Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice.

Untapped Endophytic Colonization and Plant Growth-Promoting Potential of the Genus Novosphingobium to Optimize Rice Cultivation.

With the aim of searching for potent diazotrophic bacteria that are free of public health concerns and optimize rice cultivation, the endophytic colonization and plant growth-promoting activities of some endophytic diazotrophic bacteria isolated from rice were evaluated. Among these bacteria, the emerging diazotrophic strains of the genus Novosphingobium effectively associated with rice plant interiors and consequently promoted the growth of rice, even with the lack of a nitrogen source. These results suggest that diazotrophic Novosphingobium is an alternative microbial resource for further development as a safe biological enhancer in the optimization of organic rice cultivation.

nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth.

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

Genome analysis of a novel Bradyrhizobium sp. DOA9 carrying a symbiotic plasmid.

Bradyrhizobium sp. DOA9 isolated from the legume Aeschynomene americana exhibited a broad host range and divergent nodulation (nod) genes compared with other members of the Bradyrhizobiaceae. Genome analysis of DOA9 revealed that its genome comprised a single chromosome of 7.1 Mbp and a plasmid of 0.7 Mbp. The chromosome showed highest similarity with that of the nod gene-harboring soybean symbiont B. japonicum USDA110, whereas the plasmid showed highest similarity with pBBta01 of the nod gene-lacking photosynthetic strain BTAi1, which nodulates Aeschynomene species. Unlike in other bradyrhizobia, the plasmid of DOA9 encodes genes related to symbiotic functions including nodulation, nitrogen fixation, and type III/IV protein secretion systems. The plasmid has also a lower GC content (60.1%) than the chromosome (64.4%). These features suggest that the plasmid could be the origin of the symbiosis island that is found in the genome of other bradyrhizobia. The nod genes of DOA9 exhibited low similarity with those of other strains. The nif gene cluster of DOA9 showed greatest similarity to those of photosynthetic bradyrhizobia. The type III/IV protein secretion systems of DOA9 are similar to those of nod gene-harboring B. elkanii and photosynthetic BTAi1. The DOA9 genome exhibited intermediate characteristics between nod gene-harboring bradyrhizobia and nod gene-lacking photosynthetic bradyrhizobia, thus providing the evidence for the evolution of the Bradyrhizobiaceae during ecological adaptation. Bradyrhizobium sp. DOA9 isolated from the legume Aeschynomene americana exhibited a broad host range and divergent nodulation (nod) genes compared with other members of the Bradyrhizobiaceae. Genome analysis of DOA9 revealed that its genome comprised a single chromosome of 7.1 Mbp and a plasmid of 0.7 Mbp. The chromosome showed highest similarity with that of the nod gene-harboring soybean symbiont B. japonicum USDA110, whereas the plasmid showed highest similarity with pBBta01 of the nod gene-lacking photosynthetic strain BTAi1, which nodulates Aeschynomene species. Unlike in other bradyrhizobia, the plasmid of DOA9 encodes genes related to symbiotic functions including nodulation, nitrogen fixation, and type III/IV protein secretion systems. The plasmid has also a lower GC content (60.1%) than the chromosome (64.4%). These features suggest that the plasmid could be the origin of the symbiosis island that is found in the genome of other bradyrhizobia. The nod genes of DOA9 exhibited low similarity with those of other strains. The nif gene cluster of DOA9 showed greatest similarity to those of photosynthetic bradyrhizobia. The type III/IV protein secretion systems of DOA9 are similar to those of nod gene-harboring B. elkanii and photosynthetic BTAi1. The DOA9 genome exhibited intermediate characteristics between nod gene-harboring bradyrhizobia and nod gene-lacking photosynthetic bradyrhizobia, thus providing the evidence for the evolution of the Bradyrhizobiaceae during ecological adaptation.

Divergent nod-containing Bradyrhizobium sp. DOA9 with a megaplasmid and its host range.

Bradyrhizobium sp. DOA9, a non-photosynthetic bacterial strain originally isolated from the root nodules of the legume Aeschynomene americana, is a divergent nod-containing strain. It exhibits a broad host range, being able to colonize and efficiently nodulate the roots of most plants from the Dalbergioid, Millettioid, and Robinioid tribes (7 species of Papilionoideae). In all cases, nodulation was determinate. The morphology and size of DOA9 bacteroids isolated from the nodules of various species of Papilionoideae were indistinguishable from the free-living form. However, they were spherical in Arachis hypogaea nodules. GusA-tagged DOA9 also colonized rice roots as endophytes. Since broad-host-range legume symbionts often carry multiple replicons in their genome, we analyzed the replicons for symbiosis genes by electrophoresis. DOA9 carried two replicons, a chromosome (cDOA9) and single megaplasmid (pDOA9) larger than 352 kb. The genes for nodulation (nodA, B, C) and nitrogen fixation (nifH) were localized on the megaplasmid. Southern blot hybridization revealed two copies of nodA on the megaplasmid, single copies of nodB and C on the megaplasmid, and one copy each of nifH on the chromosome and megaplasmid. These results suggested that Bradyrhizobium sp. DOA9 may have the unusual combination of a broad host range, bacteroid differentiation, and symbiosis-mediating replicons.

Genetic diversity, symbiotic evolution, and proposed infection process of Bradyrhizobium strains isolated from root nodules of Aeschynomene americana L. in Thailand.

The diversity of bacteria nodulating Aeschynomene americana L. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK, recA, and glnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genus Bradyrhizobium, in which B. yuanmingense was the dominant species. Some of the other species, including B. japonicum, B. liaoningense, and B. canariense, were minor species. These isolated strains were divided into 2 groups-nod-containing and divergent nod-containing strains-based on Southern blot hybridization and PCR amplification of nodABC genes. The divergent nod genes could not be PCR amplified and failed to hybridize nod gene probes designed from B. japonicum USDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity of nod genes was well correlated with the grouping based on that of nifH gene, in which the nod-containing and divergent nod-containing strains were obviously distinguished. The divergent nod-containing strains and photosynthetic bradyrhizobia shared close nifH sequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated from A. americana could nodulate Aeschynomene plants that belong to different cross-inoculation (CI) groups, including A. afraspera and A. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic and nod-containing strain) originating from CI group 1 nodulating roots of A. indica (CI group 3). An infection process used to establish symbiosis on Aeschynomene different from the classical one is proposed.

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand.

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn(2+) and Co(2+) resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595(T)=BCC 40155(T)).