RESUMO
Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.
Assuntos
Azospirillum brasilense/fisiologia , Bacillus pumilus/fisiologia , Chlorella/crescimento & desenvolvimento , Microalgas/crescimento & desenvolvimento , Azospirillum brasilense/efeitos dos fármacos , Bacillus pumilus/efeitos dos fármacos , Metabolismo dos Carboidratos/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Chlorella/efeitos dos fármacos , Chlorella/metabolismo , Clorofila/metabolismo , Clorofila A , Escherichia coli/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Microalgas/metabolismo , Compostos Orgânicos Voláteis/farmacologiaRESUMO
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
Extracytoplasmic function (ECF) sigma factors (σ(E)) are known to bring about changes in gene expression to enable bacteria to adapt to different stresses. The Azospirillum brasilense Sp245 genome harbours nine genes encoding σ(E), of which two are adjacent to the genes encoding ChrR-type zinc-binding anti-sigma (ZAS) factors. We describe here the role and regulation of a new pair of rpoE-chrR, which was found in the genome of A. brasilense Sp7 in addition to the previously described rpoE-chrR pair (designated rpoE1-chrR1). The rpoE2-chrR2 pair is also cotranscribed, and their products show protein-protein interaction. The -10 and -35 promoter elements of rpoE2-chrR2 and rpoE1-chrR1 were similar but not identical. Unlike the promoter of rpoE1-chrR1, the rpoE2-chrR2 promoter was neither autoregulated nor induced by oxidative stress. Inactivation of chrR2 or overexpression of rpoE2 in A. brasilense Sp7 resulted in an overproduction of carotenoids. It also conferred resistance to oxidative stresses and antibiotics. By controlling the synthesis of carotenoids, initiation and elongation of translation, protein folding and purine biosynthesis, RpoE2 seems to play a crucial role in preventing and repairing the cellular damage caused by oxidative stress. Lack of autoregulation and constitutive expression of rpoE2-chrR2 suggest that RpoE2-ChrR2 may provide a rapid mechanism to cope with oxidative stress, wherein singlet oxygen ((1)O(2))-mediated dissociation of the RpoE2-ChrR2 complex might release RpoE2 to drive the expression of its target genes.
Assuntos
Antibacterianos/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Viabilidade Microbiana/efeitos dos fármacos , Oxidantes/toxicidade , Estresse Oxidativo , Fatores de Transcrição/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/fisiologia , Carotenoides/biossíntese , Farmacorresistência Bacteriana , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ(32), σ(H) or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.
Assuntos
Azospirillum brasilense/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Estresse Oxidativo , Fator sigma/metabolismo , Estresse Fisiológico , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/genética , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Proteínas de Choque Térmico/genética , Azul de Metileno/metabolismo , Oxidantes/metabolismo , Proteoma/análise , Rhodobacter sphaeroides/fisiologia , Fator sigma/genética , Sais de Tetrazólio/metabolismoRESUMO
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
Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ(E)) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5' rapid amplification of cDNA ends showed that the rpoE-chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.
Assuntos
Antibacterianos/toxicidade , Azospirillum brasilense/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/biossíntese , Fator sigma/biossíntese , Estresse Fisiológico , Transcrição Gênica , Antibacterianos/metabolismo , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Etanol/metabolismo , Etanol/toxicidade , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Azul de Metileno/metabolismo , Azul de Metileno/toxicidade , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética , Fator sigma/genética , Cloreto de Sódio/metabolismo , Cloreto de Sódio/toxicidade , Técnicas do Sistema de Duplo-HíbridoRESUMO
Pseudomonads producing the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) can control soil-borne phytopathogens, but their impact on other plant-beneficial bacteria remains poorly documented. Here, the effects of synthetic Phl and Phl(+) Pseudomonas fluorescens F113 on Azospirillum brasilense phytostimulators were investigated. Most A. brasilense strains were moderately sensitive to Phl. In vitro, Phl induced accumulation of carotenoids and poly-ß-hydroxybutyrate-like granules, cytoplasmic membrane damage and growth inhibition in A. brasilense Cd. Experiments with P. fluorescens F113 and a Phl(-) mutant indicated that Phl production ability contributed to in vitro growth inhibition of A. brasilense Cd and Sp245. Under gnotobiotic conditions, each of the three strains, P. fluorescens F113 and A. brasilense Cd and Sp245, stimulated wheat growth. Co-inoculation of A. brasilense Sp245 and Pseudomonas resulted in the same level of phytostimulation as in single inoculations, whereas it abolished phytostimulation when A. brasilense Cd was used. Pseudomonas Phl production ability resulted in lower Azospirillum cell numbers per root system (based on colony counts) and restricted microscale root colonization of neighbouring Azospirillum cells (based on confocal microscopy), regardless of the A. brasilense strain used. Therefore, this work establishes that Phl(+) pseudomonads have the potential to interfere with A. brasilense phytostimulators on roots and with their plant growth promotion capacity.
Assuntos
Antibacterianos/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/metabolismo , Controle Biológico de Vetores , Pseudomonas fluorescens/metabolismo , Triticum/crescimento & desenvolvimento , Antibacterianos/síntese química , Antibacterianos/metabolismo , Azospirillum brasilense/crescimento & desenvolvimento , Carotenoides/metabolismo , Carotenoides/farmacologia , Hidroxibutiratos/metabolismo , Hidroxibutiratos/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/síntese química , Floroglucinol/metabolismo , Floroglucinol/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Poliésteres/metabolismo , Poliésteres/farmacologia , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crescimento & desenvolvimento , Triticum/efeitos dos fármacos , Triticum/microbiologiaRESUMO
Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245 did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation, i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50-60 degrees C, 10 min) as compared with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7 had higher heat resistance (50, 55, 60 degrees C). The viability maintenance and the portion of heat-resistant cells depended on the conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations at -20 degrees C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.
Assuntos
Azospirillum brasilense/crescimento & desenvolvimento , Azospirillum brasilense/ultraestrutura , Azospirillum brasilense/efeitos dos fármacos , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Nitrogênio/metabolismo , Fósforo/metabolismoRESUMO
The plant-beneficial bacterium Azospirillum brasilense can swim in liquids and swarm or migrate with the formation of microcolonies in soft media. To get closer to understanding the influence of natural environments on A. brasilense motility, we studied the individual and social movement of the bacterium in the presence of various plant lectins. The lectins with specificity for N-acetyl-beta-d-glucosamine oligomers (wheat germ, Solanum tuberosum and Ulex europeus agglutinins) decreased A. brasilense swimming speed and induced the formation of branched-granular colonies instead of the swarming rings. These effects seemed to be a consequence of specific interactions between the agglutinins and the lectin-binding polymers present in the A. brasilense cell envelope. Concanavalin A (with an affinity for terminal alpha-d-mannosyl and alpha-d-glucosyl residues) and Phaseolus vulgaris phytohemagglutinin P (with unknown specificity) almost did not affect the motility of A. brasilense.
Assuntos
Azospirillum brasilense/fisiologia , Quimiotaxia/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Azospirillum brasilense/efeitos dos fármacosRESUMO
Azospirillum brasilense Sp7 has been shown to overproduce carotenoids if the anti-sigma factor (anti-sigma(E))-encoding gene is inactivated. The anti-sigma mutant (Car-1) of A. brasilense Sp7 was more tolerant to the stresses generated by elevated temperature (40 degrees C), PEG-200 (30 mg mL(-1)) and the antibacterial agent Polymyxin-B (PMB, 25 microg mL(-1)) but not to elevated salinity (15 mg mL(-1)). Inhibition of carotenoid synthesis by diphenylamine inhibited the ability of the mutant to tolerate all the three stresses. Out of the four stress agents, only elevated temperature and salinity induced the rpoE promoter and increased the carotenoid content in Sp7 as well as in the Car-1 mutant. Comparison of the membrane permeability of the parent and the mutant by a PMB-N-phenyl-1-naphthylamine coupled assay showed that the presence of carotenoids in the mutant reduced the permeability of their membranes. Our study indicates that the carotenoid synthesis, which is under the control of extracytoplasmic function sigma factor (sigma(E)) in A. brasilense Sp7, plays a positive role in tolerating elevated temperature, the antibacterial peptide and PEG-200.
Assuntos
Azospirillum brasilense/efeitos dos fármacos , Proteínas de Bactérias/genética , Carotenoides/metabolismo , Tolerância a Medicamentos , Mutação , Polietilenoglicóis/farmacologia , Polimixina B/farmacologia , Fator sigma/antagonistas & inibidores , Antibacterianos/farmacologia , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Permeabilidade da Membrana Celular , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Fator sigma/metabolismo , TemperaturaRESUMO
For the rhizobacterium Azospirillum brasilense, the optimal nutritional range of C:N ratios corresponds to the presence of malate (ca. 3 to 5 g l(-1) of its sodium salt) and ammonium (ca. 0.5 to 3 g l(-1) of NH4Cl) as preferred carbon and nitrogen sources, respectively. This microaerophilic aerotactic bacterium is known to have a narrow optimal oxygen concentration range of ca. 3 to 5 microM, which is 1.2% to 2% of oxygen solubility in air-saturated water under normal conditions. In this work, the effects of stress conditions (bound-nitrogen deficiency related to a high C:N ratio in the medium; excess of oxygen) on aerobically grown A. brasilense Sp245, a native wheat-associated endophyte, were investigated in the absence and presence of wheat germ agglutinin (WGA, plant stress protein and a molecular host-plant signal for the bacterium) using FTIR spectroscopy of whole cells in the diffuse reflectance mode (DRIFT). The nutritional stress resulted in the appearance of prominent spectroscopic signs of poly-3-hydroxybutyrate (PHB) accumulation in the bacterial cells; in addition, splitting of the amide I band related to bacterial cellular proteins indicated some stress-induced alterations in their secondary structure components. Similar structural changes were observed in the presence of nanomolar WGA both in stressed A. brasilense cells and under normal nutritional conditions. Comparative analysis of the data obtained and the relevant literature data indicated that the stress conditions applied (which resulted in the accumulation of PHB involved in stress tolerance) and/or the presence of nanomolar concentrations of WGA induced synthesis of bacterial cell-surface (glyco)proteins rich in beta-structures, that could be represented by hemagglutinin and/or porin.
Assuntos
Azospirillum brasilense/efeitos dos fármacos , Nitrogênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Aglutininas do Germe de Trigo/farmacologia , Aerobiose , Azospirillum brasilense/crescimento & desenvolvimento , Azospirillum brasilense/metabolismo , Hidroxibutiratos/metabolismo , Nitrogênio/farmacologia , Poliésteres/metabolismoRESUMO
Azospirillum brasilense is a nitrogen-fixing, root-colonizing bacterium that brings about plant-growth-promoting effects mainly because of its ability to produce phytohormones. Ethylenediamine (EDA)-resistant mutants of A. brasilense were isolated and screened for their higher ability to decrease acetylene and release ammonia in the medium. One of the mutants showed considerably higher levels of acetylene decrease and ammonia excretion. Nitrogenase activity of this mutant was relatively resistant to inhibition by NH(4)Cl. Adenosine triphosphate ribosylation of dinitrogenase reductase in the mutant did not increase even in presence of 10 mM NH(4)Cl. Although the mutant showed decreased glutamine synthetase (GS) activity, neither the levels of GS synthesized by the mutant nor the NH (4) (+) -binding site in the GS differed from those of the parent. The main reason for the release of ammonia by the mutant seems to be the fixation of higher levels of nitrogen than its GS can assimilate, as well as higher levels of adenylylation of GS, which may decrease ammonia assimilation.
Assuntos
Amônia/metabolismo , Apirase/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Glutamato-Amônia Ligase/metabolismo , Adenosina Difosfato Ribose/metabolismo , Cloreto de Amônio/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/crescimento & desenvolvimento , Dinitrogenase Redutase/metabolismo , Farmacorresistência Bacteriana , Etilenodiaminas/farmacologia , Mutação , Fixação de NitrogênioRESUMO
O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.
Assuntos
Azospirillum brasilense/genética , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/isolamento & purificação , Telúrio/metabolismo , Telúrio/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Sequência de Bases , Carbono-Oxigênio Liases/química , Carbono-Oxigênio Liases/metabolismo , Cromatografia , Clonagem Molecular , Farmacorresistência Fúngica , Teste de Complementação Genética , Mutação , Oxirredução , FenótipoRESUMO
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.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Sequência de Aminoácidos , Azospirillum brasilense/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Nitratos/metabolismo , Fixação de Nitrogênio/genética , Nitrogenase/genética , Nitrogenase/metabolismo , Nitrosoguanidinas/farmacologia , Óperon , Proteínas PII Reguladoras de Nitrogênio , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Azospirillum brasilense/fisiologia , Proteínas de Bactérias/fisiologia , Células Quimiorreceptoras/fisiologia , Raízes de Plantas/microbiologia , Sequência de Aminoácidos , Azospirillum brasilense/efeitos dos fármacos , Benzoquinonas/farmacologia , Quimiotaxia , Metabolismo Energético , Dados de Sequência MolecularRESUMO
The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied. EPS of A. brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%). Under saline stress, A. brasilense produced a totally different EPS, composed mainly of galactose. Root exudates induced changes in A. brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates. No changes were induced by root exudates when A. brasilense was grown under saline stress. Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.
Assuntos
Azospirillum brasilense/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Raízes de Plantas/microbiologia , Polissacarídeos Bacterianos/metabolismo , Cloreto de Sódio/farmacologia , Triticum/microbiologia , Azospirillum brasilense/metabolismo , Azospirillum brasilense/fisiologia , Meios de Cultura , Resposta ao Choque Térmico , Lipopolissacarídeos/química , Pressão Osmótica , Raízes de Plantas/metabolismo , Polissacarídeos Bacterianos/química , Microbiologia do SoloRESUMO
To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/patogenicidade , Glucuronidase/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Triticum/microbiologia , Silicatos de Alumínio/farmacologia , Azospirillum brasilense/citologia , Azospirillum brasilense/efeitos dos fármacos , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Marcadores Genéticos/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Raízes de Plantas/microbiologia , Sensibilidade e Especificidade , SimbioseRESUMO
Azospirillum brasilense is a microaerophilic, plant growth-promoting bacterium, whose nitrogenase activity has been shown to be sensitive to salinity stress. Growth of A. brasilense in semi-solid medium showed that diazotrophic growth in N-free medium was relatively less sensitive to high NaCl concentrations (200-400 mM) than that in presence of NH4+. Increase in salinity stress to diazotrophic A. brasilense in the semi-solid medium led to the migration of the pellicle to deeper anaerobic zones. Assays of acetylene reduction and nifH- lacZ and nifA- lacZ fusions indicated that salinity stress inhibited nitrogenase biosynthesis more strongly than nitrogenase activity. Under salt stress, the amount of dinitrogenase reductase inactivated by ADP-ribosylation was strongly reduced, indicating that the dinitrogenase reductase ADP ribosyl transferase (DRAT) activity was also inhibited by increased NaCl concentrations. Movement of the pellicle to the anaerobic zone and inhibition of DRAT might be adaptive responses of A. brasilense to salinity stress under diazotrophic conditions. Supplementation of glycine betaine, which alleviates salt stress, partially reversed both responses.
Assuntos
Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Nitrogenase/biossíntese , Nitrogenase/metabolismo , Cloreto de Sódio/farmacologia , Anaerobiose , Proteínas de Bactérias/metabolismo , Dinitrogenase Redutase/metabolismo , Relação Dose-Resposta a Droga , Oxirredutases/metabolismo , Cloreto de Sódio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.