Morphocultural characterization, autoinducer biosynthesis and biofilm formation in rhizobacteria isolated from vegetable crops

O objetivo deste trabalho foi caracterizar e agrupar rizobactérias, isoladas de hortaliças, quanto à morfologia cultural, riqueza e diversidade e avaliar a biossíntese de autoindutores N-acil lactonas homoserinas (ALH) e a capacidade de formação de biofilmes. Sete estirpes também foram avaliadas quanto ao potencial de promoção de crescimento de Brassica oleraceae var. acephala em casa de vegetação. Para verificar a produção de ALH, foram realizados ensaios com Agrobacterium tumefaciens estirpe NT1 como sistema repórter. A formação de biofilme foi avaliada pelo cultivo do isolado em meio líquido. A promoção do crescimento foi avaliada após inoculação das estirpes em plantas de couve-de-folha pela determinação da produção de massa de matérias fresca e seca. A maior diversidade morfocultural foi encontrada entre as estirpes isoladas de couve-de-folha. De um total de 112 estirpes testadas, 13% foram positivas quanto à produção de ALH; de 91 estirpes, 96% foram capazes de formar biofilmes; e de 79 estirpes, 11% foram positivas para ambas as características. Foram observadas diferenças significativas na massa de matéria seca das raízes com inoculação de 109 UFC mL-1 da estirpe R142, que incrementou em 55% a massa de matéria seca das raízes de couve, em relação ao controle. Não há relação entre a capacidade de formar biofilme e a detecção de ALH produzidos pelas rizobactérias avaliadas.The objective of this work was to characterize and group rhizobacteria isolated from vegetable crops for culture morphology, richness and diversity, and to evaluate the biosynthesis of N-acyl homoserine lactone (AHL) autoinducers and the capacity to form biofilms. Seven strains were also assessed for their potential to promote plant growth of Brassica oleraceae var. acephala in greenhouse. To test the production of AHL, the indicator strain Agrobacterium tumefaciens NT1 was used. The formation of biofilms was evaluated by cultivating the isolates in liquid medium. Growth promotion was evaluated after the inoculation of the strains in kale plants and through determination of the fresh and dry mass production. The largest morphocultural diversity was found among the strains isolated from kale. From a total of 112 tested strains, 13% were positive for AHL production, among 91 strains, 96% were capable to form biofilms, and among 79 strains, 11% were positive for both characteristics. Significant differences were observed in root dry mass of plants inoculated with 109 UFC mL-1 of strain R142 that increased in 55% the root dry mass in comparison to the control. There is no relationship between the capacity to form biofilms and the detection of the AHL produced by the evaluated rhizobacteria

Modelagem molecular de proteínas: o caso de uma glucoronosiltransferase (GumK) de Gluconacetobacter diazotrophicus PAL5

A β-1,2-glucuronosiltransferase (GumK) de Gluconacetobacter diazotrophicus é uma proteína de 41 kDa que está envolvida na biossíntese de exopolissacarídeos. GumK é o membro da família de enzimas ativadoras de glicosiltransferases e é responsável pela adição de um resíduo de ácido UDP-glucurônico para um resíduo de manosil-(alfa-1,3)-difosfopoliprenol-celobiose para produzir glucuronil-(beta-1,2)-manosil-(difosfopoliprenol-cellobiose alfa-1,3). Para compreender melhor o mecanismo de ação das glicosiltransferases bacterianas desta família, a modelagem por homologia de GumK foi executado. A estrutura tridimensional da enzima GumK foi construída usando o programa MODELLER 9v8 e validadas com o programa Procheck. Para construir o modelo de GumK, sequência semelhanças foram encontradas no Protein Data Bank quando comparada à GumK de Xanthomonas campestris. A enzima possui dois domínios Rossmann bem definidos com uma fenda catalítica entre eles, que é uma característica típica da superfamília de glicosiltransferase B. O alinhamento de sequências entre GumK e outras glicosiltransferases mostrou várias regiões conservadas incluindo o motivo presente no sítio de ligação do substrato, todos os resíduos que entram em contato com o ligante UDP são conservadas na família GT70, o que indica que, como esperado, a região de ligação com substrato é conservada. Os dados biológicos e estruturais aqui relatados lançam luz sobre a base molecular para a seletividade nesta família de glicosiltransferases. Estas análises enfatizam o modelo tridimensional construído para GumK que representa a primeira informação estrutural para enzimas envolvidas na síntese da goma de G. diazotrophicus

The importance of denitrification performed by nitrogen-fixing bacteria used as inoculants in South America

Replacing synthetic fertilizers by biological nitrogen fixation (BNF) is regarded as an environmentally sound practice, but some diazotrophic bacteria are capable of emitting N2O by denitrification. The ability to use nitrate represents an ecological advantage for the survival of some microorganisms under O2-limiting conditions, but may contribute to increased N2O emissions. Scope The importance of denitrification performed by N2-fixing bacteria used as inoculants in South America is discussed, especially the possibility of these bacteria act as N2O source or sink. Conclusions There is no doubt of the importance of BNF as a sustainable N source for plants. Through genome investigation, we demonstrated that some strains widely used as inoculants for BNF harbor the entire denitrification pathway to reduce nitrate to N2. Others contain none, or only some of the denitrification genes, resulting in complete absence of denitrification or production of intermediates such as NO2−, NO or N2O. Evidence of differential effects of bacterial strains on soil N2O were reported, but more studies are still needed to affirm crop inoculation can be a driver for source or sink of this gas. Finally, considerations were made about BNF as an indispensable resource to indirectly mitigate greenhouse gas emissions in agroecosystems.Fil: Zilli, Jerri Édson. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Alves, Bruno Jose Rodrigues. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Rouws, Luc Felicianus Marie. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Simões-Araujo, Jean Luiz. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: de Barros Soares, Luis Henrique. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Obando Castellanos, Dolly Melissa. Universidad Nacional de Río Cuarto, Facultad de Ciencias Exactas, Físicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: O'Hara, Graham. Murdoch University; Australi

Identification of genes involved in indole-3-acetic acid biosynthesis by Gluconacetobacter diazotrophicus PAL5 strain using transposon mutagenesis

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds that the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao, cccA and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects in development of rice roots