RESUMO
The association of conventional fertilizers with fertilizers of organic origin is a well-known practice in agriculture for achieving high productivity and also with the adoption of inoculants, composed of bacteria for promoting better plant development of crops. Thus, the objective of the work was to investigate the growth and productivity of radish with organic fertilization and the influence of the use of nitrogen fixing bacteria of the genus Azospirillum spp.radish (Raphanus sativus L.) Hybrid Margaret Queen. The treatments were the use of different doses of organic fertilizer and the application of Azospirillum brasilense, besides the control. At the end of the cycle, plant height, tuber diameter, tuber fresh mass, tuber dry mass, and total tuber fresh mass were evaluated. With and without application of A. brasilense,increases of 16.5% and 6.40% in tuber diameter were obtained, respectively. Without application of A. brasilense, there was an increase of 0.37% in leaf dry mass when the maximum concentration of 8 kg m² of organic fertilizer was used. For root fresh mass and root dry mass, with foliar application of A. brasilense, there was an increase of 9.57% and 0.67%, while without A. brasilensethere was an increase of 2.43% and 0.22%. There were increases of 12.83% for total fresh mass with the management of A. brasilenseand 3.4% without A. brasilense, using the maximum concentration of 8 kg m² of organic fertilizer. For height, there was an increase of 5.12% without A. brasilense.It was concluded that organic fertilization alone or combined with A. brasilenseis an alternative in radish production.(AU)
A associação de fertilizantes convencionais com fertilizantes de origem orgânica é uma prática bastante conhecida na agricultura por alcançar alta produtividade e também com a adoção de inoculantes, compostos por bactérias por promoverem melhor desenvolvimento vegetal das lavouras. Assim, o objetivo do trabalho foi investigar o crescimento e a produtividade do rabanete com adubação orgânica e a influência do uso de bactérias fixadoras de nitrogênio do gênero Azospirillum spp. rabanete (Raphanus sativus L.) Híbrido Margaret Queen. Os tratamentos foram o uso de diferentes doses de adubo orgânico e a aplicação de Azospirillum brasilense, além da testemunha. Ao final do ciclo, foram avaliados altura da planta, diâmetro do tubérculo, massa fresca do tubérculo, massa seca do tubérculo e massa fresca total do tubérculo. Com e sem aplicação de A. brasilense, foram obtidos aumentos de 16,5% e 6,40% no diâmetro do tubérculo, respectivamente. Sem aplicação de A. brasilense, houve aumento de 0,37% na massa seca foliar quando utilizada a concentração máxima de 8 kg m² de adubo orgânico. Para massa fresca de raiz e massa seca de raiz, com aplicação foliar de A. brasilense, houve aumento de 9,57% e 0,67%, enquanto sem A.brasilense houve aumento de 2,43% e 0,22%. Houve incrementos de 12,83% para massa fresca total com manejo de A. brasilense e de 3,4% sem A. brasilense, com a utilização da concentração máxima de 8 kg m² de adubo orgânico. Para altura, houve aumento de 5,12% sem A. brasilense. Concluiu-se que a adubação orgânica isolada ou combinada com A. brasilense é uma alternativa na produção de rabanete.(AU)
Assuntos
Compostagem/métodos , Raphanus/crescimento & desenvolvimento , Agricultura Orgânica/métodos , Azospirillum brasilense/enzimologiaRESUMO
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.(AU)
A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.(AU)
Assuntos
Glutamato-Amônia Ligase/biossíntese , Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Fixação de Nitrogênio , Escherichia coliRESUMO
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.
A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.
Assuntos
Proteínas de Bactérias/genética , Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Compostos de Amônio , Glutamato-Amônia Ligase/genética , Escherichia coli/genéticaRESUMO
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.
A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.
Assuntos
Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Escherichia coli , Fixação de Nitrogênio , Glutamato-Amônia Ligase/biossínteseRESUMO
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.
Assuntos
Compostos de Amônio , Azospirillum brasilense , Proteínas de Bactérias , Glutamato-Amônia Ligase , Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Glutamato-Amônia Ligase/genéticaRESUMO
The genome of Azospirillum brasilense encodes five RpoH sigma factors: two OxyR transcription regulators and three catalases. The aim of this study was to understand the role they play during oxidative stress and their regulatory interconnection. Out of the 5 paralogs of RpoH present in A. brasilense, inactivation of only rpoH1 renders A. brasilense heat sensitive. While transcript levels of rpoH1 were elevated by heat stress, those of rpoH3 and rpoH5 were upregulated by H2O2 Catalase activity was upregulated in A. brasilense and its rpoH::km mutants in response to H2O2 except in the case of the rpoH5::km mutant, suggesting a role for RpoH5 in regulating inducible catalase. Transcriptional analysis of the katN, katAI, and katAII genes revealed that the expression of katN and katAII was severely compromised in the rpoH3::km and rpoH5::km mutants, respectively. Regulation of katN and katAII by RpoH3 and RpoH5, respectively, was further confirmed in an Escherichia coli two-plasmid system. Regulation of katAII by OxyR2 was evident by a drastic reduction in growth, KatAII activity, and katAII::lacZ expression in an oxyR2::km mutant. This study reports the involvement of RpoH3 and RpoH5 sigma factors in regulating oxidative stress response in alphaproteobacteria. We also report the regulation of an inducible catalase by a cascade of alternative sigma factors and an OxyR. Out of the three catalases in A. brasilense, those corresponding to katN and katAII are regulated by RpoH3 and RpoH5, respectively. The expression of katAII is regulated by a cascade of RpoE1âRpoH5 and OxyR2.IMPORTANCEIn silico analysis of the A. brasilense genome showed the presence of multiple paralogs of genes involved in oxidative stress response, which included 2 OxyR transcription regulators and 3 catalases. So far, Deinococcus radiodurans and Vibrio cholerae are known to harbor two paralogs of OxyR, and Sinorhizobium meliloti harbors three catalases. We do not yet know how the expression of multiple catalases is regulated in any bacterium. Here we show the role of multiple RpoH sigma factors and OxyR in regulating the expression of multiple catalases in A. brasilense Sp7. Our work gives a glimpse of systems biology of A. brasilense used for responding to oxidative stress.
Assuntos
Azospirillum brasilense/enzimologia , Catalase/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Catalase/metabolismo , Proteínas de Choque Térmico/genética , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Fator sigma/genética , Fatores de Transcrição/genéticaRESUMO
Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.
Assuntos
3-Oxoacil-(Proteína Carreadora de Acil) Redutase/genética , Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Flagelos/genética , Plasmídeos/genética , Dobramento de ProteínaRESUMO
Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent -12/-24 promoter consensus. The expression of an exaA::lacZ fusion was induced maximally by glycerol and was dependent on σ54 Bioinformatic analysis of the sequence flanking the -12/-24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA EraR also showed a positive interaction with RpoN in two-hybrid and pulldown assays.IMPORTANCE Quinoprotein alcohol dehydrogenase (ExaA) plays an important role in the catabolism of alcohols in bacteria. Although exaA expression is thought to be regulated by a two-component system consisting of EraS and EraR, the mechanism of regulation was not known. This study shows the details of the regulation of expression of the exaA gene in A. brasilense We have shown here that exaA of A. brasilense is maximally induced by glycerol and harbors a σ54-dependent promoter. The response regulator EraR binds to an inverted repeat located upstream of the exaA promoter. This study shows that a LuxR-type response regulator (EraR) binds upstream of the exaA gene and physically interacts with σ54 The unique feature of this regulation is that EraR is a LuxR-type transcription regulator that lacks the GAFTGA motif, a characteristic feature of the enhancer binding proteins that are known to interact with σ54 in other bacteria.
Assuntos
Oxirredutases do Álcool/metabolismo , Azospirillum brasilense/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicerol/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Repressoras/genética , Transativadores/genéticaRESUMO
3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, α-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD+. Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde- and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Azospirillum brasilense/enzimologia , Ácido Láctico/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Ácido Láctico/biossíntese , Ácido Láctico/química , Simulação de Acoplamento Molecular , NAD/metabolismo , NADP/metabolismo , Eletricidade Estática , Especificidade por SubstratoRESUMO
OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.
Assuntos
Azospirillum brasilense/enzimologia , Proteínas de Bactérias/genética , Regulação Enzimológica da Expressão Gênica , Peroxirredoxinas/genética , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Peroxirredoxinas/metabolismo , Proteínas Repressoras/genética , Fator sigma/genéticaRESUMO
BACKGROUND: Azopirillum brasilense is a plant-growth promoting nitrogen-fixing bacteria that is used as bio-fertilizer in agriculture. Since nitrogen fixation has a high-energy demand, the reduction of N2 to NH4+ by nitrogenase occurs only under limiting conditions of NH4+ and O2. Moreover, the synthesis and activity of nitrogenase is highly regulated to prevent energy waste. In A. brasilense nitrogenase activity is regulated by the products of draG and draT. The product of the draB gene, located downstream in the draTGB operon, may be involved in the regulation of nitrogenase activity by an, as yet, unknown mechanism. RESULTS: A deep in silico analysis of the product of draB was undertaken aiming at suggesting its possible function and involvement with DraT and DraG in the regulation of nitrogenase activity in A. brasilense. In this work, we present a new artificial intelligence strategy for protein classification, named ProClaT. The features used by the pattern recognition model were derived from the primary structure of the DraB homologous proteins, calculated by a ProClaT internal algorithm. ProClaT was applied to this case study and the results revealed that the A. brasilense draB gene codes for a protein highly similar to the nitrogenase associated NifO protein of Azotobacter vinelandii. CONCLUSIONS: This tool allowed the reclassification of DraB/NifO homologous proteins, hypothetical, conserved hypothetical and those annotated as putative arsenate reductase, ArsC, as NifO-like. An analysis of co-occurrence of draB, draT, draG and of other nif genes was performed, suggesting the involvement of draB (nifO) in nitrogen fixation, however, without the definition of a specific function.
Assuntos
Azospirillum brasilense/química , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/química , Biologia Computacional/métodos , Nitrogenase/química , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional/instrumentação , Genes Bacterianos , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , ÓperonRESUMO
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.
Assuntos
Azospirillum brasilense/enzimologia , Azospirillum brasilense/fisiologia , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Azospirillum brasilense/genética , Técnicas Bacteriológicas , Coenzimas/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Flavina-Adenina Dinucleotídeo/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Microscopia Confocal , Fósforo-Oxigênio Liases/isolamento & purificação , Coloração e RotulagemRESUMO
The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.
Assuntos
Antibacterianos/farmacologia , Arseniato Redutases/metabolismo , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Farmacorresistência Bacteriana , Metiltransferases/metabolismo , Antibacterianos/metabolismo , Arseniato Redutases/genética , Azospirillum brasilense/genética , Cloranfenicol/metabolismo , Cloranfenicol/farmacologia , Clindamicina/metabolismo , Clindamicina/farmacologia , Clonagem Molecular , Diterpenos/metabolismo , Diterpenos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Metiltransferases/genética , Mutagênese Insercional , Ribossomos/metabolismoRESUMO
AIM: To develop co-aggregated bacterial inoculant comprising of Methylobacterium oryzae CBMB20/Methylobacterium suomiense CBMB120 strains with Azospirillum brasilense (CW903) strain and testing their efficiency as inoculants for plant growth promotion (PGP). METHODS AND RESULTS: Biofilm formation and co-aggregation efficiency was studied between A. brasilense CW903 and methylobacterial strains M. oryzae CBMB20 and M. suomiense CBMB120. Survival and release of these co-aggregated bacterial strains entrapped in alginate beads were assessed. PGP attributes of the co-aggregated bacterial inoculant were tested in tomato plants under water-stressed conditions. Results suggest that the biofilm formation efficiency of the CBMB20 and CBMB120 strains increased by 15 and 34%, respectively, when co-cultivated with CW903. Co-aggregation with CW903 enhanced the survivability of CBMB20 strain in alginate beads. Water stress index score showed least stress index in plants inoculated with CW903 and CBMB20 strains maintained as a co-aggregated inoculant. CONCLUSIONS: This study reports the development of co-aggregated cell inoculants containing M. oryzae CBMB20 and A. brasilense CW903 strains conferred better shelf life and stress abatement in inoculated tomato plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings could be extended to other PGP bacterial species to develop multigeneric bioinoculants with multiple benefits for various crops.
Assuntos
Alginatos/química , Azospirillum brasilense/fisiologia , Biofilmes/crescimento & desenvolvimento , Methylobacterium/fisiologia , Solanum lycopersicum/crescimento & desenvolvimento , Azospirillum brasilense/enzimologia , Azospirillum brasilense/ultraestrutura , Desidratação/prevenção & controle , Secas , Etilenos/metabolismo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Hidrólise , Peroxidação de Lipídeos , Solanum lycopersicum/microbiologia , Malondialdeído/metabolismo , Methylobacterium/enzimologia , Methylobacterium/ultraestrutura , Microscopia Eletrônica de Varredura , Microesferas , Peroxidase/metabolismo , Solo/químicaRESUMO
The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/enzimologia , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.
Assuntos
Trifosfato de Adenosina/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Ácidos Cetoglutáricos/metabolismo , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismo , Azospirillum brasilense/metabolismo , Vetores Genéticos , PlasmídeosRESUMO
Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.
Assuntos
Trifosfato de Adenosina/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Ácidos Cetoglutáricos/metabolismo , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismo , Azospirillum brasilense/metabolismo , Vetores Genéticos , PlasmídeosRESUMO
Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.
Assuntos
Azospirillum brasilense , Proteínas de Bactérias/biossíntese , Peroxidases/biossíntese , Microbiologia do Solo , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Azospirillum brasilense/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Benzotiazóis/farmacologia , Catecóis/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Cinética , Peso Molecular , Peroxidases/isolamento & purificação , Ácidos Sulfônicos/farmacologiaRESUMO
The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.
Assuntos
Proteínas de Bactérias/química , Cromatografia de Afinidade/métodos , Proteínas PII Reguladoras de Nitrogênio/química , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas PII Reguladoras de Nitrogênio/isolamento & purificação , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura de TransiçãoRESUMO
In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.