Genome-based reclassification of azospirillum brasilense SP245 as the type strain of azospirillum baldaniorum sp. nov

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T, revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4–68.6%. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).Fil: Ferreira, Natalia Dos Santos. Universidade Federal Rural Do Rio de Janeiro; BrasilFil: Sant´Anna, Fernando Hayashi. Universidade Federal do Rio Grande do Sul; BrasilFil: Reis, Veronica Massena. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Ambrosini, Adriana. Universidade Federal do Rio Grande do Sul; BrasilFil: Volpiano, Camila Gazolla. Universidade Federal do Rio Grande do Sul; BrasilFil: Rothballer, Michael. Helmholtz Center Munich German Research Center For Environmental Health; AlemaniaFil: Schwab, Stefan. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Baura, Valter Antonio. Universidade Federal do Paraná; BrasilFil: Balsanelli, Eduardo. Universidade Federal do Paraná; BrasilFil: Pedrosa, Fabio de Oliveira. Universidade Federal do Paraná; BrasilFil: Passaglia, Luciane Maria Pereira. Universidade Federal do Rio Grande do Sul; BrasilFil: de Souza, Emanuel Maltempi. Universidade Federal do Paraná; BrasilFil: Hartmann, Anton. Ludwig Maximilians Universitat; AlemaniaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Zilli, Jerri Edson. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasi

The genomes of three Bradyrhizobium sp. isolated from root nodules of Lupinus albescens grown in extremely poor soils display important genes for resistance to environmental stress

Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops

The genomes of three Bradyrhizobium sp. isolated from root nodules of Lupinus albescens grown in extremely poor soils display important genes for resistance to environmental stress

Abstract Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops

The genomes of three Bradyrhizobium sp. isolated from root nodules of Lupinus albescens grown in extremely poor soils display important genes for resistance to environmental stress

Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops

Classification of the inoculant strain of cowpea UFLA03-84 and of other strains from soils of the Amazon region as Bradyrhizobium viridifuturi (symbiovar tropici)

Cowpea (Vigna unguiculata L.) is a legume species that considerably benefits from inoculation with nitrogen fixing bacteria of the genus Bradyrhizobium. One of the strains recommended for inoculation in cowpea in Brazil is UFLA03-84 (Bradyrhizobium sp.). The aim of our study was to define the taxonomic position of the UFLA03-84 strain and of two other strains of Bradyrhizobium (UFLA03-144 and INPA237B), all belonging to the same phylogenetic group and isolated from soils of the Brazilian Amazon. Multilocus sequence analysis (MLSA) of the housekeeping genes atpD, gyrB, recA, and rpoB grouped (with similarity higher than 99%) the three strains with Bradyrhizobium viridifuturi SEMIA 690(T). The analyses of average nucleotide identity and digital DNA-DNA hybridization supported classification of the group as Bradyrhizobium viridifuturi. The three strains exhibited similar behavior in relation to the most of the phenotypic characteristics evaluated. However, some characteristics exhibited variation, indicating phenotypic diversity within the species. Phylogenetic analysis of the nodC and nifH genes showed that the three strains are members of the same symbiovar (tropici) that contains type strains of Bradyrhizobium species coming from tropical soils (SEMIA 690(T)B. viridifuturi, CNPSo 1112(T)B. tropiciagri, CNPSo 2833(T)B. embrapense, and B. brasilense UFLA03-321(T))

Maize Root Lectins Mediate the Interaction with <i>Herbaspirillum seropedicae</i> via N-Acetyl Glucosamine Residues of Lipopolysaccharides

<div><p><i>Herbaspirillum seropedicae</i> is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for <i>H. seropedicae</i> attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene <i>waaL</i> (codes for the O-antigen ligase) is induced during rhizosphere colonization by <i>H. seropedicae</i>. Furthermore a <i>waaL</i> mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits <i>H. seropedicae</i> attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type <i>H. seropedicae</i>, but failed to agglutinate the <i>waaL</i> mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in <i>H. seropedicae</i> attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.</p></div

Maize root colonization by different amounts of <i>H. seropedicae</i> wild-type (black bars) and <i>waaL</i> mutant (gray bars) strains.

<p>Panel A: maize was inoculated separately with the indicated amount of each bacterial strain. Panel B: maize was inoculated with a 1∶1 mixture of both strains. The total number of bacterial cells inoculated is indicated in the x axis. Results are shown as means of log₁₀ (number of bacteria.g⁻¹ of fresh root) ± standard deviation. Different letters indicate significant difference at p<0.01 (Duncan multiple range test).</p

Identification of isolated maize root lectin-like (MRL) proteins by MALDI-TOF spectrometry.

a<p>Protein alignment performed with NCBI PSI-Blastp (<a href="http://ncbi.nlm.gov/BLAST/" target="_blank">http://ncbi.nlm.gov/BLAST/</a>).</p><p>Peptide mass fingerprint (PMF) comparisons to the database and sequence coverage data were obtained using the MASCOT program.</p

Effect of proteins on <i>H. seropedicae</i> attachment onto root surface.

<p><i>H. seropedicae</i> wild type (black bars), <i>waaL</i> (gray bars) strains or maize roots were incubated with 5 µg.mL⁻¹ proteinase-K for 20 minutes at 30°C before attachment assay where indicated. Purified LPS, N-acetyl glucosamine (NAcGlc), WGA or MRLs (0.5 mg.mL⁻¹) were added as indicated. Results are shown as means of log₁₀ (number of bacteria.g⁻¹ of fresh root) ± standard deviation. Asterisks indicate significant difference at p<0.01 (Duncan multiple range test) of wild-type attachment between control and test conditions.</p

Electrophoretic pattern of LPS isolated from <i>H. seropedicae</i> SmR1 (wild type, lane A) and LPSEB (<i>waaL</i>, lane B).

<p>SDS-PAGE was performed with total LPS extracted from 10⁷ cells grown in NFbHPN medium, and LPS was visualized by periodate oxidation and silver staining.</p