Obtenção e avaliação de mutantes exoZ- e phbAB- envolvidos no metabolismo de polímeros de carbono em Rhizobium tropici SEMIA 4080 com potencial biotecnológico

Rhizobium tropici e outras bactérias pertencentes à ordem Rhizobiales são produtores de polissacarídeos extracelulares (EPS), que possuem a função de molécula receptora do micro simbionte, fazendo uma interação célula/célula e desencadeando o processo de nodulação. A diversidade de estruturas e composição química apresentadas pelas moléculas de EPS é refletida pela diversidade de enzimas responsáveis pela sua síntese. Neste trabalho, buscou-se a inativação através da mutagênese insercional por transposição in vitro dos genes exoZ da estirpe selvagem Rhizobium tropici SEMIA 4080 envolvidas na síntese das subunidades repetitivas do EPS e no tanden phbAB, responsáveis pela síntese do PHB, com o intuito de obter microrganismos com aspecto altamente mucoso, produtor superior de EPS em relação a estirpe selvagem. Os mutantes obtidos apresentaram colônias mucosas quando cultivados nos meios de culturas PSYA, demonstrando que estirpes de rizóbio mutantes nos genes exoZ e phbAB são capazes de formar biofilme “in vitro” e, ao avaliar a eficiência relativa na produção de EPS, o mutante 4080 Z03 apresentou o melhor resultado. Para as três amostras de EPS foi possível observar um comportamento de fluxo de líquidos pseudoplástico e, também, a influência da concentração de EPS sobre a viscosidade aparente das soluções aquosas. A estirpe selvagem e todos os mutantes induziram a formação de nódulos em feijoeiro (fenótipo Fix+), sugerindo que os genes exoZ e phbAB não estão envolvidos nos processos de infecção e formação nodular mas, sendo necessários para a fixação biológica de nitrogênioRhizobium tropici and other bacteria belonging to the order Rhizobiales are extracellular polysaccharides (EPS) producers, which possess the function of the receptor molecule to function as micro-symbiont, making an interaction cell / cell and triggering the nodulation process. The diversity of structures and chemical composition of EPS presented by molecules is reflected by the diversity of enzymes responsible for its synthesis. In this study, we sought to inactivation by insertional mutagenesis by “in vitro” transposition of genes from wild type exoZ Rhizobium tropici SEMIA 4080 involved in the synthesis of the EPS repeating subunits and phbAB tanden, responsible for synthesis of PHB, in order to obtain microrganisms with high mucosal aspect producer of EPS compared to wild type. The mutants showed mucous colonies when grown in culture media PsyA, demonstrating that mutant strains in genes exoZ and phbAB from rhizobia are able to form biofilm “in vitro”, and to evaluate the relative efficiency in the production of EPS, the mutant 4080 Z03 showed the best result. For the three samples of EPS was possible to observe a flow behavior of pseudoplastic fluids, and also the influence of EPS concentration on the apparent viscosity of aqueous solutions. The wild type and mutants induced the formation of nodules in common bean (Fix+ phenotype), suggesting that genes exoZ and phbAB are not involved in the processes of infection and nodule formation, but are necessary for nitrogen fixationCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Análise de polissacarídeos essenciais para a nodulação do feijoeiro por Rhizobium tropici cultivados em diferentes fontes de carbono

O estabelecimento da simbiose é baseado em um complexo diálogo entre o rizóbio e a planta hospedeira. Polissacarídeos de superfície de origem rizobiana aparentam ser essenciais para o processo de infecção. A composição dos EPSs das estirpes de Rhizobium tropici SEMIA 40]7 e SEMIA 4080 diferiram quantitativamente. Os quatro tipos de fontes de carbono têm efeitos diferentes na concentração da galactose e glicose, sendo maior para SEMIA 4080 cultivada em meio contendo sacarose. Mesmo a estirpe SEMIA 4080, apresentando grande quantidade de exopolissacarídeos liberado na cultura, não apresenta diferenças significativas no número de nódulos em relação às inoculações com a estirpe SEMIA 4077, sendo esta última, a que apresenta pior desempenho quanto à massa seca dos nódulos.The establishment of symbiosis is based on a complex molecular dialogue between rhizobia and host plant. Rhizobial surface polysaccharides appear to be essential for the infection processo The EPSs composition produced by Rhizobium tropici SEMIA 4077 and SEMIA 4080, differed quantitatively from one another. . Four types •of culture. media showed differential effects on the concentrations of the galactose and glucose, particularly the SEMIA 4080 strain, which was cultivated in sucrose medium. Even though the SEM IA 4080 strain produced a large quantity of EPS in culture, it was not significantly different from the number of nodules related to inoculations using strain SEMIA 4077.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Exopolysaccharides composition produced by rhizobia under different carbon sources

O objetivo deste trabalho foi avaliar o efeito de diferentes fontes de carbono na composição dos exopolissacarídeos produzidos por Rhizobium tropici SEMIA 4077 e SEMIA 4080, e o efeito da inoculação dessas estirpes na nodulação em feijoeiro. As fontes de carbono utilizadas interferiram nas concentrações de galactose e glicose dos exopolissacarídeos, que foram maior na SEMIA 4080 em meio com sacarose. Embora a SEMIA 4080 tenha liberado grande quantidade de exopolissacarídeos na cultura, não houve diferença entre as estirpes quanto ao número de nódulos do feijoeiro, independentemente da fonte de carbono utilizada no preparo do inóculo. No parâmetro matéria seca de nódulos, houve diferença entre as estirpes, independentemente da fonte de carbono utilizada no preparo do inóculo.The objective of this work was to evaluate the effect of different carbon souces on the exopolysaccharides composition produced by Rhizobium tropici SEMIA 4077 and SEMIA 4080, and the effect of these strains inoculation on the nodulation in common bean. The carbon sources used interfered on the concentrations of the galactose and glucose of exopolysaccharides, being higher in SEMIA 4080 strain when cultivated in sucrose medium. Even though SEMIA 4080 strain produced a large quantity of exopolysaccharides in culture, there was no difference between strains regarding the number of nodules, independently of the carbon source used for inoculum preparation. However, in the parameter dry weight of nodules there was significant difference between the strains, independently of the carbon sources used for inoculum preparation

Exopolysaccharides composition produced by rhizobia under different carbon sources

O objetivo deste trabalho foi avaliar o efeito de diferentes fontes de carbono na composição dos exopolissacarídeos produzidos por Rhizobium tropici SEMIA 4077 e SEMIA 4080, e o efeito da inoculação dessas estirpes na nodulação em feijoeiro. As fontes de carbono utilizadas interferiram nas concentrações de galactose e glicose dos exopolissacarídeos, que foram maior na SEMIA 4080 em meio com sacarose. Embora a SEMIA 4080 tenha liberado grande quantidade de exopolissacarídeos na cultura, não houve diferença entre as estirpes quanto ao número de nódulos do feijoeiro, independentemente da fonte de carbono utilizada no preparo do inóculo. No parâmetro matéria seca de nódulos, houve diferença entre as estirpes, independentemente da fonte de carbono utilizada no preparo do inóculo.The objective of this work was to evaluate the effect of different carbon souces on the exopolysaccharides composition produced by Rhizobium tropici SEMIA 4077 and SEMIA 4080, and the effect of these strains inoculation on the nodulation in common bean. The carbon sources used interfered on the concentrations of the galactose and glucose of exopolysaccharides, being higher in SEMIA 4080 strain when cultivated in sucrose medium. Even though SEMIA 4080 strain produced a large quantity of exopolysaccharides in culture, there was no difference between strains regarding the number of nodules, independently of the carbon source used for inoculum preparation. However, in the parameter dry weight of nodules there was significant difference between the strains, independently of the carbon sources used for inoculum preparation

Characterization of Exopolysaccharides Produced by Rhizobia Species

ABSTRACT Increasing attention has been given, over the past decades, to the production of exopolysaccharides (EPS) from rhizobia, due to their various biotechnological applications. Overall characterization of biopolymers involves evaluation of their chemical, physical, and biological properties; this evaluation is a key factor in understanding their behavior in different environments, which enables researchers to foresee their potential applications. Our focus was to study the EPS produced by Mesorhizobium huakuii LMG14107, M. loti LMG6125, M. plurifarium LMG11892,Rhizobium giardini bv. giardiniH152T, R. mongolense LMG19141, andSinorhizobium (= Ensifer)kostiense LMG19227 in a RDM medium with glycerol as a carbon source. These biopolymers were isolated and characterized by reversed-phase high-performance liquid chromatography (RP-HPLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopies. Maximum exopolysaccharide production was 3.10, 2.72, and 2.50 g L-1for the strains LMG6125, LMG19227, and LMG19141, respectively. The purified EPS revealed prominent functional reactive groups, such as hydroxyl and carboxylic, which correspond to a typical heteropolysaccharide. The EPS are composed primarily of galactose and glucose. Minor components found were rhamnose, glucuronic acid, and galacturonic acid. Indeed, from the results of techniques applied in this study, it can be noted that the EPS are species-specific heteropolysaccharide polymers composed of common sugars that are substituted by non-carbohydrate moieties. In addition, analysis of these results indicates that rhizobial EPS can be classified into five groups based on ester type, as determined from the 13C NMR spectra. Knowledge of the EPS composition now facilitates further investigations relating polysaccharide structure and dynamics to rheological properties

Production of exopolysaccharide from rhizobia with potential biotechnological and bioremediation applications

The potential use of rhizobia under controlled fermentation conditions may result in the production of new extracellular polymeric substances (EPS) having novel and superior properties that will open up new areas of industrial applications and thus increase their demand. The production of EPS and the stability of emulsions formed with soybean oil, diesel oil and toluene using different concentrations of purified EPS derived from wild-type and mutant strains of Rhizobium tropici SEMIA 4077 was investigated. The EPS was defined as a heteropolysaccharide composed of six constituent monosaccharides that displayed higher intrinsic viscosity and pseudoplastic non-Newtonian fluid behavior in an aqueous solution. The ratio between the total EPS production and the cellular biomass was 76.70 for the 4077:Z04 mutant strain and only 8.10 for the wild-type strain. The EPS produced by the wild-type R. tropici SEMIA 4077 resulted in more stable emulsions with the tested toluene than xanthan gum, and the emulsification indexes with hydrocarbons and soybean oil were higher than 50%, indicating strong emulsion-stabilizing capacity. These results demonstrate that the EPS of R. tropici strains could be attractive for use in industrial and environmental applications, as it had higher intrinsic viscosity and good emulsification activity. (C) 2015 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evaluation of the biotechnological potential of Rhizobium tropici strains for exopolysaccharide production

Rhizobium tropici, a member of the Rhizobiaceae family, has the ability to synthesize and secrete extracellular polysaccharides (EPS). Rhizobial EPS have attracted much attention from the scientific and industrial communities. Rhizobial isolates and R. tropici mutants that produced higher levels of EPS than the wildtype strain SEMIA4080 were used in the present study. The results suggested a heteropolymer structure for these EPS composed by glucose and galactose as prevailing monomer unit. All EPS samples exhibited a typical non-Newtonian and pseudoplastic fluid flow, and the aqueous solutions apparent viscosities increased in a concentration-dependent manner. These results serve as a foundation for further studies aimed at enhancing interest in the application of the MUTZC3, JAB1 and JAB6 strains with high EPS production and viscosity can be exploited for the large-scale commercial production of Rhizobial polysaccharides. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/)