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1.  Genome Sequence of Paraburkholderia nodosa Strain CNPSo 1341, a N2-Fixing Symbiont of the Promiscuous Legume Phaseolus vulgaris 
Genome Announcements  2016;4(6):e01073-16.
Paraburkholderia nodosa CNPSo 1341 is a N2-fixing symbiont of Phaseolus vulgaris isolated from an undisturbed soil of the Brazilian Cerrado. Its draft genome contains 8,614,032 bp and 8,068 coding sequences (CDSs). Nodulation and N2-fixation genes were clustered in the genome that also contains several genes of secretion systems and quorum sensing.
doi:10.1128/genomeA.01073-16
PMCID: PMC5095457  PMID: 27811087
2.  Genome of Rhizobium leucaenae strains CFN 299T and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions 
BMC Genomics  2016;17:534.
Background
Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici “group” are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian “Cerrados” soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299T and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions.
Results
The genomes of R. leucaenae strains CFN 299T and CPAO 29.8 were estimated at 6.7–6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299T is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different.
Conclusion
A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899T and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2859-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-016-2859-z
PMCID: PMC4971678  PMID: 27485828
Stress tolerance; Biological nitrogen fixation; Nodulation; Nod factors; Symbioses; Secretion systems
3.  Genome Sequence of Bradyrhizobium stylosanthis Strain BR 446T, a Nitrogen-Fixing Symbiont of the Legume Pasture Stylosanthes guianensis 
Genome Announcements  2016;4(3):e00631-16.
Bradyrhizobium stylosanthis BR 446T is a nitrogen-fixing symbiont of the tropical legume pasture Stylosanthes guianensis. Its draft genome contains 8,801,717 bp and 8,239 coding sequences (CDSs). Several putative genes that might confer high competitiveness and saprophytic capacity under the stressful conditions of tropical soils were identified in the genome.
doi:10.1128/genomeA.00631-16
PMCID: PMC4929517  PMID: 27365354
4.  Draft Genome Sequence of Pseudomonas fluorescens Strain ET76, Isolated from Rice Rhizosphere in Northwestern Morocco 
Genome Announcements  2016;4(3):e00356-16.
Pseudomonas fluorescens ET76 was isolated from rice rhizosphere in northwestern Morocco. Its draft genome was estimated to be 6,681,652 bp with 5,789 coding sequences (CDSs). Genes encoding for type I to VI secretion systems, PvdQ, proteases, siderophores, hydrogen cyanide synthase, ACC-deaminase, among others, highlight its potential use in biological control of plant pathogens.
doi:10.1128/genomeA.00356-16
PMCID: PMC4889006  PMID: 27198014
5.  Shifts in taxonomic and functional microbial diversity with agriculture: How fragile is the Brazilian Cerrado? 
BMC Microbiology  2016;16:42.
Background
The Cerrado—an edaphic type of savannah— comprises the second largest biome of the Brazilian territory and is the main area for grain production in the country, but information about the impact of land conversion to agriculture on microbial diversity is still scarce. We used a shotgun metagenomic approach to compare undisturbed (native) soil and soils cropped for 23 years with soybean/maize under conservation tillage—“no-till” (NT)—and conventional tillage (CT) systems in the Cerrado biome.
Results
Soil management and fertilizer inputs with the introduction of agriculture improved chemical properties, but decreased soil macroporosity and microbial biomass of carbon and nitrogen. Principal coordinates analyses confirmed different taxonomic and functional profiles for each treatment. There was predominance of the Bacteria domain, especially the phylum Proteobacteria, with higher numbers of sequences in the NT and CT treatments; Archaea and Viruses also had lower numbers of sequences in the undisturbed soil. Within the Alphaproteobacteria, there was dominance of Rhizobiales and of the genus Bradyrhizobium in the NT and CT systems, attributed to massive inoculation of soybean, and also of Burkholderiales. In contrast, Rhizobium, Azospirillum, Xanthomonas, Pseudomonas and Acidobacterium predominated in the native Cerrado. More Eukaryota, especially of the phylum Ascomycota were detected in the NT. The functional analysis revealed lower numbers of sequences in the five dominant categories for the CT system, whereas the undisturbed Cerrado presented higher abundance.
Conclusion
High impact of agriculture in taxonomic and functional microbial diversity in the biome Cerrado was confirmed. Functional diversity was not necessarily associated with taxonomic diversity, as the less conservationist treatment (CT) presented increased taxonomic sequences and reduced functional profiles, indicating a strategy to try to maintain soil functioning by favoring taxa that are probably not the most efficient for some functions. Our results highlight that underneath the rustic appearance of the Cerrado vegetation there is a fragile soil microbial community.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-016-0657-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-016-0657-z
PMCID: PMC4794851  PMID: 26983403
Shotgun metagenome; Soil microbiome; Functional biodiversity; Soil management; No-tillage; Cerrado
6.  RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt 
BMC Genomics  2016;17:198.
Background
Rhizobium tropici strain CIAT 899 establishes effective symbioses with several legume species, including Phaseolus vulgaris and Leucaena leucocephala. This bacterium synthesizes a large variety of nodulation factors in response to nod-gene inducing flavonoids and, surprisingly, also under salt stress conditions. The aim of this study was to identify differentially expressed genes in the presence of both inducer molecules, and analyze the promoter regions located upstream of these genes.
Results
Results obtained by RNA-seq analyses of CIAT 899 induced with apigenin, a nod gene-inducing flavonoid for this strain, or salt allowed the identification of 19 and 790 differentially expressed genes, respectively. Fifteen of these genes were up-regulated in both conditions and were involved in the synthesis of both Nod factors and indole-3-acetic acid. Transcription of these genes was presumably activated through binding of at least one of the five NodD proteins present in this strain to specific nod box promoter sequences when the bacterium was induced by both apigenin and salt. Finally, under saline conditions, many other transcriptional responses were detected, including an increase in the transcription of genes involved in trehalose catabolism, chemotaxis and protein secretion, as well as ribosomal genes, and a decrease in the transcription of genes involved in transmembrane transport.
Conclusions
To our knowledge this is the first time that a transcriptomic study shows that salt stress induces the expression of nodulation genes in the absence of flavonoids. Thus, in the presence of both nodulation inducer molecules, apigenin and salt, R. tropici CIAT 899 up-regulated the same set of symbiotic genes. It could be possible that the increases in the transcription levels of several genes related to nodulation under saline conditions could represent a strategy to establish symbiosis under abiotic stressing conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2543-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-016-2543-3
PMCID: PMC4782375  PMID: 26951045
RNA-seq; Rhizobium tropici CIAT 899; Nodulation; Nod factors; Lipochitooligosaccharides; Apigenin; Salt stress
7.  Draft Genome Sequence of Pantoea ananatis Strain AMG521, a Rice Plant Growth-Promoting Bacterial Endophyte Isolated from the Guadalquivir Marshes in Southern Spain 
Genome Announcements  2016;4(1):e01681-15.
The rice endophyte Pantoea ananatis AMG521 shows several plant growth-promoting properties and promotes rice yield increases. Its draft genome was estimated at 4,891,568 bp with 4,704 coding sequences (CDS). The genome encodes genes for N-acylhomoserine lactone (AHL) synthases, AHL hydrolases, hyperadherence (yidQ, yidP, and yidR), fusaric acid resistance, and oxidation of lignin, highlighting its biotechnological potential.
doi:10.1128/genomeA.01681-15
PMCID: PMC4759065  PMID: 26893418
8.  Accessing inoculation methods of maize and wheat with Azospirillum brasilense 
AMB Express  2016;6:3.
The utilization of inoculants containing Azospirillum is becoming more popular due to increasing reports of expressive gains in grain yields. However, incompatibility with pesticides used in seed treatments represents a main limitation for a successful inoculation. Therefore, in this study we searched for alternatives methods for seed inoculation of maize and wheat, aiming to avoid the direct contact of bacteria with pesticides. Different doses of inoculants containing Azospirillum brasilense were employed to perform inoculation in-furrow, via soil spray at sowing and via leaf spray after seedlings had emerged, in comparison to seed inoculation. Experiments were conducted first under greenhouse controlled conditions and then confirmed in the field at different locations in Brazil. In the greenhouse, most parameters measured responded positively to the largest inoculant dose used in foliar sprays, but benefits could also be observed from both in-furrow and soil spray inoculation. However, our results present evidence that field inoculation with plant-growth promoting bacteria must consider inoculant doses, and point to the need of fine adjustments to avoid crossing the threshold of growth stimulation and inhibition. All inoculation techniques increased the abundance of diazotrophic bacteria in plant tissues, and foliar spray improved colonization of leaves, while soil inoculations favored root and rhizosphere colonization. In field experiments, inoculation with A. brasilense allowed for a 25 % reduction in the need for N fertilizers. Our results have identified alternative methods of inoculation that were as effective as the standard seed inoculation that may represent an important strategy to avoid the incompatibility between inoculant bacteria and pesticides employed for seed treatment.
doi:10.1186/s13568-015-0171-y
PMCID: PMC4710622  PMID: 26759120
Azospirillum brasilense; Biological nitrogen fixation; Leaf spray; Plant growth promoting bacteria; Soil spray; Triticum aestivum L.; Zea mays L.
9.  Genome Sequence of Bradyrhizobium viridifuturi Strain SEMIA 690T, a Nitrogen-Fixing Symbiont of Centrosema pubescens 
Genome Announcements  2015;3(6):e01481-15.
SEMIA 690T is a nitrogen-fixing symbiont of Centrosema pubescens, and comprises the recently described species Bradyrhizobium viridifuturi. Its draft genome indicates that it belongs to the Bradyrhizobium elkanii superclade. SEMIA 690T carries two copies of the regulatory nodD gene, and the nod and nif operons resemble those of Bradyrhizobium diazoefficiens.
doi:10.1128/genomeA.01481-15
PMCID: PMC4683235  PMID: 26679590
10.  Genome Sequence of Bradyrhizobium tropiciagri Strain CNPSo 1112T, Isolated from a Root Nodule of Neonotonia wightii 
Genome Announcements  2015;3(6):e01482-15.
CNPSo 1112T is a nitrogen-fixing symbiont of perennial soybean, a tropical legume forage. Its draft genome indicates a large genome with a circular chromosome and 9,554 coding sequences (CDSs). Operons of nodulation, nitrogen fixation, and uptake hydrogenase were present in the symbiotic island, and the genome encompasses several CDSs of stress tolerance.
doi:10.1128/genomeA.01482-15
PMCID: PMC4683236  PMID: 26679591
11.  Maize growth promotion by inoculation with Azospirillum brasilense and metabolites of Rhizobium tropici enriched on lipo-chitooligosaccharides (LCOs) 
AMB Express  2015;5:71.
There is an increasing interest in the development and use of inoculants carrying plant growth-promoting bacteria (PGPB) in crops of agronomic interest. The great majority of the inoculants commercialized worldwide contain rhizobia for legume crops, but the use of PGPB as Azospirillum spp. for non-legume is expanding, as well as of inoculants combining microorganisms and microbial metabolites. In this study we evaluated the effects of inoculants containing Azospirillum brasilense with or without metabolites of Rhizobium tropici strain CIAT 899 highly enriched in lipo-chitooligosaccharides (LCOs) in six field experiments performed for three summer crop seasons in Brazil with maize (Zea mays L.). Inoculants and metabolites were applied either at sowing by seed inoculation, or by leaf spray at the V3 stage of plant growth. Improvement in shoot dry weight (SDW) and total N accumulated in shoots (TNS) by single, but especially by dual inoculation was observed in some of the experiments. Statistically significant increases in grain yield in relation to the non-inoculated control were observed in five out of six experiments when maize was inoculated with Azospirillum supplied with enriched metabolites of R. tropici applied by seed or leaf spray inoculation. The results give strength to the development of a new generation of inoculants carrying microorganisms and microbial molecules.
doi:10.1186/s13568-015-0154-z
PMCID: PMC4644132  PMID: 26567001
Nod factor; Zea mays; Inoculant; PGPB
12.  Opening the “black box” of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899 
BMC Genomics  2015;16:864.
Background
Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants.
Methods
The three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.).
Results
Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.
Conclusions
We report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-2033-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-2033-z
PMCID: PMC4624370  PMID: 26502986
Biological nitrogen fixation; LCO; nodD gene; Nod factors; Symbiosis
13.  Genome Sequence of Rhizobium ecuadorense Strain CNPSo 671T, an Indigenous N2-Fixing Symbiont of the Ecuadorian Common Bean (Phaseolus vulgaris L.) Genetic Pool 
Genome Announcements  2015;3(5):e01058-15.
Rhizobium ecuadorense CNPSo 671T was isolated from a common bean nodule in Ecuador. The draft genome brings novelty about indigenous rhizobial species in centers of genetic diversity of the legume.
doi:10.1128/genomeA.01058-15
PMCID: PMC4574372  PMID: 26383667
14.  Genome Sequence of Bradyrhizobium pachyrhizi Strain PAC48T, a Nitrogen-Fixing Symbiont of Pachyrhizus erosus (L.) Urb. 
Genome Announcements  2015;3(5):e01074-15.
Bradyrhizobium pachyrhizi PAC48T has been isolated from a jicama nodule in Costa Rica. The draft genome indicates high similarity with that of Bradyrhizobium elkanii. Several coding sequences (CDSs) of the stress response might help in survival in the tropics. PAC48T carries nodD1 and nodK, similar to Bradyrhizobium (Parasponia) ANU 289 and a particular nodD2 gene.
doi:10.1128/genomeA.01074-15
PMCID: PMC4574380  PMID: 26383651
15.  A database for the taxonomic and phylogenetic identification of the genus Bradyrhizobium using multilocus sequence analysis 
BMC Genomics  2015;16(Suppl 5):S10.
Background
Biological nitrogen fixation, with an emphasis on the legume-rhizobia symbiosis, is a key process for agriculture and the environment, allowing the replacement of nitrogen fertilizers, reducing water pollution by nitrate as well as emission of greenhouse gases. Soils contain numerous strains belonging to the bacterial genus Bradyrhizobium, which establish symbioses with a variety of legumes. However, due to the high conservation of Bradyrhizobium 16S rRNA genes - considered as the backbone of the taxonomy of prokaryotes - few species have been delineated. The multilocus sequence analysis (MLSA) methodology, which includes analysis of housekeeping genes, has been shown to be promising and powerful for defining bacterial species, and, in this study, it was applied to Bradyrhizobium, species, increasing our understanding of the diversity of nitrogen-fixing bacteria.
Description
Classification of bacteria of agronomic importance is relevant to biodiversity, as well as to biotechnological manipulation to improve agricultural productivity. We propose the construction of an online database that will provide information and tools using MLSA to improve phylogenetic and taxonomic characterization of Bradyrhizobium, allowing the comparison of genomic sequences with those of type and representative strains of each species.
Conclusion
A database for the taxonomic and phylogenetic identification of the Bradyrhizobium, genus, using MLSA, will facilitate the use of biological data available through an intuitive web interface. Sequences stored in the on-line database can be compared with multiple sequences of other strains with simplicity and agility through multiple alignment algorithms and computational routines integrated into the database. The proposed database and software tools are available at http://mlsa.cnpso.embrapa.br, and can be used, free of charge, by researchers worldwide to classify Bradyrhizobium, strains; the database and software can be applied to replicate the experiments presented in this study as well as to generate new experiments. The next step will be expansion of the database to include other rhizobial species.
doi:10.1186/1471-2164-16-S5-S10
PMCID: PMC4460661  PMID: 26040196
Bradyrhizobium database; Taxonomic of Prokaryotes; Phylogeny of Prokaryotes; Multilocus Sequence Analysis; 16S rRNA Gene; Bioinformatics; Pattern Recognition
16.  Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation 
BMC Genomics  2015;16(1):251.
Background
Nodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes—with an emphasis on common bean (Phaseolus vulgaris)—and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated.
Results
Phenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes.
Conclusion
Our proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1458-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1458-8
PMCID: PMC4393855  PMID: 25880529
nodD gene; Nod factors; Nodulation; Symbiosis; Nitrogen fixation; Rhizobium tropici
17.  Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis 
BMC Genomics  2014;15(1):643.
Background
Strain CPAC 7 (=SEMIA 5080) was recently reclassified into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.) Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoefficiens inoculant strains is well recognized, few data on their protein expression are available.
Results
We provided a two-dimensional proteomic reference map of CPAC 7 obtained under free-living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment (e.g. inositol monophosphatase, IMPase), fixation of atmospheric nitrogen (N2) (e.g. NifH) and defenses against stresses (e.g. chaperones). By using bioinformatic tools, it was possible to attribute probable functions to ten hypothetical proteins. For another ten proteins classified as “NO related COG” group, we analyzed by RT-qPCR the relative expression of their coding-genes in response to the nodulation-gene inducer genistein. Six of these genes were up-regulated, including blr0227, which may be related to polyhydroxybutyrate (PHB) biosynthesis and competitiveness for nodulation.
Conclusions
The proteomic map contributed to the identification of several proteins of B. diazoefficiens under free-living conditions and our approach—combining bioinformatics and gene-expression assays—resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-643) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-643
PMCID: PMC4287336  PMID: 25086822
Symbiosis; Nitrogen fixation; Two-dimensional proteomics; RT-qPCR; Bradyrhizobium
18.  The complete genome of Burkholderia phenoliruptrix strain BR3459a, a symbiont of Mimosa flocculosa: highlighting the coexistence of symbiotic and pathogenic genes 
BMC Genomics  2014;15(1):535.
Background
Burkholderia species play an important ecological role related to xenobiosis, the promotion of plant growth, the biocontrol of agricultural diseases, and symbiotic and non-symbiotic biological nitrogen fixation. Here, we highlight our study as providing the first complete genome of a symbiotic strain of B. phenoliruptrix, BR3459a (=CLA1), which was originally isolated in Brazil from nodules of Mimosa flocculosa and is effective in fixing nitrogen in association with this leguminous species.
Results
Genomic comparisons with other pathogenic and non-pathogenic Burkholderia strains grouped B. phenoliruptrix BR3459a with plant-associated beneficial and environmental species, although it shares a high percentage of its gene repertoire with species of the B. cepacia complex (Bcc) and "pseudomallei" group. The genomic analyses showed that the bce genes involved in exopolysaccharide production are clustered together in the same genomic region, constituting part of the Group III cluster of non-pathogenic bacteria. Regarding environmental stresses, we highlight genes that might be relevant in responses to osmotic, heat, cold and general stresses. Furthermore, a number of particularly interesting genes involved in the machinery of the T1SS, T2SS, T3SS, T4ASS and T6SS secretion systems were identified. The xenobiotic properties of strain BR3459a were also investigated, and some enzymes involved in the degradation of styrene, nitrotoluene, dioxin, chlorocyclohexane, chlorobenzene and caprolactam were identified. The genomic analyses also revealed a large number of antibiotic-related genes, the most important of which were correlated with streptomycin and novobiocin. The symbiotic plasmid showed high sequence identity with the symbiotic plasmid of B. phymatum. Additionally, comparative analysis of 545 housekeeping genes among pathogenic and non-pathogenic Burkholderia species strongly supports the definition of a new genus for the second branch, which would include BR3459a.
Conclusions
The analyses of B. phenoliruptrix BR3459a showed key property of fixing nitrogen that together with genes for high tolerance to environmental stresses might explain a successful strategy of symbiosis in the tropics. The strain also harbours interesting sets of genes with biotechnological potential. However, the resemblance of certain genes to those of pathogenic Burkholderia raise concerns about large-scale applications in agriculture or for bioremediation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-535) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-535
PMCID: PMC4101177  PMID: 24972629
Burkholderia phenoliruptrix; Comparative genomics; Nitrogen-fixing and pathogenic Burkholderia
19.  Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean 
BMC Genomics  2014;15(1):420.
Background
The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6T and B. diazoefficiens USDA 110T.
Results
Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15.
Conclusions
Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N2 fixation, nodulation and competitiveness.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-420) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-420
PMCID: PMC4070871  PMID: 24888481
Symbiosis; Nodulation; Nitrogen fixation; Competitiveness; Secretion systems; Horizontal gene transfer; Membrane transporters; Surface polysaccharides; Secondary metabolism; Phytohormone synthesis
20.  Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture 
AMB Express  2014;4:26.
Six endophytic bacteria of corn roots were identified as Bacillus sp. and as Enterobacter sp, by sequencing of the 16S rRNA gene. Four of the strains, CNPSo 2476, CNPSo 2477, CNPSo 2478 and CNPSo 2480 were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Two Bacillus strains (CNPSo 2477 and CNPSo 2478) showed outstanding skills for the production of IAA, siderophores and lytic enzymes, but were not good candidates as growth promoters, because they reduced seed germination. However, the same strains were antagonists against the pathogenic fungi Fusarium verticillioides, Colletotrichum graminicola, Bipolaris maydis and Cercospora zea-maydis. As an indication of favorable bacterial action, Enterobacter sp. CNPSo 2480 and Bacillus sp. CNPSo 2481 increased the root volume by 44% and 39%, respectively, and the seed germination by 47% and 56%, respectively. Therefore, these two strains are good candidates for future testing as biological inoculants for corn.
doi:10.1186/s13568-014-0026-y
PMCID: PMC4052694  PMID: 24949261
Molecular phylogeny; 16S rRNA; nifH; Plant growth promotion; Antagonism
21.  Proteomic Analysis of Soybean [Glycine max (L.) Merrill] Roots Inoculated with Bradyrhizobium japonicum Strain CPAC 15 
Proteomics Insights  2013;6:7-11.
This research intended to analyze the expression pattern of proteins in roots of the Brazilian soybean cultivar Conquista when inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. At ten days after bacterial inoculation, whole-cell proteins were extracted from roots and separated by 2-D gel electrophoresis. Comparative analysis revealed significant changes in the intensity of 37 spots due to the inoculation (17 up-regulated and 20 down-regulated proteins), identified by MALDI-TOF/TOF-TOF. Identified proteins were associated with COG functional categories of information storage and processing, cellular processes and signaling, metabolism, and also in the “poorly characterized” and “not in COG” categories. Among the up-regulated proteins, we identified sucrose synthase (nodulin-100), β-tubulin, rubisco activase, glutathione-S-transferase, a putative heat-shock 70-kDa protein, pyridine nucleotide-disulphideoxidoreductase and a putative transposase. Proteomic analysis allowed for the identification of some putative symbiotic functions and confirmed the main biological processes triggered in the nitrogen-fixing symbiosis with soybean.
doi:10.4137/PRI.S13288
PMCID: PMC4147754  PMID: 25288888
rhizobium; 2-D; proteomics; soybean roots; symbiosis
22.  The Genome of Anopheles darlingi, the main neotropical malaria vector 
Marinotti, Osvaldo | Cerqueira, Gustavo C. | de Almeida, Luiz Gonzaga Paula | Ferro, Maria Inês Tiraboschi | Loreto, Elgion Lucio da Silva | Zaha, Arnaldo | Teixeira, Santuza M. R. | Wespiser, Adam R. | Almeida e Silva, Alexandre | Schlindwein, Aline Daiane | Pacheco, Ana Carolina Landim | da Silva, Artur Luiz da Costa | Graveley, Brenton R. | Walenz, Brian P. | Lima, Bruna de Araujo | Ribeiro, Carlos Alexandre Gomes | Nunes-Silva, Carlos Gustavo | de Carvalho, Carlos Roberto | Soares, Célia Maria de Almeida | de Menezes, Claudia Beatriz Afonso | Matiolli, Cleverson | Caffrey, Daniel | Araújo, Demetrius Antonio M. | de Oliveira, Diana Magalhães | Golenbock, Douglas | Grisard, Edmundo Carlos | Fantinatti-Garboggini, Fabiana | de Carvalho, Fabíola Marques | Barcellos, Fernando Gomes | Prosdocimi, Francisco | May, Gemma | de Azevedo Junior, Gilson Martins | Guimarães, Giselle Moura | Goldman, Gustavo Henrique | Padilha, Itácio Q. M. | Batista, Jacqueline da Silva | Ferro, Jesus Aparecido | Ribeiro, José M. C. | Fietto, Juliana Lopes Rangel | Dabbas, Karina Maia | Cerdeira, Louise | Agnez-Lima, Lucymara Fassarella | Brocchi, Marcelo | de Carvalho, Marcos Oliveira | Teixeira, Marcus de Melo | Diniz Maia, Maria de Mascena | Goldman, Maria Helena S. | Cruz Schneider, Maria Paula | Felipe, Maria Sueli Soares | Hungria, Mariangela | Nicolás, Marisa Fabiana | Pereira, Maristela | Montes, Martín Alejandro | Cantão, Maurício E. | Vincentz, Michel | Rafael, Miriam Silva | Silverman, Neal | Stoco, Patrícia Hermes | Souza, Rangel Celso | Vicentini, Renato | Gazzinelli, Ricardo Tostes | Neves, Rogério de Oliveira | Silva, Rosane | Astolfi-Filho, Spartaco | Maciel, Talles Eduardo Ferreira | Ürményi, Turán P. | Tadei, Wanderli Pedro | Camargo, Erney Plessmann | de Vasconcelos, Ana Tereza Ribeiro
Nucleic Acids Research  2013;41(15):7387-7400.
Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
doi:10.1093/nar/gkt484
PMCID: PMC3753621  PMID: 23761445
23.  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.
doi:10.1128/JB.01821-12
PMCID: PMC3497515  PMID: 23144415
24.  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.
doi:10.1128/JB.01756-12
PMCID: PMC3510596  PMID: 23209196
25.  Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicum strain CPAC 15 
BMC Genomics  2013;14:153.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-14-153
PMCID: PMC3608089  PMID: 23497193
Subtractive library; Differential expression of genes; Nodulation

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