Identification and characterization of VapBC toxin-antitoxin system in Bosea sp. PAMC 26642 isolated from Arctic lichens.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.).

Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.

FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties.

Fluorescence-activated cell sorting (FACS) has rarely been applied to screening of microorganisms because of poor detection resolution, which is compromised by poor stability, toxicity, or interference from background fluorescence of the fluorescence sensors used. Here, a fluorescence-based rapid high-throughput cell sorting method was first developed using a fluorescence resonance energy transfer (FRET) fluorescent nanoprobe NP-RA, which was constructed by coating a silica nanoparticle with Rhodamine B and methyl-red (an azo dye). Rhodamine B (inner layer) is the FRET donor and methyl-red (outer layer) is the acceptor. This ready-to-use NP-RA is non-fluorescent, but fluoresces once the outer layer is degraded by microorganisms. In our experiment, NP-RA was ultrasensitive to model strain Shewanella decolorationis S12, showing a broad detection range from 8.0 cfu/mL to 8.7 × 108 cfu/mL under confocal laser scanning microscopy, and from 1.1 × 107 to 9.36 × 108 cfu/mL under a fluorometer. In addition, NP-RA bioimaging can clearly identify other azo-respiring cells in the microbial community, including Bosea thiooxidans DSM 9653 and Lysinibacillus pakistanensis NCCP-54. Furthermore, the fluorescent probe NP-RA is compatible with downstream FACS so that azo-respiring cells can be rapidly sorted out directly from an artificial microbial community. To our knowledge, no fluorescent nanoprobe has yet been designed for tracking and sorting azo-respiration functional microorganisms.

Bosea caraganae sp. nov. a new species of slow-growing bacteria isolated from root nodules of the relict species Caragana jubata (Pall.) Poir. originating from Mongolia.

Two Gram-stain-negative strains, RCAM04680T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379T, Bosea vaviloviae LMG 28367T, Bosea massiliensis LMG 26221T and Bosea lupini LMG 26383T (the rrs-similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680T showed the highest sequence similarity to the type strain B. lupini LMG 26383T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C18 : 1ω7c (40.8 %). The whole genomes of strains RCAM04680T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680T and B. vaviloviae LMG 28367T. The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680T (=LMG 31125T), is proposed.

Bosea psychrotolerans sp. nov., a psychrotrophic alphaproteobacterium isolated from Lake Michigan water.

Three strains of a Gram-stain negative bacterium were isolated from Lake Michigan water. 16S rRNA gene sequence analysis revealed that strain 1131 had sequence similarities to Bosea vaviloviae LMG 28367T, Bosea lathyri LMG 26379T, Bosea lupini LMG 26383T, Bosea eneae CCUG 43111T, Bosea vestrisii CCUG 43114T and Boseamassiliensis CCUG 43117T of 99.8, 99.1, 98.4, 98.4, 98.4 and 98.2 %, respectively. The average nucleotide identity value between strain 1131T and Bosea vaviloviae Vaf-18T was 93.4 % and the DNA relatedness was 38 %. The primary cellular fatty acids of strain 1131T were C16 : 1ω7c and C18 : 1ω7c. The primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major compound in the quinone system was ubiquinone Q-10 and in the polyamine pattern sym-homospermidine was predominant. Additional phenotypic characteristics included growth at 5-35 °C, pH values of pH 5.5-8.0, a salt tolerance range of 0.0-1.2 % (w/v), and production of an unknown water soluble brown pigment. After phenotypic, chemotaxonomic and genomic analyses, this isolate was identified as a novel species for which the name Bosea psychrotolerans is proposed. The type strain is 1131T (NRRL B-65405=LMG 30034).

Diversity of endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining.

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.

Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant.

The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L(-1) Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L(-1); and biomass dose, 2.0 g·L(-1). Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution.

Extra-slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Preferential association of endophytic bradyrhizobia with different rice cultivars and its implications for rice endophyte evolution.

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice (Oryza sativa) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro (Macroptilium atropurpureum). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia. On the other hand, non-PB strains were able to nodulate Aeschynomene americana, Vigna radiata, and M. atropurpureum but unable to nodulate either A. indica or A. evenia. Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.

Bosea vaviloviae sp. nov., a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia formosa (Stev.) Fed.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

Cloning of a novel 6-chloronicotinic acid chlorohydrolase from the newly isolated 6-chloronicotinic acid mineralizing Bradyrhizobiaceae strain SG-6C.

A 6-chloronicotinic acid mineralizing bacterium was isolated from enrichment cultures originating from imidacloprid-contaminated soil samples. This Bradyrhizobiaceae, designated strain SG-6C, hydrolytically dechlorinated 6-chloronicotinic acid to 6-hydroxynicotinic acid, which was then further metabolised via the nicotinic acid pathway. This metabolic pathway was confirmed by growth and resting cell assays using HPLC and LC-MS studies. A candidate for the gene encoding the initial dechlorination step, named cch2 (for 6-chloronicotinic acid chlorohydrolase), was identified using genome sequencing and its function was confirmed using resting cell assays on E. coli heterologously expressing this gene. The 464 amino acid enzyme was found to be a member of the metal dependent hydrolase superfamily with similarities to the TRZ/ATZ family of chlorohydrolases. We also provide evidence that cch2 was mobilized into this bacterium by an Integrative and Conjugative Element (ICE) that feeds 6-hydroxynicotinic acid into the existing nicotinic acid mineralization pathway.

[Identification of a new heterotrophic nitrobacterium strain Colloides sp. JZ1-1 and its nitrifying capability].

A heterotrophic nitrobacterium strain JZ1-1 with higher nitrifying capability was isolated and mutagenized from an acclimated activated sludge. The JZ1-1 was identified as Colloides sp., according to its morphological and physiological features. The factors affecting the nitrifying capability of JZ1-1 were investigated, including medium carbon source, C/N ratio, pH value, dissolved oxygen, temperature, and ammonium nitrogen concentration. The optimal carbon source was sodium citrate, and the nitrification was favored when the C/N ratio was from 10 to 14, temperature was 30 degrees C, and pH value was 6-9 when cultured at a rotating speed of more than 150 r x min(-1). JZ1-1 could degrade ammonium nitrogen effectively when the initial concentration of ammonium nitrogen was from 100 mg x L(-1) to 500 mg x L(-1). JZ1-1 was stable after 5 generations of subculture.

Tardiphaga robiniae gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from Robinia pseudoacacia in Flanders (Belgium).

Gram-negative, rod-shaped bacteria were isolated from Robinia pseudoacacia root nodules. On the basis of the 16S rRNA gene phylogeny, they are closely related to Bradyrhizobium, Rhodopseudomonas and Nitrobacter species (97% sequence similarity), belonging to the class Alphaproteobacteria and family Bradyrhizobiaceae. The results of physiological and biochemical tests together with sequence analysis of housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) allowed differentiation of this group from other validly published Bradyrhizobiaceae genera. NodA, nodC and nifH genes could not be amplified. On the basis of genotypic and phenotypic data, these organisms represent a novel genus and species for which the name Tardiphaga robiniae gen. nov., sp. nov. (LMG 26467(T)=CCUG 61473(T)), is proposed.

Multilocus sequence analysis of Bosea species and description of Bosea lupini sp. nov., Bosea lathyri sp. nov. and Bosea robiniae sp. nov., isolated from legumes.

Gram-negative, rod-shaped bacteria were isolated from root nodules of Lupinus polyphyllus, Lathyrus latifolius and Robinia pseudoacacia. Based on the 16S rRNA gene phylogeny, they were closely related to Bosea species (100-97 % similarity), belonging to the class Alphaproteobacteria, family Bradyrhizobiaceae. The closest relatives of LMG 26383(T), LMG 26379(T) and LMG 26381(T) were respectively the type strains of Bosea thiooxidans (99.6 %), B. eneae (98.3 %) and B. minatitlanensis (99.0 %). Chemotaxonomic data, including major fatty acid profiles, supported the assignment of our strains to the genus Bosea. Analysis of the concatenated sequences of five housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) and the results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from each other and from the five Bosea species with validly published names. No nodA or nodC genes could be amplified, while nifH PCR gave non-specific products. On the basis of genotypic and phenotypic data, three novel species, Bosea lupini sp. nov. (type strain LMG 26383(T)  = CCUG 61248(T)  = R-45681(T)), Bosea lathyri sp. nov. (type strain LMG 26379(T)  = CCUG 61247(T)  = R-46060(T)) and Bosea robiniae sp. nov. (type strain LMG 26381(T)  = CCUG 61249(T)  = R-46070(T)), are proposed.

Genome sequence of the newly isolated chemolithoautotrophic Bradyrhizobiaceae strain SG-6C.

Strain SG-6C (DSM 23264, CCM 7827) is a chemolithoautotrophic bacterium of the family Bradyrhizobiaceae. It can also grow heterotrophically under appropriate environmental conditions. Here we report the annotated genome sequence of this strain in a single 4.3-Mb circular scaffold.

Complete genome sequences of the chemolithoautotrophic Oligotropha carboxidovorans strains OM4 and OM5.

We report on genome sequencing of Oligotropha carboxidovorans strain OM4 and resequencing of strain OM5. The genomes of both are composed of one chromosome and two plasmids. The presence of two plasmids in the OM5 genome is inconsistent with the previously published sequence, for which only one plasmid was described (D. Paul, S. Bridges, S. Burgess, Y. Dandass, and M. Lawrence, BMC Genomics 11:511, 2010).

Rhizobium rosettiformans sp. nov., isolated from a hexachlorocyclohexane dump site, and reclassification of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium aggregatum comb. nov.

A Gram-negative, rod-shaped, motile, aerobic bacterial strain, W3(T), was isolated from hexachlorocyclohexane (HCH)-contaminated groundwater from Lucknow, India, and its taxonomic position was determined using a polyphasic approach. Strain W3(T) shared highest 16S rRNA gene sequence similarity of 97.8 % with Rhizobium selenitireducens B1(T), followed by Rhizobium daejeonense L61(T) (97.7 %), Rhizobium radiobacter ATCC 19358(T) (97.5 %) and Blastobacter aggregatus IFAM 1003(T) (97.2 %). Strain W3(T) formed a monophyletic clade with Blastobacter aggregatus IFAM 1003(T) ( = DSM 1111(T)) in the cluster of species of the genus Rhizobium. Phylogenetic analyses of strain W3(T) using atpD and recA gene sequences confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Hence, the taxonomic characterization of B. aggregatus DSM 1111(T) was also undertaken. Strains W3(T) and B. aggregatus DSM 1111(T) contained summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c; 65.4 and 70.8 %, respectively) as the major fatty acid, characteristic of the genus Rhizobium. DNA-DNA relatedness of strain W3(T) with Rhizobium selenitireducens LMG 24075(T), Rhizobium daejeonense DSM 17795(T), Rhizobium radiobacter DSM 30147(T) and B. aggregatus DSM 1111(T) was 42, 34, 30 and 34 %, respectively. The DNA G+C contents of strain W3(T) and B. aggregatus DSM 1111(T) were 62.3 and 62.7 mol%, respectively. A nifH gene encoding dinitrogenase reductase was detected in strain W3(T) but not in B. aggregatus DSM 1111(T). Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization and DNA-DNA hybridization, it is concluded that strain W3(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium rosettiformans sp. nov. is proposed (type strain W3(T)  = CCM 7583(T)  = MTCC 9454(T)). It is also proposed that Blastobacter aggregatus Hirsch and Müller 1986 be transferred to the genus Rhizobium as Rhizobium aggregatum comb. nov. (type strain IFAM 1003(T)  = DSM 1111(T)  = ATCC 43293(T)).