RESUMO
Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the alpha subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that "M. nodulans" is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that "M. nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.
Assuntos
Fabaceae/microbiologia , Metanol/metabolismo , Methylobacterium/classificação , Methylobacterium/fisiologia , Fixação de Nitrogênio/fisiologia , Plantas Medicinais , Simbiose , Aciltransferases/genética , Oxirredutases do Álcool/genética , Proteínas de Bactérias , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Methylobacterium/genética , Methylobacterium/isolamento & purificação , Dados de Sequência Molecular , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.
Assuntos
Bradyrhizobium/classificação , Impressões Digitais de DNA/métodos , DNA Intergênico , Fabaceae/microbiologia , Plantas Medicinais , Técnicas de Tipagem Bacteriana , Bradyrhizobium/genética , Análise por Conglomerados , DNA Bacteriano/genética , DNA Ribossômico/genética , Genótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , SenegalRESUMO
The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.
Assuntos
Acacia/microbiologia , Bradyrhizobium/classificação , Impressões Digitais de DNA/métodos , Fabaceae/microbiologia , Variação Genética , Genoma Bacteriano , Plantas Medicinais , Bradyrhizobium/genética , Bradyrhizobium/isolamento & purificação , Genótipo , Polimorfismo de Fragmento de RestriçãoRESUMO
Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).
Assuntos
Fabaceae/microbiologia , Fixação de Nitrogênio , Plantas Medicinais , Rhizobium/classificação , Proteínas de Bactérias/análise , DNA Bacteriano/genética , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Enzimas/análise , Gâmbia , Genes de RNAr , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Rhizobium/genética , Rhizobium/isolamento & purificação , Rhizobium/fisiologia , SenegalRESUMO
SDS-PAGE of total bacterial proteins was applied to the classification of 25 Sudanese and five Kenyan strains isolated from the root nodules of Acacia senegal and Prosopis chilensis. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridization, genomic fingerprinting using repetitive sequence-based PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus Sinorhizobium. Two of the strains grouped with Sinorhizobium saheli and seven with Sinorhizobium terangae, while the rest did not cluster with any of the established species. The majority of the strains formed two phenotypically and genotypically distinct groups and we therefore propose that these strains should be classified as two new species, Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov.
Assuntos
Acacia/microbiologia , Fabaceae/microbiologia , Plantas Medicinais , Sinorhizobium/classificação , Sinorhizobium/isolamento & purificação , Proteínas de Bactérias/química , Técnicas de Tipagem Bacteriana , Eletroforese/métodos , Eletroforese em Gel de Poliacrilamida , Enzimas/análise , Genes de RNAr , Quênia , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sinorhizobium/genética , SudãoRESUMO
We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.
Assuntos
Bradyrhizobium/classificação , Bradyrhizobium/fisiologia , Fabaceae/microbiologia , Fotossíntese , Plantas Medicinais , RNA Ribossômico 16S/genética , Bradyrhizobium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes Bacterianos , Genes de RNAr , Dados de Sequência Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , Padrões de Referência , Mapeamento por Restrição , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization. It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96.2% sequence homology). The name Allorhizobium undicola gen. nov., sp. nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).