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A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95–96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, the authors identify a thermosensitive enzyme that synthesizes c-di-GMP and modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa.

The contribution of biological nitrogen fixation to the total N requirement of food and feed crops diminished in importance with the advent of synthetic N fertilizers, which fueled the “green revolution”. Despite being environmentally unfriendly, the synthetic versions gained prominence primarily due to their low cost, and the fact that most important staple crops never evolved symbiotic associations with bacteria. In the recent past, advances in our knowledge of symbiosis and nitrogen fixation and the development and application of recombinant DNA technology have created opportunities that could help increase the share of symbiotically-driven nitrogen in global consumption. With the availability of molecular biology tools, rapid improvements in symbiotic characteristics of rhizobial strains became possible. Further, the technology allowed probing the possibility of establishing a symbiotic dialogue between rhizobia and cereals. Because the evolutionary process did not forge a symbiotic relationship with the latter, the potential of molecular manipulations has been tested to incorporate a functional mechanism of nitrogen reduction independent of microbes. In this review, we discuss various strategies applied to improve rhizobial strains for higher nitrogen fixation efficiency, more competitiveness and enhanced fitness under unfavorable environments. The challenges and progress made towards nitrogen self-sufficiency of cereals are also reviewed. An approach to integrate the genetically modified elite rhizobia strains in crop production systems is highlighted.

Cosmids carrying genes involved in utilization of rhamnose, sorbitol, and adonitol were isolated from a genomic library of Rhizobium leguminosarum by complementation of plasmid-cured derivatives of strain Rlt100 that were unable to grow on these carbon sources. Transposon mutagenesis was used to identify regions of each cosmid necessary for catabolism of the respective carbon source; partial DNA sequencing, as well as analysis of gene fusions created with transposon Tn5-B20, helped to determine the orientation and possible function of genes required for growth on the three substrates. Representative Tn5 insertions in the cosmids were recombined into the wild-type strain Rlt100 by gene replacement to generate isogenic strains unable to use either rhamnose, sorbitol, or adonitol. These strains were tested for their nodulation competitiveness compared with Rlt100 in co-inoculation experiments on clover plants. While sorbitol and adonitol catabolic mutants were unaltered in their competitive behavior, the nodulation competitiveness of three different rhamnose utilization mutants was significantly impaired. This result, coupled with the fact that the rhamnose catabolic genes were inducible by clover root extracts, suggests an important role for rhamnose catabolism in the early stages of the interaction of R. leguminosarum with clover plants. Hybridization studies with probes derived from the rhamnose, sorbitol, and adonitol catabolic loci demonstrated that these genes are plasmid encoded in virtually all R. leguminosarum strains, including representatives from all three biovars from a variety of different geographic locations.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the hemecopper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixNgusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation.

This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures. The pairs of plasmids p42f-pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of p42c with pRL10 are shown to be similar, whereas the symbiotic plasmids (p42d and pRL10) are structurally unrelated and seem to follow distinct evolutionary paths. Even though purifying selection is acting on the whole genome, the accessory component is evolving more rapidly. This component is constituted largely for proteins for transport of diverse metabolites and elements of external origin. The present analysis allows us to conclude that a heterogeneous and quickly diversifying group of plasmids co-exists in a common genomic framework.