Molecular diversity of rhizobia-nodulating native Mimosa of Brazilian protected areas.

Symbiotic Paraburkholderia have been increasingly studied in the past 20 years, especially when associated with Mimosa; however, studies with native/endemic species are still scarce. In this study, thirty strains were isolated from root nodules of native Mimosa paranapiacabae and M. micropteris in two locations of the Campos Gerais. The BOX-PCR fingerprinting revealed high genomic diversity, and the 16S rRNA phylogeny clustered the strains in three distinct groups (GI, GII, GIII), with one strain occupying an isolated position. Phylogenetic analysis with four concatenated housekeeping genes (atpD + gltB + gyrB + recA) confirmed the same clusters of 16S rRNA, and the closest species were P. nodosa BR 3437T and P. guartelaensis CNPSo 3008T; this last one isolated from another Mimosa species of the Campos Gerais. The phylogenies of the symbiotic genes nodAC and nifH placed all strains in a well-supported branch with the other species of the symbiovar mimosae. The phylogenetic analyses indicated that the strains represent novel lineages of sv. mimosae and that endemic Mimosa coevolved with indigenous Paraburkholderia in their natural environments.

Phylogeny of symbiotic genes reveals symbiovars within legume-nodulating Paraburkholderia species.

Bacteria belonging to the genus Paraburkholderia are capable of establishing symbiotic relationships with plants belonging to the Fabaceae (=Leguminosae) family and fixing the atmospheric nitrogen in specialized structures in the roots called nodules, in a process known as biological nitrogen fixation (BNF). In the nodulation and BNF processes several bacterial symbiotic genes are involved, but the relations between symbiotic, core genes and host specificity are still poorly studied and understood in Paraburkholderia. In this study, eight strains of nodulating nitrogen-fixing Paraburkholderia isolated in Brazil, together with described species and other reference strains were used to infer the relatedness between core (16S rDNA, recA) and symbiotic (nod, nif, fix) genes. The diversity of genes involved in the nodulation (nodAC) and nitrogen fixation (nifH) abilities was investigated. Only two groups, one containing three Paraburkholderia species symbionts of Mimosa, and another one with P. ribeironis strains presented similar phylogenetic patterns in the analysis of core and symbiotic genes. In three other groups events of horizontal gene transfer of symbiotic genes were detected. Paraburkholderia strains with available genomes were used in the complementary analysis of nifHDK and fixABC and confirmed well-defined phylogenetic positions of symbiotic genes. In all analyses of nod, nif and fix genes the strains were distributed into five clades with high bootstrap support, allowing the proposal of five symbiovars in nodulating nitrogen-fixing Paraburkholderia, designated as mimosae, africana, tropicalis, atlantica and piptadeniae. Phylogenetic inferences within each symbiovar are discussed.

Paraburkholderia atlantica sp. nov. and Paraburkholderia franconis sp. nov., two new nitrogen-fixing nodulating species isolated from Atlantic forest soils in Brazil.

A polyphasic study was conducted with 11 strains trapped by Mimosa pudica and Phaseolus vulgaris grown in soils of the Brazilian Atlantic Forest. In the phylogenetic analysis of the 16S rRNA gene, one clade of strains (Psp1) showed higher similarity with Paraburkholderia piptadeniae STM7183T (99.6%), whereas the second (Psp6) was closely related to Paraburkholderia tuberum STM678T (99%). An MLSA (multilocus sequence analysis) with four (recA, gyrB, trpB and gltB) housekeeping genes placed both Psp1 and Psp6 strains in new clades, and BOX-PCR profiles indicated high intraspecific genetic diversity within each clade. Values of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of the whole genome sequences were of 56.9 and 94.4% between the Psp1 strain CNPSo 3157T and P. piptadeniae; and of 49.7% and 92.7% between the Psp6 strain CNPSo 3155T and P. tuberum, below the threshold for species delimitation. In the nodC analysis, Psp1 strains clustered together with P. piptadeniae, while Psp6 did not group with any symbiotic Paraburkholderia. Other phenotypic, genotypic and symbiotic properties were evaluated. The polyphasic analysis supports that the strains represent two novel species, for which the names Paraburkholderia franconis sp. nov. with type strain CNPSo 3157T (= ABIP 241, = LMG 31644) and Paraburkholderia atlantica sp. nov. with type strain CNPSo 3155T (= ABIP 236, = LMG 31643) are proposed.

Paraburkholderia guartelaensis sp. nov., a nitrogen-fixing species isolated from nodules of Mimosa gymnas in an ecotone considered as a hotspot of biodiversity in Brazil.

A polyphasic approach was used to infer the phylogenetic position of six nitrogen-fixing symbiotic bacteria isolated from Mimosa gymnas nodules grown in an ecotone between the Brazilian biomes of Atlantic Forest and Cerrado, considered as a hotspot of biodiversity. The 16S rRNA gene phylogeny indicated the highest similarity with Paraburkholderia oxyphila (98.7-98.9%), but similar values were found with other Paraburkholderia species. The multilocus sequence analysis (MLSA) of five (recA, gyrB, trpB, gltB, and atpD) housekeeping genes indicated that the CNPSo strains represent a novel lineage, sharing less than 95.7% of nucleotide identity (NI) with other Paraburkholderia species, being more closely related to P. nodosa. Genome parameters were analyzed for strain CNPSo 3008T, and DNA-DNA hybridization revealed a maximum of 55.9% of DNA-DNA relatedness with P. nodosa, while average nucleotide identity with the two closest species was of 93.84% with P. nodosa and of 87.93% with P. mimosarum, both parameters confirming that the strain represents a new species. In the analysis of the nodulation nodC gene, all CNPSo strains showed the highest similarity with P. nodosa, and nodulation tests indicated host specificity with Mimosa. Other phylogenetic, physiological, and chemotaxonomic properties were evaluated. All data obtained support the description of the novel species Paraburkholderia guartelaensis sp. nov., with CNPSo 3008T (= U13000T = G29.01T) indicated as the type strain.

Phylogenetic diversity of rhizobia nodulating native Mimosa gymnas grown in a South Brazilian ecotone.

Floristic surveys performed in "Campos Gerais" (Paraná, Brazil), an ecotone of Mata Atlântica and Cerrado biomes, highlights the richness and relative abundance of the family Fabaceae and point out the diversity and endemism of Mimosa spp. Our study reports the genetic diversity of rhizobia isolated from root nodules of native/endemic Mimosa gymnas Barneby in three areas of Guartelá State Park, an important conservation unit of "Campos Gerais". Soils of the sample areas were characterized as sandy, acid, poor in nutrients and organic matter. The genetic variability among the isolates was revealed by BOX-PCR genomic fingerprinting. Phylogeny based on 16S rRNA gene grouped the strains in a large cluster including Paraburkholderia nodosa and P. bannensis, while recA-gyrB phylogeny separated the strains in two groups: one including P. nodosa and the other without any described Paraburkholderia species. MLSA confirmed the separate position of this second group of strains within the genus Paraburkholderia and the nucleotide identity of the five concatened housekeeping genes was 95.9% in relation to P. nodosa BR 3437T. Phylogram based on symbiosis-essential nodC gene was in agreement with 16S rRNA analysis. Our molecular phylogenetic analysis support that Paraburkholderia are the main symbionts of native Mimosa in specific edaphic conditions found in South America and reveal the importance of endemic/native leguminous plants as reservoirs of novel rhizobial species.

Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis.

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.

A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots.

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.

Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains.

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 µM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ(EcfG) regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.

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 N(2)-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.