RESUMO
Climate change has caused major changes in abiotic factors, with water stress as the greatest threat to agricultural production. The measures aimed at alleviating the problems caused by this limiting production factor have occurred through the adoption of sustainable strategies, especially microbial biotechnology, which uses the interactions between the microorganism and the plant, ensuring productive quality and inducing plant resistance to stresses biotic and abiotic. The objective of the present work was to evaluate the biological nitrogen fixation and the development of bean seedlings, with co-inoculation of two types of inoculants, which were subjected to water stress by different pot capacities. The experiment was conducted in a greenhouse, at Universidade Paranaense - UNIPAR, from April to June 2019. The experimental design was completely randomized (DIC), with 5 replications, 16 treatments and 80 experimental units. The cultivar used was SCS Riqueza. The parameters evaluated were pot capacity (25%, 50%, 75% and 90%); small, large and total nodules, shoot and root length, dry and fresh weight, total carbon and nitrogen. The evaluation of the morphological parameters of the bean seedlings indicated that the co- inoculation technique promoted beneficial effects for the dry mass parameters of shoot, nodule and root. The analysis of the percentage of carbon and nitrogen in the tissues of the seedlings provided an increase in the concentration of these elements in treatments that involved co-inoculation (Azospirillum brasilensis and Rhizobium tropici) with pot capacities of 25 and 75% (CV), demonstrating that the association of microorganisms is beneficial in the limiting water situation.(AU)
A mudança climática tem causado grandes mudanças nos fatores abióticos, sendo o estresse hídrico a maior ameaça à produção agrícola. As medidas destinadas a aliviar os problemas causados por este fator limitante de produção ocorreram através da adoção de estratégias sustentáveis, especialmente a biotecnologia microbiana, que utiliza as interações entre o microorganismo e a planta, garantindo a qualidade produtiva e induzindo a resistência da planta ao estresse biótico e abiótico. O objetivo do presente trabalho foi avaliar a fixação biológica de nitrogênio e o desenvolvimento de mudas de feijão, com co-inoculação de dois tipos de inoculantes, que foram submetidos ao estresse hídrico por diferentes capacidades de vaso. A experiência foi realizada em uma estufa, na Universidade Paranaense - UNIPAR, de abril a junho de 2019. O projeto experimental foi completamente randomizado (DIC), com 5 réplicas, 16 tratamentos e 80 unidades experimentais. A cultivar utilizada foi a SCS Riqueza. Os parâmetros avaliados foram a capacidade do vaso (25%, 50%, 75% e 90%); nódulos pequenos, grandes e totais, comprimento do rebento e da raiz, peso seco e fresco, carbono total e nitrogênio. A avaliação dos parâmetros morfológicos das mudas de feijão indicou que a técnica de co-inoculação promoveu efeitos benéficos para os parâmetros de massa seca do turião, nódulo e raiz. A análise da porcentagem de carbono e nitrogênio nos tecidos das mudas proporcionou um aumento na concentração destes elementos nos tratamentos que envolveram a co-inoculação (Azospirillum brasilensis e Rhizobium tropici) com capacidades de vaso de 25 e 75% (CV), demonstrando que a associação de microorganismos é benéfica na situação limite da água.(AU)
El cambio climático ha provocado importantes cambios en los factores abióticos, siendo el estrés hídrico la mayor amenaza para la producción agrícola. Las medidas encaminadas a paliar los problemas causados por este factor limitante de la producción se han producido mediante la adopción de estrategias sostenibles, especialmente la biotecnología microbiana, que utiliza las interacciones entre el microorganismo y la planta, asegurando la calidad productiva e induciendo la resistencia de la planta a los estreses bióticos y abióticos. El objetivo del presente trabajo fue evaluar la fijación biológica de nitrógeno y el desarrollo de plántulas de frijol, con la co-inoculación de dos tipos de inoculantes, que fueron sometidos a estrés hídrico por diferentes capacidades de maceta. El experimento se realizó en un invernadero, en la Universidade Paranaense - UNIPAR, de abril a junio de 2019. El diseño experimental fue completamente al azar (DIC), con 5 repeticiones, 16 tratamientos y 80 unidades experimentales. El cultivar utilizado fue SCS Riqueza. Los parámetros evaluados fueron capacidad de maceta (25%, 50%, 75% y 90%); nódulos pequeños, grandes y totales, longitud de brotes y raíces, peso seco y fresco, carbono y nitrógeno total. La evaluación de los parámetros morfológicos de las plántulas de frijol indicó que la técnica de coinoculación promovió efectos beneficiosos para los parámetros de masa seca de brotes, nódulos y raíces. El análisis del porcentaje de carbono y nitrógeno en los tejidos de las plántulas proporcionó un aumento en la concentración de estos elementos en los tratamientos que involucraron la coinoculación (Azospirillum brasilensis y Rhizobium tropici) con capacidades de maceta de 25 y 75% (CV), demostrando que la asociación de microorganismos es beneficiosa en la situación de agua limitante.(AU)
Assuntos
Azospirillum brasilense/fisiologia , Phaseolus/fisiologia , Rhizobium tropici/fisiologia , Desidratação , Fixação de Nitrogênio/fisiologiaRESUMO
Association of rhizobia with other plant growth-promoting bacteria (PGPB), such as Azospirillum, have the potential to increase crop yields. This work aimed to assess how Rhizobium tropici and Azospirillum brasilense alone or in combination, affect the growth and yields of common bean grains (Phaseolus vulgaris L.). In a field experiment, R. tropici and A. brasilense were inoculated on seeds, alone or in combination, associated or not with foliar spraying of A. brasilense. Shoot biomass, nitrogen accumulation, thousand-grain weight, and grain yield were evaluated. Application of A. brasilense, on seed or by foliar spraying, and seed inoculation of R. tropici, had an additive effect, increasing biomass and accumulated nitrogen, thousand-grain weight, and grain yield.
Assuntos
Azospirillum brasilense/fisiologia , Phaseolus/microbiologia , Folhas de Planta/microbiologia , Rhizobium tropici/fisiologia , Sementes/microbiologia , Biomassa , Nitrogênio/metabolismo , Desenvolvimento Vegetal , Folhas de Planta/crescimento & desenvolvimentoRESUMO
BACKGROUND: Reactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA-I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris. RESULTS: To corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots. CONCLUSIONS: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.
Assuntos
Perfilação da Expressão Gênica , Glomeromycota/fisiologia , NADPH Oxidases/genética , Phaseolus/genética , Phaseolus/microbiologia , Raízes de Plantas/genética , Rhizobium tropici/fisiologia , Parede Celular/metabolismo , Phaseolus/citologia , Phaseolus/enzimologia , Raízes de Plantas/microbiologia , Transdução de Sinais/genética , SimbioseRESUMO
Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.
Assuntos
Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Phaseolus/microbiologia , Rhizobium tropici/fisiologia , Proteínas de Bactérias/genética , Cobre/toxicidade , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mutagênese Insercional , Mutação , Phaseolus/efeitos dos fármacos , Phaseolus/crescimento & desenvolvimento , Phaseolus/metabolismo , Nodulação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rhizobium tropici/genética , Rhizobium tropici/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , SimbioseRESUMO
Allelopathy is a phenomenon that involves the production of secondary metabolites that influence the growth of plants and microorganisms; however, this alellopathic effect has been scarcely studied on the rhizobia-legume symbiosis. The aims of this research were 1) to assess the allelopathic potential of aqueous extracts of Ipomoea purpurea L. Roth on seed germination and root length of common bean seedlings (Phaseolus vulgaris L.), 2) to determine its effects on the in vitro growth of Rhizobium tropici CIAT899, and 3) to evaluate the allelopathic potential of I. purpurea on the growth, nodulation and physiology of common bean plants inoculated with R. tropici. After 48h, 15% of the aqueous root extract of I. purpurea stimulated seed germination, whereas 4% of the aqueous shoot extracts stimulated such germination. Both the root or shoot extracts stimulated seed germination and e root length. In vitro growth of R. tropici was inhibited as a result of the application of both aqueous extracts. The presence of I. purpurea negatively affected both the growth and physiological responses of common bean plants, and this effect was attenuated after the inoculation of R. tropici; nevertheless, this allelopathic plant affected root nodulation. Our results suggest that the symbiosis of rhizobia and roots of common bean plants is an important element for attenuating the negative effects caused by the allelopathic plant.
Assuntos
Alelopatia , Ipomoea , Phaseolus/efeitos dos fármacos , Phaseolus/microbiologia , Extratos Vegetais/farmacologia , Rhizobium tropici/fisiologia , Simbiose , Phaseolus/fisiologiaRESUMO
Bax-inhibitor 1 (BI-1) is a cell death suppressor conserved in all eukaryotes that modulates cell death in response to abiotic stress and pathogen attack in plants. However, little is known about its role in the establishment of symbiotic interactions. Here, we demonstrate the functional relevance of an Arabidopsis thaliana BI-1 homolog (PvBI-1a) to symbiosis between the common bean (Phaseolus vulgaris) and Rhizobium tropici. We show that the changes in expression of PvBI-1a observed during early symbiosis resemble those of some defence response-related proteins. By using gain- and loss-of-function approaches, we demonstrate that the overexpression of PvBI-1a in the roots of common bean increases the number of rhizobial infection events (and therefore the final number of nodules per root), but induces the premature death of nodule cells, affecting their nitrogen fixation efficiency. Nodule morphological alterations are known to be associated with changes in the expression of genes tied to defence, autophagy, and vesicular trafficking. Results obtained in the present work suggest that BI-1 has a dual role in the regulation of programmed cell death during symbiosis, extending our understanding of its critical function in the modulation of host immunity while responding to beneficial microbes.
Assuntos
Proteínas de Membrana/genética , Phaseolus/genética , Proteínas de Plantas/genética , Rhizobium tropici/fisiologia , Apoptose/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Phaseolus/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Simbiose/genéticaRESUMO
BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.
Assuntos
Adaptação Fisiológica/genética , Inoculantes Agrícolas/genética , Meio Ambiente , Genômica , Phaseolus/microbiologia , Rhizobium tropici/genética , Rhizobium tropici/fisiologia , Inoculantes Agrícolas/citologia , Inoculantes Agrícolas/metabolismo , Inoculantes Agrícolas/fisiologia , Antibacterianos/farmacologia , Transporte Biológico/genética , Sequência Conservada/genética , Farmacorresistência Bacteriana/genética , Genoma de Planta/genética , Concentração de Íons de Hidrogênio , Hidrogenase/genética , Ferro/metabolismo , Metais/farmacologia , Família Multigênica/genética , Fixação de Nitrogênio/genética , Nitrosação/genética , Pressão Osmótica , Estresse Oxidativo/genética , Phaseolus/fisiologia , Filogenia , Reguladores de Crescimento de Plantas/biossíntese , Nodulação/genética , Plasmídeos/genética , Polissacarídeos/genética , Rhizobium tropici/citologia , Rhizobium tropici/metabolismo , Especificidade da Espécie , Estresse Fisiológico/genética , Simbiose/genética , TemperaturaRESUMO
BACKGROUND: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28°C and 35°C. RESULTS: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. CONCLUSIONS: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.
Assuntos
Proteínas de Bactérias/análise , Proteoma/análise , Rhizobium tropici/química , Rhizobium tropici/fisiologia , Estresse Fisiológico , Eletroforese em Gel Bidimensional , Proteômica , Rhizobium tropici/crescimento & desenvolvimento , Rhizobium tropici/efeitos da radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
To study the interactions between a Rhizobium tropici strain and lectins isolated from the seeds of Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), a lectin fluorescence assay was performed. In addition, an experiment was designed to evaluate the effect of the two lectins on bacterial growth. Both lectins were found to bind to R. tropici cells, but the interactions were inhibited by D-mannose. Interestingly, only ConBr stimulated bacterial growth in proportion to the concentrations used (15.6-500 µg/mL), and the bacterial growth stimulation was inhibited by D-mannose as well. Structure/Function analyses by bioinformatics were carried out to evaluate the volume and carbohydrate recognition domain (CRD) configuration of ConA and ConBr. The difference of spatial arrangement and volume of CRD may indicate the variation between biological activities of both lectins. The results suggest that ConBr could be a promising tool for studies focusing on the interactions between rhizobia and host plants.
Assuntos
Canavalia/química , Lectinas de Plantas/farmacologia , Rhizobium tropici/efeitos dos fármacos , Sementes/química , Bioensaio , Relação Dose-Resposta a Droga , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Manose/farmacologia , Modelos Moleculares , Fixação de Nitrogênio/fisiologia , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Rhizobium tropici/fisiologia , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Rhizobium tropici is a well-studied legume symbiont characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH. However, high phenetic and genetic variabilities among R. tropici strains have been largely reported, with two subgroups, designated type A and B, already defined within the species. A polyphasic study comprising multilocus sequence analysis, phenotypic and genotypic characterizations, including DNA-DNA hybridization, strongly supported the reclassification of R. tropici type A strains as a novel species. Type A strains formed a well-differentiated clade that grouped with R. tropici, Rhizobium multihospitium, Rhizobium miluonense, Rhizobium lusitanum and Rhizobium rhizogenes in the phylogenies of the 16S rRNA, recA, gltA, rpoA, glnII and rpoB genes. Several phenotypic traits differentiated type A strains from all related taxa. The novel species, for which the name Rhizobium leucaenae sp. nov. is proposed, is a broad host range rhizobium being able to establish effective root-nodule symbioses with Leucaena leucocephala, Leucaena esculenta, common beans (Phaseolus vulgaris) and Gliricidia sepium. Strain CFN 299(T) (â=âUSDA 9039(T)â=âLMG 9517(T)â=âCECT 4844(T)â=âJCM 21088(T)â=âIAM 14230(T)â=âSEMIA 4083(T)â=âCENA 183(T)â=âUMR1026(T)â=âCNPSo 141(T)) is designated the type strain of Rhizobium leucaenae sp. nov.
Assuntos
Rhizobium tropici/classificação , Rhizobium tropici/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S/genética , Rhizobium tropici/fisiologia , Análise de Sequência de DNARESUMO
Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.
Assuntos
Phaseolus/fisiologia , Nodulação/genética , Raízes de Plantas/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismo , Rhizobium tropici/fisiologia , Membrana Celular/ultraestrutura , Proliferação de Células , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Morfogênese , Phaseolus/genética , Phaseolus/crescimento & desenvolvimento , Phaseolus/microbiologia , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteína Quinase C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhizobium tropici/genética , Rhizobium tropici/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Transdução de SinaisRESUMO
Ornithine lipids (OLs) are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. OLs can be hydroxylated within the secondary fatty acyl moiety and this modification has been related to increased stress tolerance. Rhizobium tropici, a nodule-forming α-proteobacterium known for its stress tolerance, forms four different OLs. Studies of the function of these OLs have been hampered due to lack of knowledge about their biosynthesis. Here we describe that OL biosynthesis increases under acid stress and that OLs are enriched in the outer membrane. Using a functional expression screen, the OL hydroxylase OlsE was identified, which in combination with the OL hydroxylase OlsC is responsible for the synthesis of modified OLs in R. tropici. Unlike described OL hydroxylations, the OlsE-catalysed hydroxylation occurs within the ornithine moiety. Mutants deficient in OlsE or OlsC and double mutants deficient in OlsC/OlsE were characterized. R. tropici mutants deficient in OlsC-mediated OL hydroxylation are more susceptible to acid and temperature stress. All three mutants lacking OL hydroxylases are affected during symbiosis.
Assuntos
Ornitina/análogos & derivados , Rhizobium tropici/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidroxilação , Lipídeos/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Mutação , Ornitina/química , Ornitina/metabolismo , Rhizobium tropici/química , Rhizobium tropici/enzimologia , Rhizobium tropici/genética , Estresse FisiológicoRESUMO
Rhizobium tropici is representative of the diversity of tropical rhizobia, besides comprising strains very effective in fixing N(2) in symbiosis with the common bean (Phaseolus vulgaris L.). The genome of a Brazilian commercial inoculant R. tropici strain (PRF 81, =SEMIA 4088), estimated at 7.85 Mb, was analyzed through a total of 9,026 shotgun reads, assembled in 1,668 phrap contigs, and covering approximately 30% of the genome. Annotation identified 2,135 coding DNA sequences (CDS), and only 57.2% have possible functions. The genome comprises a mosaic of genes, with CDS showing the highest similarities with 134 microorganisms, none of which represents more than 19% of the CDS with putative known functions. The high saprophytic capacity of PRF 81 may reside in a variety of genes related to transport, biodegradation of xenobiotics, defense, and secretion proteins, many of which were reported for the first time in the present study. Novelty was also found in nodulation (nodG, a double nodIJ system, nodT, nolF, nolG) and capsular polysaccharide genes, showing stronger similarities with Sinorhizobium (=Ensifer) than with the main symbionts of the common bean -- R. etli and R. leguminosarum -- suggesting that the original host of R. tropici might be another tropical legume or emphasizing the highly promiscuous nature of this rhizobial species.
Assuntos
Genes Bacterianos , Nodulação/genética , Rhizobium tropici/genética , Rhizobium tropici/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Genoma Bacteriano , Fixação de Nitrogênio/genética , Phaseolus/microbiologia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Simbiose/genéticaRESUMO
The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.