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1.  Complete Genome Sequence of Burkholderia phenoliruptrix BR3459a (CLA1), a Heat-Tolerant, Nitrogen-Fixing Symbiont of Mimosa flocculosa 
Journal of Bacteriology  2012;194(23):6675-6676.
The genus Burkholderia represents a challenge to the fields of taxonomy and phylogeny and, especially, to the understanding of the contrasting roles as either opportunistic pathogens or bacteria with biotechnological potential. Few genomes of nonpathogenic strains, especially of diazotrophic symbiotic bacteria, have been sequenced to improve understanding of the genus. Here, we contribute with the complete genome sequence of Burkholderia phenoliruptrix strain BR3459a (CLA1), an effective diazotrophic symbiont of the leguminous tree Mimosa flocculosa Burkart, which is endemic to South America.
PMCID: PMC3497515  PMID: 23144415
2.  Genome Sequences of Burkholderia sp. Strains CCGE1002 and H160, Isolated from Legume Nodules in Mexico and Brazil 
Journal of Bacteriology  2012;194(24):6927.
The genome sequences of Burkholderia sp. strains CCGE1002 from Mexico and H160 from Brazil, isolated from legume nodules, are reported. Their gene contents in relation to plant-microbe interactions and xenobiotic degradation are discussed.
PMCID: PMC3510596  PMID: 23209196
3.  Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicum strain CPAC 15 
BMC Genomics  2013;14:153.
Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant’s needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood.
This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time.
During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.
PMCID: PMC3608089  PMID: 23497193
Subtractive library; Differential expression of genes; Nodulation
4.  Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.) 
BMC Genomics  2012;13:735.
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.
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.
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.
PMCID: PMC3557214  PMID: 23270491
Nodulation; Nitrogen fixation; Plant-microbe interactions; Antimicrobial resistance
5.  A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots 
Genetics and Molecular Biology  2012;35(1 Suppl):348-352.
Sample preparation is a critical step in two-dimensional gel electrophoresis (2-DE) of plant tissues. Here we describe a phenol/SDS procedure that, although greatly simplified, produced well-resolved and reproducible 2-DE profiles of protein extracts from soybean [Glycine max (L.) Merril] roots. Extractions were made in three replicates using both the original and simplified procedure. To evaluate the quality of the extracted proteins, ten spots were randomly selected and identified by mass spectrometry (MS). The 2-DE gels were equally well resolved, with no streaks or smears, and no significant differences were observed in protein yield, reproducibility, resolution or number of spots. Mass spectra of the ten selected spots were compared with database entries and allowed high-quality identification of proteins. The simplified protocol described here presents considerable savings of time and reagents without compromising the quality of 2-DE protein profiles and compatibility with MS analysis, and may facilitate the progress of proteomics studies of legume-rhizobia interactions.
PMCID: PMC3392887  PMID: 22802720
2-DE; protein extraction; proteomics; soybean roots; symbiosis
6.  Proteomic profiling of Rhizobium tropici PRF 81: identification of conserved and specific responses to heat stress 
BMC Microbiology  2012;12:84.
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.
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.
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.
PMCID: PMC3502158  PMID: 22647150
7.  Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria 
Brazilian Journal of Microbiology  2012;43(2):698-710.
Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.
PMCID: PMC3768805  PMID: 24031882
Biological nitrogen fixation; Bradyrhizobium; multilocus sequence analysis; phylogeny; taxonomy
8.  The Genetics of Symbiotic Nitrogen Fixation: Comparative Genomics of 14 Rhizobia Strains by Resolution of Protein Clusters  
Genes  2012;3(1):138-166.
The symbiotic relationship between legumes and nitrogen fixing bacteria is critical for agriculture, as it may have profound impacts on lowering costs for farmers, on land sustainability, on soil quality, and on mitigation of greenhouse gas emissions. However, despite the importance of the symbioses to the global nitrogen cycling balance, very few rhizobial genomes have been sequenced so far, although there are some ongoing efforts in sequencing elite strains. In this study, the genomes of fourteen selected strains of the order Rhizobiales, all previously fully sequenced and annotated, were compared to assess differences between the strains and to investigate the feasibility of defining a core ‘symbiome’—the essential genes required by all rhizobia for nodulation and nitrogen fixation. Comparison of these whole genomes has revealed valuable information, such as several events of lateral gene transfer, particularly in the symbiotic plasmids and genomic islands that have contributed to a better understanding of the evolution of contrasting symbioses. Unique genes were also identified, as well as omissions of symbiotic genes that were expected to be found. Protein comparisons have also allowed the identification of a variety of similarities and differences in several groups of genes, including those involved in nodulation, nitrogen fixation, production of exopolysaccharides, Type I to Type VI secretion systems, among others, and identifying some key genes that could be related to host specificity and/or a better saprophytic ability. However, while several significant differences in the type and number of proteins were observed, the evidence presented suggests no simple core symbiome exists. A more abstract systems biology concept of nitrogen fixing symbiosis may be required. The results have also highlighted that comparative genomics represents a valuable tool for capturing specificities and generalities of each genome.
PMCID: PMC3899959
nitrogen fixation; symbiosis; Rhizobiales; genome; comparative genomics; nodulation genes; secretion systems
9.  Limited vegetative compatibility as a cause of somatic recombination in Trichoderma pseudokoningii 
Brazilian Journal of Microbiology  2011;42(4):1625-1637.
With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.
PMCID: PMC3768707  PMID: 24031797
Somatic recombination; Limited vegetative compatibility; Trichoderma pseudokoningii; Parasexuality
10.  Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses 
Pedrosa, Fábio O. | Monteiro, Rose Adele | Wassem, Roseli | Cruz, Leonardo M. | Ayub, Ricardo A. | Colauto, Nelson B. | Fernandez, Maria Aparecida | Fungaro, Maria Helena P. | Grisard, Edmundo C. | Hungria, Mariangela | Madeira, Humberto M. F. | Nodari, Rubens O. | Osaku, Clarice A. | Petzl-Erler, Maria Luiza | Terenzi, Hernán | Vieira, Luiz G. E. | Steffens, Maria Berenice R. | Weiss, Vinicius A. | Pereira, Luiz F. P. | Almeida, Marina I. M. | Alves, Lysangela R. | Marin, Anelis | Araujo, Luiza Maria | Balsanelli, Eduardo | Baura, Valter A. | Chubatsu, Leda S. | Faoro, Helisson | Favetti, Augusto | Friedermann, Geraldo | Glienke, Chirlei | Karp, Susan | Kava-Cordeiro, Vanessa | Raittz, Roberto T. | Ramos, Humberto J. O. | Ribeiro, Enilze Maria S. F. | Rigo, Liu Un | Rocha, Saul N. | Schwab, Stefan | Silva, Anilda G. | Souza, Eliel M. | Tadra-Sfeir, Michelle Z. | Torres, Rodrigo A. | Dabul, Audrei N. G. | Soares, Maria Albertina M. | Gasques, Luciano S. | Gimenes, Ciela C. T. | Valle, Juliana S. | Ciferri, Ricardo R. | Correa, Luiz C. | Murace, Norma K. | Pamphile, João A. | Patussi, Eliana Valéria | Prioli, Alberto J. | Prioli, Sonia Maria A. | Rocha, Carmem Lúcia M. S. C. | Arantes, Olívia Márcia N. | Furlaneto, Márcia Cristina | Godoy, Leandro P. | Oliveira, Carlos E. C. | Satori, Daniele | Vilas-Boas, Laurival A. | Watanabe, Maria Angélica E. | Dambros, Bibiana Paula | Guerra, Miguel P. | Mathioni, Sandra Marisa | Santos, Karine Louise | Steindel, Mario | Vernal, Javier | Barcellos, Fernando G. | Campo, Rubens J. | Chueire, Ligia Maria O. | Nicolás, Marisa Fabiana | Pereira-Ferrari, Lilian | da Conceição Silva, José L. | Gioppo, Nereida M. R. | Margarido, Vladimir P. | Menck-Soares, Maria Amélia | Pinto, Fabiana Gisele S. | Simão, Rita de Cássia G. | Takahashi, Elizabete K. | Yates, Marshall G. | Souza, Emanuel M. | Richardson, Paul M.
PLoS Genetics  2011;7(5):e1002064.
The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.
Author Summary
In this work we describe the genome of H. seropedicae SmR1, a bacterium capable of fixing nitrogen and promoting the growth of important plant crops such as maize, rice, and sugar cane. Several investigations have shown that H. seropedicae supplies fixed nitrogen to the associated plant and increases grain productivity up to 50%. In the genome of H. seropedicae, we identified all the genes involved in the nitrogen fixation process and its regulation and, in addition, genes potentially involved in the establishment of efficient interaction with the host plant. Our analyses also revealed that this bacterium has a highly versatile metabolism capable of synthesizing and degrading a large number of organic and inorganic compounds. We believe that the knowledge of the genome of this bacterium will direct research to a better understanding of this important endophytic organism and allow the construction of new strains with enhanced agronomic efficiency.
PMCID: PMC3093359  PMID: 21589895
11.  Genomic and evolutionary comparisons of diazotrophic and pathogenic bacteria of the order Rhizobiales 
BMC Microbiology  2010;10:37.
Species belonging to the Rhizobiales are intriguing and extensively researched for including both bacteria with the ability to fix nitrogen when in symbiosis with leguminous plants and pathogenic bacteria to animals and plants. Similarities between the strategies adopted by pathogenic and symbiotic Rhizobiales have been described, as well as high variability related to events of horizontal gene transfer. Although it is well known that chromosomal rearrangements, mutations and horizontal gene transfer influence the dynamics of bacterial genomes, in Rhizobiales, the scenario that determine pathogenic or symbiotic lifestyle are not clear and there are very few studies of comparative genomic between these classes of prokaryotic microorganisms trying to delineate the evolutionary characterization of symbiosis and pathogenesis.
Non-symbiotic nitrogen-fixing bacteria and bacteria involved in bioremediation closer to symbionts and pathogens in study may assist in the origin and ancestry genes and the gene flow occurring in Rhizobiales. The genomic comparisons of 19 species of Rhizobiales, including nitrogen-fixing, bioremediators and pathogens resulted in 33 common clusters to biological nitrogen fixation and pathogenesis, 15 clusters exclusive to all nitrogen-fixing bacteria and bacteria involved in bioremediation, 13 clusters found in only some nitrogen-fixing and bioremediation bacteria, 01 cluster exclusive to some symbionts, and 01 cluster found only in some pathogens analyzed. In BBH performed to all strains studied, 77 common genes were obtained, 17 of which were related to biological nitrogen fixation and pathogenesis. Phylogenetic reconstructions for Fix, Nif, Nod, Vir, and Trb showed possible horizontal gene transfer events, grouping species of different phenotypes.
The presence of symbiotic and virulence genes in both pathogens and symbionts does not seem to be the only determinant factor for lifestyle evolution in these microorganisms, although they may act in common stages of host infection. The phylogenetic analysis for many distinct operons involved in these processes emphasizes the relevance of horizontal gene transfer events in the symbiotic and pathogenic similarity.
PMCID: PMC2907836  PMID: 20144182
12.  Genetic diversity of indigenous common bean (Phaseolus vulgaris L.) rhizobia from the state of Minas Gerais, Brazil 
Brazilian Journal of Microbiology  2009;40(4):852-856.
We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.
PMCID: PMC3768583  PMID: 24031433
BOX-PCR; Phaseolus vulgaris; Rhizobium
13.  Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil▿  
The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N2-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.
PMCID: PMC1855619  PMID: 17308185
14.  Genetic Characterization of Soybean Rhizobia in Paraguay† 
Applied and Environmental Microbiology  2000;66(11):5099-5103.
The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.
PMCID: PMC92426  PMID: 11055970

Results 1-14 (14)