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 26383T, LMG 26379T and LMG 26381T 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 26383T = CCUG 61248T = R-45681T), Bosea lathyri sp. nov. (type strain LMG 26379T = CCUG 61247T = R-46060T) and Bosea robiniae sp. nov. (type strain LMG 26381T = CCUG 61249T = R-46070T), are proposed.
We investigated symbiotic and physiological properties, and taxonomic position, of 23 bacterial strains isolated from Lebeckia ambigua root nodules collected from the South African fynbos region. The ...capacity for nodulation and nitrogen fixation on three provenances of L. ambigua was investigated for these strains together with several physiological characters, including growth rate on peat and in betonite clay, survival on polyethylene beads and pH tolerance. Additionally, the 16S rRNA gene phylogeny was determined. The root nodule bacteria isolated clustered in five different groups belonging to the genus Burkholderia, most closely related to B. caledonica, B. graminis and B. tuberum. Moreover there was a very strong influence of collection site on the taxonomy of the Burkholderia strains. The physiological characterisation revealed two promising strains, WSM4174 and WSM4184, achieved rapid growth in normal media and reached high, stable numbers in sterile peat. However, there was a worrying susceptibility to desiccation amongst these Burkholderia. Additionally, evidence was found for isolation of non-symbiotic strains from the nodule material collected in South Africa.
► Bacteria isolated from Lebeckia ambigua root nodules from the South African fynbos. ► Belonging to the genus Burkholderia. ► Strong influence of collection site on the taxonomy of the strains. ► Two promising strains but susceptible to desiccation. ► Evidence was found for isolation of non-symbiotic strains.
Advancement of DNA sequencing technology allows the routine use of genome sequences in the various fields of microbiology. The information held in genome sequences proved to provide objective and ...reliable means in the taxonomy of prokaryotes. Here, we describe the minimal standards for the quality of genome sequences and how they can be applied for taxonomic purposes.
Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive ...and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.
We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on ...rep-PCR clustering, 16S rRNA gene and
recA gene sequence analysis, these isolates belonged to
Bradyrhizobium,
Ensifer (Sinorhizobium),
Mesorhizobium and
Rhizobium. Of the genera encountered,
Rhizobium was the most abundant (62%) and especially the species
Rhizobium leguminosarum, followed by
Ensifer (19%),
Bradyrhizobium (14%) and finally
Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and
recA genes, suggesting that these may represent a new genus with close relationship to
Rhodopseudomonas and
Bradyrhizobium. Primers for the symbiotic genes
nodC and
nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new
nodC types were assigned to strains originating from
Ononis,
Robinia and
Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.
► Diversity of rhizobia isolated from indigenous legumes in Flanders. ► They belong to
Bradyrhizobium,
Ensifer,
Mesorhizobium and
Rhizobium. ► Possibly new genus found with close relationship to
Rhodopseudomonas and
Bradyrhizobium. ► Different symbiovars encountered and three new
nodC types were assigned. ► The ecoregion did not contribute to the separation of the bacterial endosymbionts.
Five strains of Gram-stain-negative, rod-shaped bacteria were isolated from Carmichaelia and Montigena root nodules. Based on 16S rRNA gene phylogeny, they were shown to belong to the genus ...Mesorhizobium, and to be most closely related to Mesorhizobium jarvisii ATCC 33669
(100-99.6 % sequence similarity), Mesorhizobium huakuii IAM 14158
(99.9-99.6 %), Mesorhizobium japonicum MAFF303099
(99.8-99.6 %) and Mesorhizobium erdmanii USDA 3471
(99.8-99.5 %). Additionally, the strains formed distinct groups based on housekeeping gene analysis and were most closely related to M. jarvisii ATCC 33669
(89.6-89.5 and 97.6-97.3 % sequence similarity for glnII and recA, respectively), M. erdmanii USDA 3471
(94.3-94.0 and 94.9-94.1 %), M. japonicum MAFF303099
(90.0-89.9 and 96.7-96.2 %) and M. huakuii IAM 14158
(89.9-90.0 and 95.4-94.9 %). Chemotaxonomic data supported the assignment of the strains to the genus Mesorhizobium and DNA-DNA hybridizations, average nucleotide identity analysis, matrix-assisted laser desorption ionization time-of-flight MS analysis, physiological and biochemical tests differentiated them genotypically and phenotypically from their nearest neighbouring species. Therefore, these strains are considered to represent a novel species, for which the name Mesorhizobium carmichaelinearum sp. nov. is proposed. The type strain is ICMP 18942
(=MonP1N1
=LMG 28414
).
Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, ...USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with ≥96.1 % sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6T and WSM3557T was Microvirga flocculans TFBT, with 97.6–98.0 % similarity, while WSM3693T was most closely related to Microvirga aerilata 5420S-16T, with 98.8 % similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6T, WSM3557T and WSM3693T within the genus Microvirga. DNA–DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6T, WSM3557T and WSM3693T from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6T was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100 % identical nodA sequences of WSM3557T and WSM3693T clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6T, WSM3557T and WSM3693T were most closely related to that of Rhizobium etli CFN42T nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6T = LMG 26460T = HAMBI 3236T), Microvirga lotononidis sp. nov. (type strain WSM3557T = LMG 26455T = HAMBI 3237T) and Microvirga zambiensis sp. nov. (type strain WSM3693T = LMG 26454T = HAMBI 3238T).
Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown ...phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.
Seven Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene phylogeny, they were shown to belong to the ...genus Burkholderia, with the representative strain WSM5005T being most closely related to Burkholderia tuberum (98.08 % sequence similarity). Additionally, these strains formed a distinct group in phylogenetic trees based on the housekeeping genes gyrB and recA. Chemotaxonomic data including fatty acid profiles and analysis of respiratory quinones supported the assignment of the strains to the genus Burkholderia. Results of DNA–DNA hybridizations, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from the closest species of the genus Burkholderia with a validly published name. Therefore, these strains represent a novel species for which the name Burkholderia sprentiae sp. nov. (type strain WSM5005T = LMG 27175T = HAMBI 3357T) is proposed.
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