Bradyrhizobium elkanii nod regulon: insights through genomic analysis

A successful symbiotic relationship between soybean [Glycine max (L.) Merr.] and Bradyrhizobium species requires expression of the bacterial structural nod genes that encode for the synthesis of lipochitooligosaccharide nodulation signal molecules, known as Nod factors (NFs). Bradyrhizobium diazoefficiens USDA 110 possesses a wide nodulation gene repertoire that allows NF assembly and modification, with transcription of the nodYABCSUIJnolMNOnodZ operon depending upon specific activators, i.e., products of regulatory nod genes that are responsive to signaling molecules such as flavonoid compounds exuded by host plant roots. Central to this regulatory circuit of nod gene expression are NodD proteins, members of the LysR-type regulator family. In this study, publicly available Bradyrhizobium elkanii sequenced genomes were compared with the closely related B. diazoefficiens USDA 110 reference genome to determine the similarities between those genomes, especially with regards to the nod operon and nod regulon. Bioinformatics analyses revealed a correlation between functional mechanisms and key elements that play an essential role in the regulation of nod gene expression. These analyses also revealed new genomic features that had not been clearly explored before, some of which were unique for some B. elkanii genomes

Application of representational difference analysis to identify genomic differences between Bradyrhizobium elkanii and B. Japonicum species

Bradyrhizobium elkanii is successfully used in the formulation of commercial inoculants and, together with B. japonicum, it fully supplies the plant nitrogen demands. Despite the similarity between B. japonicum and B. elkanii species, several works demonstrated genetic and physiological differences between them. In this work Representational Difference Analysis (RDA) was used for genomic comparison between B. elkanii SEMIA 587, a crop inoculant strain, and B. japonicum USDA 110, a reference strain. Two hundred sequences were obtained. From these, 46 sequences belonged exclusively to the genome of B. elkanii strain, and 154 showed similarity to sequences from B. japonicum genome. From the 46 sequences with no similarity to sequences from B. japonicum, 39 showed no similarity to sequences in public databases and seven showed similarity to sequences of genes coding for known proteins. These seven sequences were divided in three groups: similar to sequences from other Bradyrhizobium strains, similar to sequences from other nitrogen-fixing bacteria, and similar to sequences from non nitrogen-fixing bacteria. These new sequences could be used as DNA markers in order to investigate the rates of genetic material gain and loss in natural Bradyrhizobium strains

Avaliação da possibilidade de ocorrência de transformação natural de Azospirillum brasilense com DNA de plantas contendo o gene de resistência à canamicina

There is a considerable concern about the possibility of a horizontal transfer of antibiotic resistance genes from genetically modified crops to soil bacterial community. One mechanism of gene transfer in bacteria that allows uptake of genetic material from diverged species is the natural transformation. In this work, the recipient strain for natural transformation was Azospirillium brasilense, a nitrogen fixing bacteria. Tobacco transgenic plants containing the nptII gene (kanamycin resistance) were used as donor of the transgenic material. A 12 months period of contact between the transgenic plants and the bacteria was evaluated each two months by PCR analysis of the DNA extracted from the bacterial pellicle grown on selective NFbHP medium. The PCR reactions were performed with oligonucleotides for nptII and nifH genes. No amplification of nptII gene was observed until the end of the experiment. A restriction analysis with TaqI of the purified DNA fragments of nifH PCR was performed to confirm the presence of A. brasilense in the cultures. The bacteria were also spread on NFbHP plates containing kanamycin and no colonies were observed after 72 h of growth. On the contrary, on the plates containing only ampicilin the colonies were similar to those obtained with the control plates. The results obtained could be useful to develop a protocol to evaluate the effects of transgenic plants on the soil microorganisms.Existe uma grande preocupação sobre a possibilidade de transferência horizontal de genes de resistência a antibióticos de plantas geneticamente transformadas para bactérias presentes nos solos. Um mecanismo de transferência gênica em bactérias, que permite a aquisição de material genético proveniente de espécies diferentes, é a transformação natural. Nesse trabalho, Azospirillum brasilense, uma bactéria fixadora de nitrogênio, foi utilizada como receptora para a transformação natural. Plantas de tabaco transgênicas, contendo o gene nptII (resistência à canamicina) foram utilizadas como doadoras de material genético. Um período de contato de 12 meses entre as plantas transgênicas e as bactérias foi avaliado, com coletas amostrais a cada dois meses, através de análises por PCR do DNA extraído de películas bacterianas formadas em meio seletivo NFbHP. As reações de PCR foram realizadas com oligonucleotídeos específicos para os genes nptII e nifH. Nenhuma banda de amplificação correspondente ao gene nptII foi observada até o final do experimento. Os fragmentos amplificados a partir da reação para o gene nifH foram clivados com TaqI, para confirmar a presença de A. brasilense nas culturas. As bactérias também foram espalhadas em placas contendo meio NFbHP e canamicina e não foram observadas colônias após um período de 72 h de incubação. Do contrário, nas placas contendo apenas ampicilina as colônias foram similares àquelas obtidas com as placas controle. Os resultados obtidos poderão ser úteis na elaboração de um protocolo de monitoramento de possíveis efeitos de plantas trasgênicas nos microrganismos do solo

Pangenome inventory of Burkholderia sensu lato, Burkholderia sensu stricto, and the Burkholderia cepacia complex reveals the uniqueness of Burkholderia catarinensis

Here the pangenome analysis of Burkholderia sensu lato (s.l.) was performed for the first time, together with an updated analysis of the pangenome of Burkholderia sensu stricto, and Burkholderia cepacia complex (Bcc) focusing on the Bcc B. catarinensis specific features of its re-sequenced genome. The pangenome of Burkholderia s.l., Burkholderia s.s., and of the Bcc was open, composed of more than 96% of accessory genes, and more than 62% of unknown genes. Functional annotations showed that secondary metabolism genes belonged to the variable portion of genomes, which might explain their production of several compounds with varied bioactivities. Taken together, this work showed the great variability and uniqueness of these genomes and revealed an underexplored unknown potential in poorly characterized genes. Regarding B. catarinensis 89T, its genome harbors genes related to hydrolases production and plant growth promotion. This draft genome will be valuable for further investigation of its biotechnological potentials

orfatrAb - Caracterização molecular do gene que codifica um ativador de transcrição da família LysR em Azospirillum brasilense

Nitrogen, which is a component of several biomolecules such as nucleic acids and amino acids, is abundant in the atmosphere where, in its gaseous form (N 2 ), it is unavailable for most organisms. However, there are microorganisms, called diazotrophs that are able to convert the atmospheric nitrogen into an assimilable form to other organisms through a process called biological nitrogen fixation. Bacteria of the genus Azospirillum are diazotrophs, free-living or endophytic that are able to associate with grasses like wheat, rice, sugar cane, etc. One of the most used tools for the identification of genes involved in the nitrogen fixation in Azospirillum was the site-direct mutagenesis using the Tn5 transposon. This methodology allowed the isolation of one A. brasilense transconjugant with a capacity of biological nitrogen fixation in vitro that was higher than that of the wild type strain. In the place of transposon insertion into the DNA of this mutant two open reading frames were identified, orfatrAb and orf281, which was interrupted by Tn5. The aim of this work was to determinate the orfatrAb complete nucleotide sequence and the corresponding amino acid sequence which showed great similarity, especially in the amino-terminus, with proteins of the LysR family of transcriptional activators. ORFAtrAb has 297 residues, molecular weight of 33,581.6 kD, isoeletric point of 7.26 and instability index of 33.29. Sequence consensus elements of gene activator proteins involved in the metabolism and in the biological nitrogen fixation were identified in the orfatrAb promoter region.O nitrogênio, elemento-chave na composição de diversas biomoléculas como aminoácidos e ácidos nucléicos, é abundante na atmosfera, onde, sob forma de N 2 , não está disponível para a maioria dos seres vivos. Entretanto, existem microrganismos, chamados de diazotróficos, que são capazes de converter o nitrogênio atmosférico em formas assimiláveis para os outros organismos, através do processo de Fixação Biológica do Nitrogênio. Bactérias do gênero Azospirillum são microrganismos diazotróficos de vida livre ou endofíticos, que se associam com gramíneas como trigo, arroz, cana-de-açúcar, entre outras. Uma das ferramentas mais utilizadas na identificação de genes envolvidos na fixação de nitrogênio em Azospirillum foi a mutagênese sítio-dirigida com o transposon Tn5. Essa metodologia possibilitou o isolamento de um transconjugante de A. brasilense, cuja capacidade de fixação de nitrogênio in vitro foi bastante elevada em relação à da linhagem de tipo selvagem. No local de inserção do transposon no DNA desse transconjugante foram encontradas duas fases abertas de leitura, orfartAb e orf281, tendo sido, essa última, interrompida pelo Tn5. O objetivo deste trabalho foi determinar a seqüência de nucleotídeos de orfatrAb, e, a partir desta, a seqüência de aminoácidos, a qual apresentou grande similaridade, principalmente em sua região amino-terminal, com proteínas da família de ativadores de transcrição LysR. ORFatrAb possui 297 resíduos, peso molecular de 33.581,6 kDa, ponto isoelétrico de 7,26 e índice de instabilidade de 33,29. Na região reguladora de orfatrAb foram iden-tificados elementos de seqüência consensuais para a ligação de proteínas ativadoras de genes envolvidos no metabolismo e na fixação biológica do nitrogênio

Efficiency of probiotic traits in plant inoculation is determined by environmental constrains

Management of the soil microbial community to increase crop productivity is one of the main challenges of modern agriculture, and bacterial inoculants can help to overcome this challenge. In this work, two plant growth-promoting (PGP) bacteria were evaluated under contrasting soil conditions, in order to test a rhizosphere ecology model. This model states that plants select for phosphate (P) solubilizers in poor nutrient soils and Indolic Compounds (ICs) producers in rich nutrient soils. Rice plants were single- or co-inoculated with strains of Burkholderia and Enterobacter genera in clayey and sandy soils. Diversity gradients were generated in each soil type using the dilution-to-extinction approach. Inoculant survival, colonization, and effect on plant biomass were evaluated, besides P solubilization and ICs production from both rhizospheric and endophytic bacterial communities. The PGP efficiency of the Burkholderia strain was highest in the sandy soil, which had the highest bacterial P solubilization potential, whereas the PGP efficiency of the Enterobacter strain was highest in the clayey soil, which had the highest bacterial ICs production potential. These behaviors occurred as hypothesized by the model, which can be useful for PGPB testing and bioprospection. We highlight a strong dependency of the Enterobacter strain on the diversity level as the most critical factor affecting PGP efficiency, possibly related to the increased influence of keystone taxa in lower diversity as indicated by network analysis

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species

Microbiota do solo: a diversidade invisível e a sua importância

Micro-organismos estão presentes em todos os lugares da Terra que suportam vida. Os solos apresentam maior quantidade e diversidade bacteriana do que a água ou o ar, devido a sua heterogeneidade de substratos. Estima-se que há um milhão de espécies de bactérias em 30 gramas de solos florestais e que haverá pelo menos um bilhão de espécies em todo o mundo. Apesar de constituírem somente de 1 a 4% do carbono total e ocuparem menos de 5% do espaço poroso do solo, a diversidade e a quantidade de micro-organismos nesse ambiente é bastante elevada. Entretanto, como o solo é normalmente um ambiente estressante, limitado por nutrientes, somente 15 a 30% das bactérias e 10% dos fungos se encontram em estado ativo. Os componentes microbianos vivos do solo são também denominados de biomassa microbiana e as bactérias e fungos respondem por cerca de 90% da atividade microbiana do solo. A diversidade microbiana se encontra diretamente relacionada com um conjunto de fatores abióticos (atmosfera, temperatura, água, pH, potencial redox, fontes nutricionais, entre outros) e bióticos (genética microbiana, a interação entre os micro-organismos, entre outros), que permite o desenvolvimento microbiano e a estruturação da comunidade viva dos solos. A interação entre esses fatores influencia diretamente a ecologia, a atividade e a dinâmica populacional de micro-organismos no solo. Muitas transformações críticas dos principais ciclos biogeoquímicos da biosfera ocorrem nos solos e são facilitadas pelos organismos que nele vivem. Apesar da importância de todos os micro-organismos que compõem a microbiota dos solos, esse trabalho tratará mais detalhadamente da comunidade bacteriana desse ambiente e o seu papel no desenvolvimento das plantas e nos ciclos biogeoquímicos. Além disso, esse trabalho aborda os principais métodos de análise de comunidades bacterianas dos solos mais utilizados atualmente

Fungistase a fusarium graminearum de um solo submetido a diferentes sistemas de manejo

Soil management, in terms of tillage and cropping systems, strongly influences the biological properties of soil involved in the suppression of plant diseases. Fungistasis mediated by soil microbiota is an important component of disease-suppressive soils. We evaluated the influence of different management systems on fungistasis against Fusarium graminearum, the relationship of fungistasis to the bacterial profile of the soil, and the possible mechanisms involved in this process. Samples were taken from a long-term experiment set up in a Paleudult soil under conventional tillage or no-tillage management and three cropping systems: black oat (Avena strigose L.) + vetch (Vicia sativa L.)/maize (Zea mays L.) + cowpea (Vigna sinensis L.), black oat/maize, and vetch/maize. Soil fungistasis was evaluated in terms of reduction of radial growth of F. graminearum, and bacterial diversity was assessed using ribosomal intergenic spacer analysis (RISA). A total of 120 bacterial isolates were obtained and evaluated for antibiosis, and production of volatile compounds and siderophores. No-tillage soil samples showed the highest level of F. graminearum fungistasis by sharply reducing the development of this pathogen. Of the cropping systems tested, the vetch + black oat/maize + cowpea system showed the highest fungistasis and the oat/maize system showed the lowest. The management system also affected the genetic profile of the bacteria isolated, with the systems from fungistatic soils showing greater similarity. Although there was no clear relationship between soil management and the characteristics of the bacterial isolates, we may conclude that antibiosis and the production of siderophores were the main mechanisms accounting for fungistasis.O manejo, em termos de preparo e sistemas de culturas, exerce forte influência sobre as propriedades do solo; e essas, por sua vez, condicionam o seu potencial supressivo a doenças de plantas. Nesse contexto, a fungistase é um dos componentes da supressividade, sendo um processo mediado pela microbiota do solo. Este trabalho teve como objetivo avaliar a influência de diferentes sistemas de manejo sobre a fungistase ao fungo fitopatogênico Fusarium graminearum, a sua relação com o perfil bacteriano do solo e os possíveis mecanismos de ação envolvidos no processo. As avaliações foram realizadas a partir de amostras de um Argissolo Vermelho distrófico, coletadas em um experimento de manejo de longa duração, conduzido sob plantio direto e preparo convencional e cultivado com os sistemas de culturas aveia + vica/milho + caupi, vica/milho e aveia/milho. A fungistase do solo foi avaliada em termos de redução do crescimento radial do fungo testado. O perfil bacteriano foi avaliado pelo polimorfismo do espaço intergênico do 16S rRNA (RISA). Foram obtidos 120 isolados bacterianos, que foram avaliados quanto à antibiose e à produção de compostos voláteis e de sideróforos. As amostras provenientes de plantio direto apresentaram maior fungistase a F. graminearum, reduzindo acentuadamente o desenvolvimento do patógeno. Também foi observada maior inibição do patógeno nas amostras de solo cultivadas sob o sistema aveia + vica/milho + caupi, enquanto o sistema aveia/milho foi o menos antagonista. O perfil genético bacteriano do solo também foi influenciado pelos sistemas de manejo, havendo semelhança entre os sistemas mais antagonistas. Embora não tenha sido identificada uma relação entre os sistemas de manejo e as características dos isolados, pode-se concluir que a antibiose e a produção de sideróforos foram os mecanismos que mais contribuíram para a fungistase