Jean Luiz Simões-Araújo

Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

ABSTRACT Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

Molecular characterisation of the diazotrophic bacterial community in uninoculated and inoculated field-grown sugarcane (Saccharum sp.)

To identify active diazotrophs in sugarcane, 16S rRNA and nifH transcript analyses were applied. This should help to better understand the basis of the biological nitrogen fixation (BNF) activity of a high nitrogen fixing sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropédica, RJ, Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with inoculation. The five-strain mixture developed by EMBRAPA-CNPAB was used as inoculum. Root and leaf sheath samples were harvested in the third year of cultivation to analyse the 16S rRNA and nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a wide diversity of nifH sequences from previously uncharacterised Ideonella/Herbaspirillum related phylotypes in sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots was found. These results were confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus and B. tropica were detected in leaf sheaths and roots. Known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using molecular analyses. Two strains of the inoculum mix were identified at the late summer harvest.

Exopolysaccharide Production Is Required for Biofilm Formation and Plant Colonization by the Nitrogen-Fixing Endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.

Microvirga vignae sp. nov., a root nodule symbiotic bacterium isolated from cowpea grown in semi-arid Brazil

16S rRNA gene sequence analysis of eight strains (BR 3299(T), BR 3296, BR 10192, BR 10193, BR 10194, BR 10195, BR 10196 and BR 10197) isolated from nodules of cowpea collected from a semi-arid region of Brazil showed 97 % similarity to sequences of recently described rhizobial species of the genus Microvirga. Phylogenetic analyses of four housekeeping genes (gyrB, recA, dnaK and rpoB), DNA-DNA relatedness and AFLP further indicated that these strains belong to a novel species within the genus Microvirga. Our data support the hypothesis that genes related to nitrogen fixation were obtained via horizontal gene transfer, as sequences of nifH genes were very similar to those found in members of the genera Rhizobium and Mesorhizobium, which are not immediate relatives of the genus Microvirga, as shown by 16S rRNA gene sequence analysis. Phenotypic traits, such as host range and carbon utilization, differentiate the novel strains from the most closely related species, Microvirga lotononidis, Microvirga zambiensis and Microvirga lupini. Therefore, these symbiotic nitrogen-fixing bacteria are proposed to be representatives of a novel species, for which the name Microvirga vignae sp. nov. is suggested. The type strain is BR3299(T) ( = HAMBI 3457(T)).

Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazonian rainforest soils

Root nodule bacteria were trapped within cowpea (Vigna unguiculata) in soils with different cultivation histories collected from the Amazonian rainforest in northern Brazil. Analysis of the 16S rRNA gene sequences of six strains (BR 3351(T), BR 3307, BR 3310, BR 3315, BR 3323 BR and BR 3361) isolated from cowpea nodules showed that they formed a distinct group within the genus Bradyrhizobium, which was separate from previously identified type strains. Phylogenetic analyses of three housekeeping genes (glnII, recA and rpoB) revealed that Bradyrhizobium huanghuaihaiense CCBAU 23303(T) was the most closely related type strain (96% sequence similarity or lower). Chemotaxonomic data, including fatty acid profiles (predominant fatty acids being C16 : 0 and summed feature 8), the slow growth rate and carbon compound utilization patterns supported the assignment of the strains to the genus Bradyrhizobium. The results of DNA-DNA hybridizations, antibiotic resistance and physiological tests differentiated these novel strains from the most closely related species of the genus Bradyrhizobium with validly published names. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped the novel strains of the genus Bradyrhizobium together with Bradyrhizobium iriomotense strain EK05(T), with 94% and 96% sequence similarity, respectively. Based on these data, these six strains represent a novel species for which the name Brabyrhizobium manausense sp. nov. (BR 3351(T) = HAMBI 3596(T)), is proposed.

Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR

Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a Threatened Species Native to the Brazilian Cerrado

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N2-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N2 fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N2-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.

Unravelling MADS-box gene family in Eucalyptus spp.: a starting point to an understanding of their developmental role in trees

MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants ranging from root to flower and fruit development. Members of the MADS-box gene family share a highly conserved sequence of approximately 180 nucleotides that encodes a DNA-binding domain. We used bioinformatics tools to investigate the information generated by the Eucalyptus Expressed Sequence Tag (FORESTs) genome project in order to identify and annotate MADS-box genes. The comparative phylogenetic analysis of the Eucalyptus MADS-box genes with Arabidopsis homologues allowed us to group them into one of the well-known subfamilies. Trends in gene expression of these putative Eucalyptus MADS-box genes were investigated by hierarchical clustering analysis. Among 24 MADS-box genes identified by our analysis, 12 are expressed in vegetative organs. Out of these, five are expressed predominately in wood. Understanding of the molecular mechanisms performed by MADS-box proteins underlying Eucalyptus growth, development and stress reactions would provide important insights into tree development and could reveal means by which tree characteristics could be modified for the improvement of industrial properties.

Rhizobium altiplani sp. nov., isolated from effective nodules on Mimosa pudica growing in untypically alkaline soil in central Brazil

Root nodule bacteria were isolated from nodules on Mimosa pudica L. growing in neutral-alkaline soils from the Distrito Federal in central Brazil. The 16S rRNA gene sequence analysis of 10 strains placed them into the genus Rhizobium with the closest neighbouring species (each with 99 % similarity) being Rhizobium grahamii, Rhizobium cauense, Rhizobium mesoamericanum and Rhizobium tibeticum. This high similarity, however, was not confirmed by multi-locus sequence analysis (MLSA) using three housekeeping genes (recA, glnII and rpoB), which revealed R. mesoamericanum CCGE 501T to be the closest type strain (92 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with majority being C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c)], DNA G+C content (57.6 mol%), and carbon compound utilization patterns supported the placement of the novel strains in the genus Rhizobium. Results of average nucleotide identity (ANI) differentiated the novel strains from the closest species of the genus Rhizobium, R. mesoamericanum, R. grahamii and R. tibeticum with 89.0, 88.1 and 87.8 % similarity, respectively. The symbiotic genes essential for nodulation (nodC) and nitrogen fixation (nifH) were most similar (99-100 %) to those of R. mesoamericanum, another Mimosa-nodulating species. Based on the current data, these 10 strains represent a novel species of the genus Rhizobium for which the name Rhizobium altiplani sp. nov. is proposed. The type strain is BR 10423T (=HAMBI 3664T).

Expressão gênica induzida por estresses abióticos em nódulos de feijão-caupi

Abstract – The objective of this work was to evaluate the isolated or concomitant effect of drought and heat stresses on the gene expression of cowpea nodules. The bacterium Bradyrhizobium japonicum (strain BR 3267) was inoculated in cowpea seeds from the cultivar IPA 206 and, 35 days after germination, the plants were subjected to different water availability regimes and to heat stress under greenhouse conditions. To identify differentially expressed genes, the cDNA‑AFLP technique was used, and 67 differentially expressed transcript‑derived fragments (TDFs) were isolated. After TDFs sequencing and similarity analyses, using the Blastx program, 14 differentially expressed genes were identified, which are involved in different metabolic processes. The expression pattern of six genes under abiotic stress conditions was confirmed by RT‑qPCR, and the induction of different functional gene categories, such as abscisic acid biosynthesis, cell signaling, proline transporter, and lipid membrane biosynthesis, was observed. The expression of these genes indicates their participation in processes related to nodule protection under abiotic stresses.Index terms: