ gene fusion in the mutant YZ4 showed that the transcriptional regulation of nif gene in the mutant YZ4 was the same as that in the wild type.
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8. |
Dosselaere F,
Lambrecht M,
Vanderleyden J,
( 2000 ) Isolation and sequence analysis of the trpBA gene cluster, encoding tryptophan synthase, from Azospirillum brasilense. PMID : 11092742 : Abstract >>
The trpBA gene cluster of Azospirillum brasilense Sp7 was isolated by complementation of an Escherichia coli trpBA mutant. Both genes code for the two subunits of tryptophan synthase, which catalyzes the last step in tryptophan biosynthesis. No structural features indicating transcriptional regulation could be identified. Upstream of the trpBA cluster an open reading frame encoding a putative periplasmic binding protein, involved in amino acid transport, was identified. Analysis of the downstream region of the trpBA cluster revealed the presence of a putative open reading frame encoding a subunit of the acetyl-coenzyme A carboxylase carboxyl transferase complex.
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9. |
Burdman S,
De Mot R,
Vanderleyden J,
Okon Y,
Jurkevitch E,
( 2000 ) Identification and characterization of the omaA gene encoding the major outer membrane protein of Azospirillum brasilense. PMID : 11092733 : Abstract >>
The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligonucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilense in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.
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10. |
Valzasina B,
Colombo C,
Morandi P,
( 2000 ) Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis. PMID : 10651638 : DOI : 10.1021/bi9920329 Abstract >>
To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.
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11. |
Marchal K,
Frazzon J,
Passaglia LM,
Revers LF,
( 2000 ) Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression. PMID : 10650197 : DOI : 10.1111/j.1574-6968.2000.tb08928.x Abstract >>
Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype. Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected. ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.
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12. |
Rigo LU,
Funayama S,
Souza EM,
Fadel-Picheth CM,
( 1999 ) Regulation of Azospirillum brasilense nifA gene expression by ammonium and oxygen. PMID : 10518727 : DOI : 10.1111/j.1574-6968.1999.tb08739.x Abstract >>
The structure and activity of the nifA promoter of Azospirillum brasilense was studied using deletion analysis. An essential region for nifA promoter activity was identified between nucleotides -67 and -47 from the identified transcription start site. A sequence resembling a sigma(70) recognition site occurs in this region and may constitute the nifA gene promoter. The regulation of the nifA gene was studied in plasmid and chromosomal nifA::lacZ fusions. Full expression was obtained under low oxygen levels and in the absence of ammonium ions. Repression of nifA expression involves a synergistic effect between oxygen and ammonium.
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13. |
Curti B,
( 1999 ) Glutamate synthase: a complex iron-sulfur flavoprotein. PMID : 10357231 : DOI : 10.1007/s000180050319 Abstract >>
Glutamate synthase is a complex iron-sulfur flavoprotein that forms L-glutamate from L-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain.
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14. |
Proost P,
Keijers V,
Van Dommelen A,
Lambrecht M,
Vermeiren H,
( 1999 ) Characterization of a sugar-binding protein from Azospirillum brasilense mediating chemotaxis to and uptake of sugars. PMID : 10361275 : DOI : 10.1046/j.1365-2958.1999.01384.x Abstract >>
Our approach to the isolation of plant-inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE-like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D-galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D-galactose, L-arabinose and D-fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.
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15. |
Liang YY,
Kaminski PA,
Elmerich C,
( 1991 ) Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia. PMID : 1779763 : DOI : 10.1111/j.1365-2958.1991.tb01982.x Abstract >>
A gene bank of Azospirillum lipoferum Br17 constructed in the vector lambda GEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA-like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA-homology region were found. Mutants with Nif- phenotype were obtained by site-directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA-like gene. Construction of a nifA-lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA-like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.
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16. |
Nagarajan T,
Vanderleyden J,
Tripathi AK,
( 2007 ) Identification of salt stress inducible genes that control cell envelope related functions in Azospirillum brasilense Sp7. PMID : 17340145 : DOI : 10.1007/s00438-007-0224-2 Abstract >>
Plant growth promoting rhizobacteria such as Azospirillum brasilense are agronomically important as they are frequently used for crop inoculation. But adverse factors such as increasing soil salinity limit their survival, multiplication and phytostimulatory effect. In order to understand the role of the genes involved in the adaptation of A. brasilense Sp7 to salt stress, a mutant library (6,800 mutants) was constructed after random integration of a mini-Transposon Tn5 derivative containing a promoterless gusA and oriV. The library was screened for salt stress inducible Gus activity on minimal malate agar medium containing NaCl and 5-bromo-4-chloro-3-indolyl-beta-D: -glucuronide. Salt stress responsiveness of the promoters was estimated by quantifying GusA activity in the presence and absence of NaCl stress using p-nitrophenyl-beta-D: -glucuronide as a substrate. In 11 mutants showing high levels of gusA expression in the presence of salt-stress, the partial nucleotide sequence of the DNA region flanking the site of Tn5 insertion was determined and analysed using the NCBI-BLAST programs. Similarity searches revealed that 10 out of the 11 genes sequenced showed notable similarity with genes involved in functions related to modulation in the composition of exopolysaccharides, capsular polysaccharides, lipopolysaccharides, peptidoglycan and lipid bilayer of the cell envelope. Induction of cell envelope related genes in response to salt stress and salt sensitive phenotype of several mutants in A. brasilense indicate a prominent role of cell envelope in salt-stress adaptation.
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17. |
Mehnaz S,
Weselowski B,
Lazarovits G,
( 2007 ) Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere. PMID : 17329796 : DOI : 10.1099/ijs.0.64804-0 Abstract >>
A free-living diazotrophic strain, DS2(T), was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2(T) was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1omega7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2(T) (=NCCB 100108(T)=LMG 23617(T)).
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18. |
Watanabe S,
Yamada M,
Ohtsu I,
Makino K,
( 2007 ) alpha-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate, and hydroxy-L-proline. Molecular and metabolic convergent evolution. PMID : 17202142 : DOI : 10.1074/jbc.M611057200 Abstract >>
Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism in which alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of alphaKGSA to alpha-ketoglutarate. In the preceding studies, we identified a set of metabolic genes of the l-arabinose pathway including the KGSADH gene (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623; Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888; Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 33521-33536). Here, we describe that A. brasilense possesses two different KGSADH isozymes from l-arabinose-related enzyme (KGSADH-I); that is, d-glucarate/d-galactarate-inducible KGSADH-II and hydroxy-l-proline-inducible KGSADH-III. They were purified homogeneously from A. brasilense cells grown on d-galactarate or hydroxy-l-proline, respectively. When compared with KGSADH-I, amino acid sequences of KGSADH-II and KGSADH-III were significantly similar but not totally identical. Physiological characterization using recombinant enzymes revealed that KGSADH-II and KGSADH-III showed similar high substrate specificity for alphaKGSA and different coenzyme specificity; that is, NAD(+)-dependent KGSADH-II and NADP(+)-dependent KGSADH-III. In the phylogenetic tree of the aldehyde dehydrogenase (ALDH) superfamily, KGSADH-II and KGSADH-III were poorly related to the known ALDH subclasses including KGSADH-I. On the other hand, ALDH-like ycbD protein involved in d-glucarate/d-galactarate operon from Bacillus subtilis is closely related to the methylmalonyl semialdehyde dehydrogenase subclass but not A. brasilense KGSADH isozymes. To estimate the correct function, the corresponding gene was expressed, purified, and characterized. Kinetic analysis revealed the physiological role as NADP(+)-dependent KGSADH. We conclude that three different types of KGSADH appeared in the bacterial evolutional stage convergently. Furthermore, even the same pathway such as l-arabinose and d-glucarate/d-galactarate metabolism also evolved by the independent involvement of KGSADH.
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19. |
de Zamaroczy M,
Delorme F,
Elmerich C,
( 1990 ) Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense. PMID : 1702507 : DOI : 10.1007/bf00262437 Abstract >>
The complete nucleotide sequence of the open reading frame (ORF) located upstream of the glnA structural gene for glutamine synthetase (GS) in Azospirillum brasilense Sp7 was determined. This ORF, which codes for a 12 kDa protein, was identified as glnB, the structural gene for the PII protein, a component of the adenylylation cascade involved in the regulation of GS activity in some gram-negative bacteria. Transcription analysis and mRNA mapping of glnB and glnA of A. brasilense was performed with bacteria grown under different physiological conditions. The glnA gene can be transcribed either as a glnB-A mRNA of 2.4 kb or as a glnA mRNA of 1.5 kb. Differential expression of the two mRNAs was found to depend on the nitrogen source. The glnB-A mRNA was the major transcript under nitrogen fixation conditions, while the synthesis of the glnA mRNA was almost completely abolished. The glnA mRNA was predominantly produced in NH4(+)-containing medium. Transcription start site analysis revealed the presence of three different types of nitrogen-regulated promoters. GlnB-A mRNA was transcribed selectively from tandem promoters. One of them is similar to the NtrA-dependent promoter and the other to the Escherichia coli sigma 70 promoter. The synthesis of glnA mRNA was regulated by a promoter, which was repressed (or non-activated) only under conditions of nitrogen fixation, when moleuclar nitrogen was the sole nitrogen source. The transcriptional initiation site in front of glnA is not preceded by a canonical E. coli sigma 70 promoter. A sequence reminiscent of the NtrA-dependent promoter consensus, except for a fundamental mismatch, was found at positions -33 to -21. This sequence overlapped a putative "weak" NtrC-binding site, similar to those identified in enteric bacteria. From these results, it is postulated that glnA mRNA is controlled by a novel type of nitrogen-regulated promoter.
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20. |
Watanabe S,
Shimada N,
Tajima K,
Kodaki T,
Makino K,
( 2006 ) Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism. PMID : 16950779 : DOI : 10.1074/jbc.M606727200 Abstract >>
Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and alpha-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S](2+) cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.
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21. |
Rodriguez H,
Mendoza A,
Cruz MA,
Holguin G,
Glick BR,
Bashan Y,
( 2006 ) Pleiotropic physiological effects in the plant growth-promoting bacterium Azospirillum brasilense following chromosomal labeling in the clpX gene. PMID : 16867140 : DOI : 10.1111/j.1574-6941.2006.00111.x Abstract >>
Azospirillum brasilense 8-I was chromosomally labeled with green fluorescent protein (gfp) genes, using either the native promoterless gfp gene or the mutant gfpmut2 gene under the transcriptional control of the neomycin phosphate transferase (npt2) promoter inserted into Tn5 suicide plasmid vectors. One A. brasilense exconjugant, showing a steady and strong fluorescence following irradiation with 365-nm UV light was characterized in detail. This strain, A. brasilense 8-I-gfp showed increased N(2)-fixation of approximately threefold, up to a twofold increase in exopolysaccharide production, and a significant decrease in indole-3-acetic acid and poly-beta-hydroxybutyrate production over the parental strain. Sequence analysis showed that the Tn5 carrying the gfp gene was inserted in the clpX gene encoding a heat-shock protein. This data is consistent with a model in which the observed physiological changes are a consequence of pleiotropic changes that occur as a consequence of impaired heat shock (stress) protein synthesis. In summary, (i) chromosomally labelled Azospirillum brasilense was obtained carrying either native or mutant gfp genes, (ii) Pleiotropic physiological effects were caused by disruption of the clpX gene as the consequence of the insertion, (iii) a new indole-3-acetic acid-attenuated mutant of A. brasilense producing only 0.25% of the indole-3-acetic acid produced by the wild-type is presented.
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22. |
Watanabe S,
Kodaki T,
Makino K,
( 2006 ) A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism. PMID : 16835232 : DOI : 10.1074/jbc.M602585200 Abstract >>
Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family.
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23. |
Cui Y,
Tu R,
Guan Y,
Ma L,
Chen S,
( 2006 ) Cloning, sequencing, and characterization of the Azospirillum brasilense fhuE gene. PMID : 16502288 : DOI : 10.1007/s00284-005-0008-z Abstract >>
The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.
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24. |
Xie B,
Xu K,
Zhao HX,
Chen SF,
( 2005 ) Isolation of transposon mutants from Azospirillum brasilense Yu62 and characterization of genes involved in indole-3-acetic acid biosynthesis. PMID : 15961260 : DOI : 10.1016/j.femsle.2005.05.020 Abstract >>
The molecular genetics of indole-3-acetic (IAA) synthesis and regulation in Azospirillum brasilense was investigated in this study. Tn5 mutagenesis was performed and five mutants with decreased IAA production were isolated. Five Tn5-inserted genes from these mutants were cloned and sequenced. Four genes were reported for the first time to be involved in IAA production, namely, atrA, ftsA, omaA and aldA that code for GntR-family transcriptional regulator, iron-binding protein component of ABC-type Fe(3+) transport system, outer membrane protein, and aldehyde dehydrogenase, respectively. In addition, two genes atrB and atrC, with predicted proteins that showed high homology to aminotransferases, were cloned from the downstream of atrA in this bacterium. Studies also showed that complementation of atrA, ftsA and omaA were able to restore the IAA production of the corresponding IAA(-) mutants. Comparison of Fe(3+) concentrations in culture supernatants of the wild-type strain, the ftsA mutant and the complemented strain revealed that the iron-uptake ability of the ftsA mutant was highly reduced. This result also points to the necessity of iron as a metal ion in IAA synthesis. Statistical analysis showed no significant difference in the IAA accumulated in cells between the omaA mutant and the wild-type strain, suggesting the omaA might not affect IAA secretion but be involved in IAA production in other unknown ways.
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25. |
Ge SM,
Xie BE,
Chen SF,
( 2006 ) Characterization of two trpE genes encoding anthranilate synthase alpha-subunit in Azospirillum brasilense. PMID : 16430864 : DOI : 10.1016/j.bbrc.2006.01.009 Abstract >>
The previous report from our laboratory has recently identified a new trpE gene (termed trpE2) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE1(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is the first report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE1(G) while these sequence features did not exist in front of trpE2. The beta-galactosidase activity of an A. brasilense strain carrying a trpE2-lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE1(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE1(G) is regulated at the transcriptional level by attenuation while trpE2 is constantly expressed. The anthranilate synthase assays with trpE1(G)- and trpE2- mutants demonstrated that TrpE1(G) fusion protein is feedback inhibited by tryptophan while TrpE2 protein is not. We also found that both trpE1(G) and trpE2 gene products were involved in IAA synthesis.
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26. |
Alahari A,
Tripathi AK,
Le Rudulier D,
( 2006 ) Cloning and characterization of a fur homologue from Azospirillum brasilense Sp7. PMID : 16450071 : DOI : 10.1007/s00284-005-0204-x Abstract >>
A homologue of the ferric uptake regulator gene, fur, was identified from a Azospirillum brasilense Sp7 genomic DNA clone. Experiments performed with transcriptional lacZ fusions demonstrated that the A. brasilense fur homologue regulated the expression of two fur regulated Escherichia coli genes: fiu (ferric iron uptake) and fhuF (ferric hydroxamate uptake). A differential regulation by the cognate Fur and the heterologous Fur homologue in response to the iron status of the growth medium was also observed.
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27. |
Watanabe S,
Kodaki T,
Kodak T,
Makino K,
( 2006 ) Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism. PMID : 16326697 : DOI : 10.1074/jbc.M506477200 Abstract >>
Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.
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28. |
Rothballer M,
Schmid M,
Fekete A,
Hartmann A,
( 2005 ) Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245. PMID : 16232298 : DOI : 10.1111/j.1462-2920.2005.00848.x Abstract >>
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.
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29. |
Zhang Y,
Burris RH,
Roberts GP,
( 1992 ) Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense. PMID : 1577701 : DOI : 10.1128/jb.174.10.3364-3369.1992 PMC : PMC206006 Abstract >>
The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.
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30. |
Vieille C,
Elmerich C,
( 1992 ) Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutrophus phbB and to Rhizobium meliloti nodG. PMID : 1538694 : DOI : 10.1007/bf00292706 Abstract >>
A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. meliloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the phbB gene and involved in poly-beta-hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli sigma 70 consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.
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31. |
Huergo LF,
Assumpção MC,
Souza EM,
Steffens MB,
Yates MG,
Chubatsu LS,
Pedrosa FO,
( 2004 ) Repressor mutant forms of the Azospirillum brasilense NtrC protein. PMID : 15466584 : DOI : 10.1128/AEM.70.10.6320-6323.2004 PMC : PMC522079 Abstract >>
The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.
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32. |
Sperotto RA,
Gross J,
Vedoy C,
Passaglia LM,
Schrank IS,
( 2004 ) The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation. PMID : 15386115 : DOI : 10.1007/s00284-004-4318-3 Abstract >>
The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.
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33. |
Greer-Phillips SE,
Stephens BB,
Alexandre G,
( 2004 ) An energy taxis transducer promotes root colonization by Azospirillum brasilense. PMID : 15375141 : DOI : 10.1128/JB.186.19.6595-6604.2004 PMC : PMC516605 Abstract >>
Motility responses triggered by changes in the electron transport system are collectively known as energy taxis. In Azospirillum brasilense, energy taxis was shown to be the principal form of locomotor control. In the present study, we have identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. The Tlp1 protein is predicted to have an N-terminal periplasmic region and a cytoplasmic C-terminal signaling module homologous to those of other chemoreceptors. The predicted periplasmic region of Tlp1 comprises a conserved domain that is found in two types of microbial sensory receptors: chemotaxis transducers and histidine kinases. However, the function of this domain is currently unknown. We characterized the behavior of a tlp1 mutant by a series of spatial and temporal gradient assays. The tlp1 mutant is deficient in (i) chemotaxis to several rapidly oxidizable substrates, (ii) taxis to terminal electron acceptors (oxygen and nitrate), and (iii) redox taxis. Taken together, the data strongly suggest that Tlp1 mediates energy taxis in A. brasilense. Using qualitative and quantitative assays, we have also demonstrated that the tlp1 mutant is impaired in colonization of plant roots. This finding supports the hypothesis that energy taxis and therefore bacterial metabolism might be key factors in determining host specificity in Azospirillum-grass associations.
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34. |
Biondi EG,
Marini F,
Altieri F,
Bonzi L,
Bazzicalupo M,
del Gallo M,
( 2004 ) Extended phenotype of an mreB-like mutant in Azospirillum brasilense. PMID : 15256588 : DOI : 10.1099/mic.0.26904-0 Abstract >>
Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant. Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation.
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35. |
Cohen MF,
Han XY,
Mazzola M,
( 2004 ) Molecular and physiological comparison of Azospirillum spp. isolated from Rhizoctonia solani mycelia, wheat rhizosphere, and human skin wounds. PMID : 15213753 : DOI : 10.1139/w04-007 Abstract >>
Four phenotypically similar bacterial strains isolated from fungal, plant, and human sources were identified as Azospirillum species. Strains RC1 and LOD4 were isolated from the mycelium of the apple root pathogen Rhizoctonia solani AG 5 and from the rhizosphere of wheat grown in apple orchard soil, respectively. Strains C610 and F4626 isolated from human wounds were previously misclassified as Roseomonas genomospecies 3 and 6. All four strains demonstrated close similarities in 16S rRNA gene sequences, having > or =97% identity to Azospirillum brasilense type strain ATCC 29145 and <90% identity to Roseomonas gilardii, the Roseomonas type strain. Extensive phenotypic similarities among the four strains included the ability of free-living cells to fix N2. Cells of strains RC1, LOD4, and C610 but not of strain F4626 could be induced to flocculate by incubation with 10 mmol.L-1 glycerol or fructose in medium containing 0.5 mmol.L-1 NO3-. Our results indicate a wide range of potential sources for Azospirillum spp. with the isolation of Azospirillum spp. from human wounds warranting further investigation.
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36. |
Vanbleu E,
Marchal K,
Lambrecht M,
Mathys J,
Vanderleyden J,
( 2004 ) Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition. PMID : 15033235 : DOI : 10.1016/S0378-1097(04)00046-1 Abstract >>
The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.
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37. |
Jofré E,
Lagares A,
Mori G,
( 2004 ) Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense. PMID : 14987774 : DOI : 10.1016/S0378-1097(04)00003-5 Abstract >>
The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant-bacteria associations (Rhizobium-legume, Pseudomonas-potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.
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38. |
Séraphin B,
( 1992 ) The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals. PMID : 1472710 : Abstract >>
By comparing the sequence of a putative translation product from a Saccharomyces cerevisiae split gene with several data-bases, I have uncovered a new protein family. Members of this family are found in prokaryotes as well as in lower and higher eukaryotes. The function of these proteins is unknown but they share a characteristic histidine triad that may be involved in zinc binding. This group of protein has been named the HIT protein family.
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39. |
Vieille C,
Elmerich C,
( N/A ) Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ. PMID : 2131098 : Abstract >>
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.
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40. |
Wu L,
Cui Y,
Hong Y,
Chen S,
( 2011 ) A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7. PMID : 21232929 : DOI : 10.1016/j.micres.2010.12.001 Abstract >>
We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense.
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41. |
Mehnaz S,
Baig DN,
Lazarovits G,
( 2010 ) Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan. PMID : 21193815 : Abstract >>
Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.
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42. |
Truan D,
Huergo LF,
Chubatsu LS,
Merrick M,
Li XD,
Winkler FK,
( 2010 ) A new P(II) protein structure identifies the 2-oxoglutarate binding site. PMID : 20493877 : DOI : 10.1016/j.jmb.2010.05.036 Abstract >>
P(II) proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P(II) proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within P(II) proteins has been controversial. We have now solved the X-ray structure, at 1.4 A resolution, of the Azospirillum brasilense P(II) protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of P(II) proteins and shows that 2-oxoglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg(2+) together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that P(II) protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by P(II) proteins.
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43. |
Shukla AK,
Vishwakarma P,
Singh RS,
Upadhyay SN,
Dubey SK,
( 2010 ) Bio-filtration of trichloroethylene using diazotrophic bacterial community. PMID : 19962302 : DOI : 10.1016/j.biortech.2009.10.094 Abstract >>
Biodegradation of TCE was studied in a biofilter packed with wood charcoal and inoculated with diazotrophic bacterial community isolated from local soil. Steady state TCE removal efficiencies higher than 85% were observed up to inlet load of 2.866 g m(-3) h(-1). The maximum elimination capacity of 5.31 g m(-3) h(-1) was observed at an inlet load of more than 7.90 g m(-3) h(-1). The biofilter was sensitive to fluctuations in the process conditions but could easily recover its performance after 10 days shutdown. Almost constant and small pressure drop per unit length and very negligible compaction was observed during the whole experimental period. The molecular analyses such as RT-PCR and gene sequencing revealed the presence of functionally active Azospirillum species in the biofilm.
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44. |
Lerner A,
Castro-Sowinski S,
Lerner H,
Okon Y,
Burdman S,
( 2009 ) Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7. PMID : 19765087 : DOI : 10.1111/j.1574-6968.2009.01773.x Abstract >>
Here we report the identification of a glycogen phosphorylase (glgP) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense, Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense, and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.
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45. |
Li XD,
Huergo LF,
Gasperina A,
Pedrosa FO,
Merrick M,
Winkler FK,
( 2009 ) Crystal structure of dinitrogenase reductase-activating glycohydrolase (DraG) reveals conservation in the ADP-ribosylhydrolase fold and specific features in the ADP-ribose-binding pocket. PMID : 19477184 : DOI : 10.1016/j.jmb.2009.05.031 Abstract >>
Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 A. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-alpha-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.
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46. |
Wasim M,
Bible AN,
Xie Z,
Alexandre G,
( 2009 ) Alkyl hydroperoxide reductase has a role in oxidative stress resistance and in modulating changes in cell-surface properties in Azospirillum brasilense Sp245. PMID : 19332821 : DOI : 10.1099/mic.0.022541-0 Abstract >>
An ahpC mutant derivative of Azospirillum brasilense Sp245 (strain SK586) that encodes an alkyl hydroperoxide reductase was found to be more sensitive to oxidative stress caused by organic hydroperoxides compared with the wild-type. In addition, the ahpC mutant strain had multiple defects in a large array of cellular functions that were consistent with alteration of cell-surface properties, such as cell morphology in stationary phase, Calcofluor White-, Congo Red- and lectin-binding abilities, as well as cell-to-cell aggregation and flocculation. All phenotypes of the ahpC mutant were complemented by in trans expression of AhpC, and overexpression of AhpC in the wild-type strain was found to affect the same set of phenotypes, suggesting that the pleiotropic effects were caused by the ahpC mutation. SK586 was also found to be fully motile, but it lost motility at a higher rate than the wild-type during growth, such that most SK586 cells were non-motile in stationary phase. Despite these defects, the mutant did not differ from the wild-type in short-term colonization of sterile wheat roots when inoculated alone, and in competition with the wild-type strain; this implied that AhpC activity may not endow the cells with a competitive advantage in colonization under these conditions. Although the exact function of AhpC in affecting these phenotypes remains to be determined, changes in cell morphology, surface properties, cell-to-cell aggregation and flocculation are common adaptive responses to various stresses in bacteria, and the data obtained here suggest that AhpC contributes to modulating such stress responses in A. brasilense.
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47. |
Katsy EI,
Prilipov AG,
( 2009 ) Mobile elements of an Azospirillum brasilense Sp245 85-MDa plasmid involved in replicon fusions. PMID : 19249329 : DOI : 10.1016/j.plasmid.2009.02.003 Abstract >>
Sequence analysis of approximately 25kb of an Azospirillum brasilense Sp245 85-MDa (approximately 142kb) plasmid, p85, identified two novel IS elements mediating p85 fusions with a suicide plasmid vector, pJFF350. These IS elements, 1465-bp ISAzba1 and 1112-bp ISAzba3, belong to the IS256 family and to the IS5 family/IS903 group, respectively. Truncated ISAzba2 from the ISL3 family was found near one of the copies of ISAzba1 that flank pJFF350 in p85::pJFF350. As another factor potentially contributing to the known genetic plasticity of p85, a phage integrase gene was identified in this plasmid.
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48. |
Jofré E,
Fischer S,
Príncipe A,
Castro M,
Ferrari W,
Lagares A,
Mori G,
( 2009 ) Mutation in a D-alanine-D-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress. PMID : 19025567 : DOI : 10.1111/j.1574-6968.2008.01421.x Abstract >>
Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.
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49. |
Pothier JF,
Prigent-Combaret C,
Haurat J,
Moënne-Loccoz Y,
Wisniewski-Dyé F,
( 2008 ) Duplication of plasmid-borne nitrite reductase gene nirK in the wheat-associated plant growth-promoting rhizobacterium Azospirillum brasilense Sp245. PMID : 18624646 : DOI : 10.1094/MPMI-21-6-0831 Abstract >>
In the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245, nitric oxide produced by denitrification could be a signal involved in stimulation of root branching, and the dissimilatory nitrite reductase gene nirK is upregulated on wheat roots. Here, it was found that Sp245 did not contain one copy of nirK but two (named nirK1 and nirK2), localized on two different plasmids, including one plasmid prone to rearrangements. Their deduced protein sequences displayed 99.2% identity but their promoter regions and upstream genetic environment differed. Phylogenetic studies revealed that nirK1 and nirK2 clustered next to most beta-proteobacterial sequences rather than in the vicinity of other Azospirillum spp. and most alpha-proteobacterial sequences, regardless of whether DNA or deduced protein sequences were used. This points to past horizontal gene transfers. Analysis of the number of nonsynonymous and synonymous substitutions per site indicated that nirK has been subjected to neutral selection in bacteria. The use of transcriptional fusions with egfp, encoding an enhanced green fluorescent protein variant, revealed that both nirK1 and nirK2 promoter regions were upregulated in vitro under microaerobiosis or the presence of nitrite as well as on wheat roots. The analysis of nirK1 and nirK2 mutants revealed that the two genes were functional. Overall, results suggest that nirK has been acquired horizontally by A. brasilense Sp245 from a distant relative and underwent subsequent duplication; however, both paralogs remained functional and retained their upregulation by the plant partner.
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50. |
Thirunavukkarasu N,
Mishra MN,
Spaepen S,
Vanderleyden J,
Gross CA,
Tripathi AK,
( 2008 ) An extra-cytoplasmic function sigma factor and anti-sigma factor control carotenoid biosynthesis in Azospirillum brasilense. PMID : 18599837 : DOI : 10.1099/mic.0.2008/016428-0 Abstract >>
Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white-cream colour on plates, colonies of strain Cd are orange-pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange-pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti-sigma factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class sigma factor (sigma(E), RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd sigma(E) was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-sigma(E) ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-sigma(E). Cloning of Sp7 anti-sigma(E) in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-sigma(E) knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 sigma(E) in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by sigma(E) with its cognate anti-sigma(E).
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51. |
Zimmer W,
Aparicio C,
Elmerich C,
( 1991 ) Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum: identification and sequencing of a trpGDC cluster. PMID : 1896020 : DOI : 10.1007/bf00264211 Abstract >>
Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb SalI-SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI-SalI fragment including the 2 kb SalI-SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.
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52. |
Khammar N,
Martin G,
Ferro K,
Job D,
Aragno M,
Verrecchia E,
( 2009 ) Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil. PMID : 18930770 : DOI : 10.1016/j.mimet.2008.09.020 Abstract >>
Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.
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53. |
Araújo LM,
Huergo LF,
Invitti AL,
Gimenes CI,
Bonatto AC,
Monteiro RA,
Souza EM,
Pedrosa FO,
Chubatsu LS,
( 2008 ) Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein. PMID : 18392451 : DOI : 10.1590/s0100-879x2008000400006 Abstract >>
Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 microM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.
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54. |
Passaglia LM,
Nunes CP,
Zaha A,
Schrank IS,
( 1991 ) The nifHDK operon in the free-living nitrogen-fixing bacteria Azospirillum brasilense sequentially comprises genes H, D, K, an 353 bp orf and gene Y. PMID : 1823284 : Abstract >>
1. The complete nucleotide sequence of the nitrogenase structural genes from Azospirillum brasilense was determined. Two additional open reading frames of 353 and 683 base pairs were detected downstream of the nifK gene, one of which shows homology to the nifY gene. 2. Structures resembling the consensus nif promoter and NifA-binding motif were found only upstream from the nifH region and an inverted repeat structure located downstream of the nifY gene may be a potential stem-and-loop transcriptional terminator. 3. The nif structural genes of Azospirillum brasilense are transcribed as a single transcription unit and organized as nifHDKorf1Y. NifH, NifD and NifK polypeptides share significant sequence identities when compared to nif structural gene products from other organisms. 4. The three polypeptides are characterized by the presence of highly conserved cysteine residues which may play a role in binding the iron-sulfur cluster.
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55. |
Malhotra M,
Srivastava S,
( 2008 ) An ipdC gene knock-out of Azospirillum brasilense strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion. PMID : 17952626 : DOI : 10.1007/s10482-007-9207-x Abstract >>
The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (~50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by approximately 50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.
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56. |
Chang Y,
Tang T,
Li JL,
( N/A ) Isolation of a flagellar operon in Azospirillum brasilense and functional analysis of FlbD. PMID : 17572072 : DOI : 10.1016/j.resmic.2007.04.005 Abstract >>
A 10 kb fragment containing fliF, fliH, fliN, motA, flbD, flhA, flhF and fleN genes was cloned from the genomic DNA of Azospirillum brasilense Yu62. These eight genes appear to be structurally organized as an operon. FlbD, encoded by flbD, has a HTH DNA binding domain and shows homology to sigma(54)-dependent transcriptional activators such as NtrC, NifA and DctD. An in-frame deletion of flbD in A. brasilense abolishes biosynthesis of lateral flagella and swarming ability when grown on semi-solid surfaces. An intact copy of flbD on a plasmid complemented the DeltaflbD mutant by restoring lateral flagellation and swarming ability. Transcriptional analysis demonstrated that FlbD is involved in the genetic regulation of flagella biosynthesis and acts as both an activator and a repressor of flagellum gene expression in A. brasilense. DNA binding assays indicated direct interaction between FlbD and the promoter regions of laf1, fliF and flgB genes. We propose that A. brasilense has a genetic regulation profile for flagella biosynthesis similar to that observed in Caulobacter crescentus.
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57. |
Pothier JF,
Wisniewski-Dyé F,
Weiss-Gayet M,
Moënne-Loccoz Y,
Prigent-Combaret C,
( 2007 ) Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245. PMID : 17906157 : DOI : 10.1099/mic.0.2007/009381-0 Abstract >>
Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1-3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.
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58. |
Shimada N,
Mikami B,
Watanabe S,
Makino K,
( 2007 ) Preliminary crystallographic analysis of L-2-keto-3-deoxyarabonate dehydratase, an enzyme involved in an alternative bacterial pathway of L-arabinose metabolism. PMID : 17565178 : DOI : 10.1107/S1744309107015102 PMC : PMC2334997 Abstract >>
L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde. L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.
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59. |
Maroniche GA,
García JE,
Salcedo F,
Creus CM,
( 2017 ) Molecular identification of Azospirillum spp.: Limitations of 16S rRNA and qualities of rpoD as genetic markers. PMID : 28024520 : DOI : 10.1016/j.micres.2016.11.009 Abstract >>
Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp.
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60. |
Jijón-Moreno S,
Marcos-Jiménez C,
Pedraza RO,
Ramírez-Mata A,
de Salamone IG,
Fernández-Scavino A,
Vásquez-Hernández CA,
Soto-Urzúa L,
Baca BE,
( 2015 ) The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense. PMID : 25842039 : DOI : 10.1007/s10482-015-0444-0 Abstract >>
Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2 �gg mg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains.
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61. |
Fani R,
Bazzicalupo M,
Damiani G,
Bianchi A,
Schipani C,
Sgaramella V,
Polsinelli M,
( 1989 ) Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene. PMID : 2664449 : DOI : 10.1007/bf00334360 Abstract >>
A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxy-terminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.
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62. |
Ramírez-Mata A,
López-Lara LI,
Xiqui-Vázquez ML,
Jijón-Moreno S,
Romero-Osorio A,
Baca BE,
( 2016 ) The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense. PMID : 26708984 : DOI : 10.1016/j.resmic.2015.12.004 Abstract >>
In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.
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63. |
de Zamaroczy M,
Delorme F,
Elmerich C,
( 1989 ) Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7. PMID : 2608030 : DOI : 10.1007/bf00260861 Abstract >>
Transcription of the structural genes for nitrogenase (nifHDK) in Azospirillum brasilense Sp7 was analysed using Northern blots of total RNA extracted from cultures grown under nitrogen-fixing conditions. Hybridization with an internal nifH probe revealed two transcripts, a major one (by concentration) of 1.1 kb corresponding to nifH and a minor one of 5.6 kb corresponding to nifHDK. Hybridization with nifD or nifK probes revealed the minor transcript of 5.6 kb. This confirms that the nifHDK genes are organized as a single transcription unit and suggests regulation at the level of termination of transcription. The complete nucleotide sequence of nifH was established and the DNA region upstream of the initiation codon was analysed for transcription and translation signals. The nifH open reading frame (ORF) is preceded by an NtrA-dependent promoter and two elements homologous to upstream activator sequences (UAS) required for NifA-mediated activation in other diazotrophs. Promoter mapping with S1 nuclease revealed two start sites located 10 bp and 40 bp downstream of the NtrA-dependent promoter.
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64. |
Fani R,
Allotta G,
Bazzicalupo M,
Ricci F,
Schipani C,
Polsinelli M,
( 1989 ) Nucleotide sequence of the gene encoding the nitrogenase iron protein (nifH) of Azospirillum brasilense and identification of a region controlling nifH transcription. PMID : 2608029 : DOI : 10.1007/bf00260860 Abstract >>
The DNA sequence was determined for the Azospirillum brasilense nifH gene and part of the nifD gene. The nifH gene is 885 bp long and encodes 293 amino acid residues. The region upstream of the nifH open reading frame contains a putative promoter whose sequence shows perfect homology with promoters of other diazotrophic bacteria and two putative upstream activator sequences. Experiments with the promoter-probe vector pAF300 showed that this region promotes transcription in response to the nitrogen and oxygen availability of the cell. The amino acid sequence was deduced from the DNA nucleotide sequence of nifH; the polypeptide contains the four cysteine residues highly conserved among other nifH products and an arginine residue at position 101 which could be the site of the modification occurring during the "switch-off" of nitrogenase. The codon usage appears to be very biased reflecting the high G + C content of the Azospirillum nifH gene. In a comparison of the amino acid sequence with the other 18 known nifH gene products, the A. brasilense nifH product showed the highest level of homology with fast-growing Rhizobia suggesting interesting evolutionary implications.
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65. |
Watanabe S,
Tanimoto Y,
Yamauchi S,
Tozawa Y,
Sawayama S,
Watanabe Y,
( 2014 ) Identification and characterization of trans-3-hydroxy-l-proline dehydratase and �G(1)-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria. PMID : 24649405 : DOI : 10.1016/j.fob.2014.02.010 PMC : PMC3958920 Abstract >>
trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to �\-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent �G(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp �� Pyr2C �� l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/�g-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and �G(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.
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66. |
Rivera D,
Revale S,
Molina R,
Gualpa J,
Puente M,
Maroniche G,
Paris G,
Baker D,
Clavijo B,
McLay K,
Spaepen S,
Perticari A,
Vazquez M,
Wisniewski-Dyé F,
Watkins C,
Martínez-Abarca F,
Vanderleyden J,
Cassán F,
( 2014 ) Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture. PMID : 25059863 : DOI : 10.1128/genomeA.00683-14 PMC : PMC4110221 Abstract >>
We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.
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67. |
Truan D,
Bjeli? S,
Li XD,
Winkler FK,
( 2014 ) Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense. PMID : 24846646 : DOI : 10.1016/j.jmb.2014.05.008 Abstract >>
The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.
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68. |
Caballero-Mellado J,
Moënne-Loccoz Y,
Comte G,
Prigent-Combaret C,
Maurhofer M,
Défago G,
von Felten A,
( 2013 ) Comparison of prominent Azospirillum strains in Azospirillum-Pseudomonas-Glomus consortia for promotion of maize growth. PMID : 22805783 : DOI : 10.1007/s00253-012-4249-z Abstract >>
Azospirillum are prominent plant growth-promoting rhizobacteria (PGPR) extensively used as phytostimulatory crop inoculants, but only few studies are dealing with Azospirillum-containing mixed inocula involving more than two microorganisms. We compared here three prominent Azospirillum strains as part of three-component consortia including also the PGPR Pseudomonas fluorescens F113 and a mycorrhizal inoculant mix composed of three Glomus strains. Inoculant colonization of maize was assessed by quantitative PCR, transcription of auxin synthesis gene ipdC (involved in phytostimulation) in Azospirillum by RT-PCR, and effects on maize by secondary metabolic profiling and shoot biomass measurements. Results showed that phytostimulation by all the three-component consortia was comparable, despite contrasted survival of the Azospirillum strains and different secondary metabolic responses of maize to inoculation. Unexpectedly, the presence of Azospirillum in the inoculum resulted in lower phytostimulation in comparison with the Pseudomonas-Glomus two-component consortium, but this effect was transient. Azospirillum's ipdC gene was transcribed in all treatments, especially with three-component consortia, but not with all plants and samplings. Inoculation had no negative impact on the prevalence of mycorrhizal taxa in roots. In conclusion, this study brought new insights in the functioning of microbial consortia and showed that Azospirillum-Pseudomonas-Glomus three-component inoculants may be useful in environmental biotechnology for maize growth promotion.
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69. |
Delledonne M,
Porcari R,
Fogher C,
( 1990 ) Nucleotide sequence of the nodG gene of Azospirillum brasilense. PMID : 2243795 : DOI : 10.1093/nar/18.21.6435 PMC : PMC332548 Abstract >>
N/A
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70. |
Rajendran C,
Gerhardt EC,
Bjelic S,
Gasperina A,
Scarduelli M,
Pedrosa FO,
Chubatsu LS,
Merrick M,
Souza EM,
Winkler FK,
Huergo LF,
Li XD,
( 2011 ) Crystal structure of the GlnZ-DraG complex reveals a different form of PII-target interaction. PMID : 22074780 : DOI : 10.1073/pnas.1108038108 PMC : PMC3223478 Abstract >>
Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P(II)family. P(II) proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P(II) and also by P(II) posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P(II) protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 ? resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P(II) complexes, the majority of contacts with the target protein do not involve the T-loop region of P(II). Hence this structure identifies a different mode of P(II) interaction with a target protein and demonstrates the potential for P(II) proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.
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71. |
Vanoni MA,
Negri A,
Zanetti G,
Ronchi S,
Curti B,
( 1990 ) Structural studies on the subunits of glutamate synthase from Azospirillum brasilense. PMID : 2198943 : DOI : 10.1016/0167-4838(90)90273-i Abstract >>
The amino acid composition and the N-terminal sequences of the two dissimilar subunits of glutamate synthase from Azospirillum brasilense have been determined along with the sequences of selected CNBr peptides. Comparison of our data with those available for Escherichia coli glutamate synthase revealed an overall good homology between the enzymes from the two sources. This is more evident for the heavy subunits where the highly conserved N-terminal sequence containing Cys-1, suggests that this region may be involved in catalysis. However, it appears that the light subunits are different with respect to both their amino acid composition and their N-terminal region, suggesting that the latter may not be part of the enzyme active site. Finally, an extinction coefficient at 444 nm of 62.66 +/- 4.61 mM-1.cm-1 was determined.
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72. |
( 1996 ) Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense. PMID : 8763942 : DOI : 10.1128/jb.178.14.4143-4149.1996 PMC : PMC178171 Abstract >>
The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.
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73. |
( 1997 ) Isolation and characterization of the Azospirillum brasilense trpE(G) gene, encoding anthranilate synthase. PMID : 8939798 : Abstract >>
The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant. DNA sequence analysis of a 3.1-kb PstI restriction fragment of A. brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein. Previously an A. brasilense clone containing trpGDC was identified (Zimmer et al. Mol Gen Genet 229:41-51, 1991). It can, therefore, be concluded that A. brasilense contains two trpG genes. A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues. Putative terminator and anti-terminator loops have also been identified. The LLESX10S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A. brasilense TrpE(G) fused protein.
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74. |
( 1993 ) Glutamate synthase genes of the diazotroph Azospirillum brasilense. Cloning, sequencing, and analysis of functional domains. PMID : 8428988 : Abstract >>
A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli. Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A. brasilense GltS. The organization of the gltBD region of A. brasilense differs from that of the corresponding region in E. coli: in A. brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB. The deduced amino acid sequences reveal a high similarity with GltS from E. coli and with the ferredoxin-dependent GltS from maize. Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins.
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75. |
( 1996 ) Acyl carrier protein of Azospirillum brasilense: properties of the purified protein and sequencing of the corresponding gene, acpP. PMID : 8760922 : DOI : 10.1099/13500872-142-8-2097 Abstract >>
Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2.0-3.0 x 10(4) molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.
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76. |
( 1993 ) Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity. PMID : 8355653 : DOI : 10.1007/bf00277056 Abstract >>
A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A brasilense. These mutants were prototrophic and Nif+. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.
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77. |
( 1994 ) Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate decarboxylase gene. PMID : 8202090 : DOI : 10.1007/bf00280477 Abstract >>
Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.
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78. |
( 1996 ) Cloning, nucleotide sequencing, and expression of the Azospirillum brasilense lon gene: involvement in iron uptake. PMID : 8655539 : DOI : 10.1128/jb.178.12.3440-3446.1996 PMC : PMC178111 Abstract >>
The lon gene of Escherichia coli encodes the lon (La) protease, which is associated with cellular protein degradation. A lon gene homolog from Azospirillum brasilense, a nitrogen-fixing soil bacterium which lives in association with the roots of cereal grasses, was cloned and characterized. The nucleotide sequence of the A. brasilense lon gene was determined. It contains an open reading frame that encodes a protein of 810 amino acids with a predicted molecular mass of about 90 kDa. The deduced amino acid sequence showed a high level of homology with the sequences of all the known lon gene products. An open reading frame homologous to the E. coli clpX gene was found in front of the lon gene. Transcriptional analysis showed that the lon gene of A. brasilense is induced by heat shock and that the mRNA is monocistronic. An A. brasilense mutant, with Tn5 inserted in the lon gene, was shown to be defective in iron uptake and failed to express two membrane proteins that are induced by iron starvation in the parental strain.
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79. |
( 1996 ) The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis. PMID : 8640606 : DOI : 10.1139/m96-064 Abstract >>
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.
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80. |
( 1994 ) Identification and sequencing of pyrG, the CTP synthetase gene of Azospirillum brasilense Sp7. PMID : 8138139 : DOI : 10.1111/j.1574-6968.1994.tb06650.x 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.
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81. |
( 1994 ) Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism. PMID : 8002571 : DOI : 10.1128/jb.176.24.7484-7490.1994 PMC : PMC197204 Abstract >>
A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.
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82. |
( 1993 ) Identification of a nifW-like gene in Azospirillum brasilense. PMID : 8504172 : DOI : 10.1016/0167-4781(93)90188-j Abstract >>
A small ORF, 5' upstream of the fixABC operon in Azospirillum brasilense Sp7, has been identified. Sequence comparison shows significant homology to the Azotobacter vinelandii and Azorhizobium caulinodans nifW gene.
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83. |
( 1994 ) Sequence analysis of the Azospirillum brasilense exoB gene, encoding UDP-glucose 4'-epimerase. PMID : 8026752 : DOI : 10.1016/0378-1119(94)90221-6 Abstract >>
The nucleotide sequence of the Azospirillum brasilense exoB gene, located on plasmid pRhico, has been determined. The A. brasilense ExoB protein shows significant homology with other prokaryotic UDP-glucose 4'-epimerases (EC 5.1.3.2).
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84. |
( 1994 ) Interdomain loops and conformational changes of glutamate synthase as detected by limited proteolysis. PMID : 8001567 : DOI : 10.1111/j.1432-1033.1994.tb20075.x Abstract >>
Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time-dependent degradation of glutamate synthase alpha and beta subunits, to yield several fragments of different stability, was observed, the alpha subunit being more sensitive than the beta to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N-terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the alpha subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099-3106]. Therefore, these protease-sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C-terminal region of glutamate synthase beta subunit from tryptic cleavage, supporting the proposal that it contains the pyridine-nucleotide-binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog L-methionine sulfone, which binds presumably to the N-terminal region of the alpha subunit, altered the sensitivity to proteolysis of the sites of the alpha subunit proposed to be part of links between domains of glutamate synthase. These results show that long-range conformational changes of glutamate synthase occur on binding of its substrates. The study of several NADPH-dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH-dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.
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85. |
Van Bastelaere E,
Keijers V,
Vanderleyden J,
( 1995 ) Cloning and sequencing of the putative Azospirillum brasilense gene encoding GTP cyclohydrolase II. PMID : 7883178 : DOI : 10.1016/0378-1119(94)00826-e Abstract >>
Sequence analysis of a fragment of Azospirillum brasilense DNA revealed the presence of a ribA homologue, of which the 3' portion encodes a putative GTP cyclohydrolase II. The 5' portion (approx. half of the ORF) does not show homology to any other sequence from the databases.
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86. |
Raina S,
Raina R,
Venkatesh TV,
Das HK,
( N/A ) Isolation and characterization of a locus from Azospirillum brasilense Sp7 that complements the tumorigenic defect of Agrobacterium tumefaciens chvB mutant. PMID : 7756697 : Abstract >>
The chromosomal virulence gene chvB of Agrobacterium tumefaciens is required for pathogenesis. A DNA fragment from the chvB locus can hybridize to DNA from Azospirillum brasilense Sp7. This DNA fragment could restore the tumorigenic activity of the chvB mutant strain A. tumefaciens A1011 towards leaf disks of Nicotiana tabacum. An NH2-terminal open reading frame, 480 codons long, was most likely responsible for the restoration of the tumorigenic activity. The A. brasilense sequence showed good homology with the NH2-terminal region of the ndvB gene of Rhizobium meliloti.
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87. |
( 1993 ) Isolation of a glutamate synthase (GOGAT)-negative, pleiotropically N utilization-defective mutant of Azospirillum brasilense: cloning and partial characterization of GOGAT structural gene. PMID : 7902833 : DOI : 10.1128/jb.175.24.8024-8029.1993 PMC : PMC206984 Abstract >>
An Azospirillum brasilense mutant (N12) pleiotropically defective in the assimilation of nitrogenous compounds (Asm-) was isolated and found lacking in the glutamate synthase (GOGAT-). The glt (GOGAT) locus of A. brasilense was identified by isolating a broad-host-range pLAFR1 cosmid clone from a gene library of the bacterium that rectified Asm- and GOGAT- defects (full recovery of activities of the nitrogenase, the assimilatory nitrate and nitrite reductases, and the glutamate synthase). A 7.5-kb EcoRI fragment of the cosmid clone that also complemented N12 was partially sequenced to identify the open reading frame for the alpha-subunit of GOGAT. The amino acid sequences deduced from the partial nucleotide sequences of the glt locus of A. brasilense showed considerable homology with that of the alpha-subunit of GOGAT coded by the gltB gene of Escherichia coli. The genetic lesion of N12 was found within the gltB gene of A. brasilense. The gltB promoter of A. brasilense showed the presence of a consensus sigma-70-like recognition site (as in E. coli) in addition to potential NtrA-RNA polymerase, IHF, and NifA binding sites.
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88. |
Novick NJ,
Tyler ME,
( 1982 ) L-arabinose metabolism in Azospirillum brasiliense. PMID : 6798025 : PMC : PMC216631 Abstract >>
An oxidative pathway by which L-arabinose is converted to alpha-ketoglutarate in crude extracts of Azospirillum brasiliense is demonstrated. Specific activities of enzymes involved in the pathway were determined, and several pathway intermediates were identified.
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89. |
Yan D,
He L,
Li J,
( 1995 ) [Cloning and sequencing of ntrBC genes from Azospirillum brasilense]. PMID : 7483580 : Abstract >>
A gene library of Azospirillum brasilense Yu62 was constructed in EMBL3. The library was screened with PCR amplified fragment as a special probe. Ten positive plaques (EA1-EA10) were selected. Detection results showed they contained two different types of clones, representing as EA4 and EA9 respectively. Southern hybridization of EA4 displayed that target gene was located in a 2.9kb EcoRI fragment. Sequence of this fragment had allowed the position and identification of ntrC gene, which encoding a protein of 53469, consisted of 480 amino acids. In the upstream of ntrC, a complete ntrB coding region was also found, which encoding a protein of 43487, consisted of 400 amino acids. Homologous analysis of the deduced amino acid sequences of ntrC and ntrB from different bacteria demonstrated that A. brasilense was closer to Rhizobia than to other free-living diazotrophs.
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90. |
Machado HB,
Yates MG,
Funayama S,
Rigo LU,
Steffens MB,
Souza EM,
Pedrosa FO,
( 1995 ) The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated. PMID : 7553451 : DOI : 10.1139/m95-093 Abstract >>
A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.
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91. |
Moens S,
Michiels K,
Keijers V,
Van Leuven F,
Vanderleyden J,
( 1995 ) Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7. PMID : 7559324 : DOI : 10.1128/jb.177.19.5419-5426.1995 PMC : PMC177346 Abstract >>
Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.
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92. |
Bozouklian H,
Elmerich C,
( N/A ) Nucleotide sequence of the Azospirillum brasilense Sp7 glutamine synthetase structural gene. PMID : 2878685 : DOI : 10.1016/s0300-9084(86)80062-1 Abstract >>
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.
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93. |
( 1998 ) Structural homologues P(II) and P(Z) of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen-dependent functions. PMID : 9720864 : DOI : 10.1046/j.1365-2958.1998.00938.x Abstract >>
P(II) (glnB) is a signal transduction protein that in Azospirillum brasilense is specifically required for nitrogen fixation. Little is known about whether and how its homologue P(Z) (glnZ) participates in the regulation of cellular functions. In this study, we have shown the regulatory action of the two proteins by analysing the relevant single and double null-mutant strains. The transcription of glnZ is monocistronic, and it starts mainly from a sigma54-dependent promoter, activated by NtrC. glnZ expression is dependent on the ntr system, even under conditions of nitrogen excess, and is greatly enhanced in the presence of aspartate. P(Z) is uridylylated in response to nitrogen limitation, like P(II), although different amounts of the two proteins are synthesized. P(II) is required for the dephosphorylation of NtrC. Thus, in the absence of P(II), the repression of nitrate assimilation is not promoted, which, in turn, leads to a high rate of ammonium excretion. Unexpectedly, P(II) and P(Z) proteins are not essential for the reversible modification of glutamine synthetase. (Methyl)ammonium transport into the cell is negatively regulated by P(Z). The growth of a double-mutant strain (glnB::kan; glnZ::omega) is drastically disabled, although wild-type growth is restored by complementation with either glnB or glnZ. We conclude that P(II) and P(Z), despite their structural similarity, are involved in different regulatory processes, except for that required for cell growth.
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94. |
( 1998 ) Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus. PMID : 9608743 : Abstract >>
The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (= IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported.
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95. |
( 1998 ) A cytochrome cbb3 (cytochrome c) terminal oxidase in Azospirillum brasilense Sp7 supports microaerobic growth. PMID : 9791120 : PMC : PMC107629 Abstract >>
Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.
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96. |
( 1997 ) Sequencing and analysis of function of the promoter region of draTG genes from Azospirillum brasilense Yu62. PMID : 9631255 : Abstract >>
From Azospirillum brasilense Yu62, we cloned and sequenced the upstream region of draTG. No identified genes were found in this region except for portion of nifH, which is transcribed divergently from draTG. However, some potential regulatory elements were found, which include downstream promoter elements (DPE), upstream activator sequences (UAS), and A + T-rich regions. This suggests that the region between draT and nifH might be a regulatory region rather than an encoding region, and the promoter of dra operon would probably be a RpoN-dependent promoter. A draT::cam transcriptional fusion plasmid pAT1 was constructed in pAF300. The expression of draT was studied in Escherichia coli and A. brasilense by Cmr assay. The result showed that draT could be transcribed in A. brasilense but not in E. coli while grown aerobically on an LD plate. This suggests that the transcription of draT needs some factors which are absent in E. coli, and the draT upstream region has the promoter function. Using the promoter-probe vector pCB182, a draT::lacZ transcriptional fusion plasmid pCT1 was constructed. beta-galactosidase activity was determined in vivo in E. coli, in the presence or absence of NifA of Klebsiella pneumoniae, respectively. The results demonstrated that NifA was not involved in the transcriptional regulation of draTG.
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97. |
( 1998 ) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. PMID : 9758798 : PMC : PMC106545 Abstract >>
A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.
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98. |
( 1998 ) A transcriptional regulator of the LuxR-UhpA family, FlcA, controls flocculation and wheat root surface colonization by Azospirillum brasilense Sp7. PMID : 9487693 : DOI : 10.1094/MPMI.1998.11.3.177 Abstract >>
Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UphA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.
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99. |
( 1998 ) Sequencing and complementation analysis of the nifUSV genes from Azospirillum brasilense. PMID : 9503607 : DOI : 10.1111/j.1574-6968.1998.tb12854.x Abstract >>
The functionality of nitrogenase in diazotrophic bacteria is dependent upon nif genes other than the structural nifH, D, and K genes which encode the enzyme subunit proteins. Such genes are involved in the activation of nif gene expression, maturation of subunit proteins, cofactor biosynthesis, and electron transport. In this work, approximately 5500 base pairs located within the major nif gene cluster of Azospirillum brasilense Sp7 have been sequenced. The deduced open reading frames were compared to the nif gene products of Azotobacter vinelandii and other diazotrophs. This analysis indicates the presence of five ORFs encoding ORF2, nifU, nifS, nifV, and ORF4 in the same sequential organization as found in other organisms. Consensus sigma 54 and NifA binding sites are present in the putative promoter region upstream of ORF2 in the A. brasilense sequence. The nifV gene of A. brasilense but not nifU or nifS complemented corresponding mutants strains of A. vinelandii.
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100. |
( 1998 ) (Methyl)ammonium transport in the nitrogen-fixing bacterium Azospirillum brasilense. PMID : 9573149 : PMC : PMC107216 Abstract >>
An ammonium transporter of Azospirillum brasilense was characterized. In contrast to most previously reported putative prokaryotic NH4+ transporter genes, A. brasilense amtB is not part of an operon with glnB or glnZ which, in A. brasilense, encode nitrogen regulatory proteins PII and PZ, respectively. Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein. Nitrogen regulates the transcription of the amtB gene in A. brasilense by the Ntr system. amtB is the first gene identified in A. brasilense whose expression is regulated by NtrC. The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH4+ transport mechanism. Growth of amtB mutants at low ammonium concentrations is reduced compared to that of the wild type. This suggests that AmtB has a role in scavenging ammonium at low concentrations.
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101. |
( 1997 ) Cloning and sequencing of draTG genes and their downstream region of Azospirillum brasilense Yu62. PMID : 9429775 : Abstract >>
An 8-kb fragment was cloned by probing the gene library of Azospirillum brasilense Yu62 with the 4.0-kb draTG fragment of A. brasilense Sp7. DNA hybridization of this fragment demonstrated that draTG genes were located in a 3.0-kb EcoR I-Kpn I fragment, and were contiguous to the nifH gene. This 3.0-kb fragment was completely sequenced on both strands. Sequence analysis of the fragment revealed that it included the full-length draTG genes and two ORFs downstream of draG (ORF3 and incomplete ORF4). The draTG and downstream ORF3 are presumed to be cotranscribed as a single operon. Promoter element analysis of the sequenced region showed that there were some elements of the sigma 54-dependent promoter (DPEs and UASs) in the upstream region of draG and ORF3. This suggests that the draG and ORF3 might be transcribed independently in addition to being cotranscribed with draT. Both the DNA and amino acid sequences of the draTG genes from A. brasilense Yu62 were compared with those of other nitrogen-fixing bacteria. The results showed the draTG genes were highly conservative. There were only a few changes among strains and/or species. Homology analysis of ORF revealed that the ORF3 immediately downstream of draG was homologous not only with the ORF at the same position in Rhodospirillum rubrum and A. lipoferum, but also with the ORF14 of Azotobacter vinelandii and the ORF4 showed extensive similarity to yafJ of Escherichia coli.
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