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Complete genome of Rhizobium leguminosarum Norway, an ineffective Lotus micro-symbiont


Rhizobia bacteria engage in nitrogen-fixing root nodule symbiosis, a mutualistic interaction with legume plants in which a bidirectional nutrient exchange takes place. Occasionally, this interaction is suboptimal resulting in the formation of ineffective nodules in which little or no atmospheric nitrogen fixation occurs. Rhizobium leguminosarum Norway induces ineffective nodules in a wide range of Lotus hosts. To investigate the basis of this phenotype, we sequenced the complete genome of Rl Norway and compared it to the genome of the closely related strain R. leguminosarum bv. viciae 3841. The genome comprises 7,788,085 bp, distributed on a circular chromosome containing 63% of the genomic information and five large circular plasmids. The functionally classified bacterial gene set is distributed evenly among all replicons. All symbiotic genes (nod, fix, nif) are located on the pRLN3 plasmid. Whole genome comparisons revealed differences in the metabolic repertoire and in protein secretion systems, but not in classical symbiotic genes.


Legume crops are central to sustainable agricultural practices and food security [1, 2]. They have a low need for synthetic nitrogen fertilizers input, as they engage in a symbiosis with a group of diazotrophic bacteria collectively known as rhizobia. This symbiotic interaction is initiated by a molecular crosstalk between rhizobia and their cognate legume host. Upon recognition of specific signals, legume plants intracellularly accommodate rhizobia inside root organs called nodules, where they engage in a bidirectional nutrient exchange [3]. Occasionally, suboptimal interactions establish between the symbiotic partners. These lead to the formation of ineffective nodules in which limited to no nitrogen fixation occurs. These ineffective symbiotic associations are characterized by the formation of small white nodules, which results in reduced or no plant growth promotion [4].

Ineffective nitrogen-fixing symbioses have been described after introduction of crop legumes into areas where previously native legumes grew. The soil microbiota associated to native species can often outcompete inoculant strains [5]. For instance, ineffective nitrogen fixation occurs in fields where perennial and annual clovers co-exist [6, 7]. In field trials, inoculant strains were unable to completely overcome indigenous R. leguminosarum bv. trifolii strains and occupied on average 50% of the nodules [8]. In extreme cases, it has been shown that endogenous rhizobia can completely block the nodulation of introduced rhizobia. For example, the nodulation of pea cultivars Afghanistan and Iran by rhizobial inoculants is suppressed in natural soils by the presence of a non-nodulating strain [9]. However, although ineffective nodulation is a limiting factor for sustainable agriculture, the molecular basis underlying it remains largely unknown [10].

Rhizobium leguminosarum (Rl) strains are cognate micro-symbionts of legumes, including Pisum , Lens , Lathyrus , Vicia , Phaseolus and Trifolium [11]. However, a R. leguminosarum strain isolated from a Lotus corniculatus nodule in Norway exhibits a different compatibility range that includes several Lotus species and ecotypes. Rl Norway does not induce effective nodules in any Lotus species tested so far [12]. Strikingly, there are host genotype specific differences in the nodulation phenotypes induced by Rl Norway, as it cannot induce nodules on L. japonicus Gifu, but induces bumps on L. japonicus Nepal, and white nodules on L. burttii and L. japonicus MG-20. This is in contrast to compatible Mesorhizobium strains that induce monomorphic phenotypes in the same plant ecotypes [12].

The striking diversity of ineffective nodulation phenotypes induced by Rl Norway in Lotus motivated us to sequence and annotate its complete genome, and to compare it to the published genome of R. leguminosarum bv. viciae 3841 (Rlv 3841), a well-characterised R. leguminosarum strain. Here, we show that the genomes are largely conserved. There are no major differences in the nif and fix clusters required for nitrogen fixation and in the nod cluster essential for the production of nodulation factor. However, differences were observed in terms of metabolic and protein secretion system genes.

Organism information

Classification and features

Rl Norway is a Gram-negative strain in the order Rhizobiales of the class Alphaproteobacteria (Table 1). Cells are rod-shaped and have dimensions of 0.84 ± 0.11 μm in width and 1.43 ± 0.31 μm in length (Fig. 1a). This strain is fast growing and forms colonies after 3 days in TY medium at 28 °C. Colonies on TY are circular and slightly domed, their surface is shiny and smooth, and their texture is moderately mucoid (Fig. 1b).

Table 1 Classification and general features of Rl Norway in accordance to the MIGS recommendations [46] published by the Genome Standards Consortium [47]
Fig. 1

Morphological characterisation of Rl Norway. a Phase contrast micrograph of Rl Norway grown in liquid TY medium. Scale bar: 1 μm. b Photomicrograph of the colony morphology of Rl Norway grown on TY medium. Scale bar: 1 mm

The phylogenetic relationship of Rl Norway was inferred based on a concatenated tree of the dnaK, recA, and rpoB house-keeping genes (Fig. 2). Based on this phylogeny Rl Norway is placed within the R. leguminosarum group. The 16S rRNA gene of Rl Norway shows more than 99.9% identity with its orthologs in other R. leguminosarum strains, such as Rlv 3841 and Rl biovar trifolii WSM1325, WSM2304, and CB782.

Fig. 2

Phylogenetic tree showing the relationship between Rl Norway and other Rhizobia. The tree was constructed by maximum likelihood using the concatenated sequences of recA, dnaK, and rpoB. The calculated bootstrap values are indicated at the nodes. Rl Norway is highlighted in bold grey. Type strains are indicated with superscript T. B. japonicum USDA6 was used as an out-group

The metabolic fingerprinting of Rl Norway was conducted with the Biolog GN2 MicroPlate. Rl Norway grew in multiple organic compounds as sole carbon source, these included Adonitol, L-Arabinose, D-Arabitol, D-Cellobiose, D-Fructose, and Glycerol, among others (Additional file 1: Figure S1). The metabolic fingerprinting of this strain was similar to the pattern described for other R. leguminosarum strains, but it was clearly distinct from the pattern of Rlv 3841 (Additional file 1: Figure S1) [13].


Rl Norway was originally co-isolated from a L. corniculatus nodule together with two Mesorhizobium strains, but does not induce nodules in L. corniculatus or L. japonicus Gifu, when inoculated alone [12]. However, it induces bumps on L. japonicus Nepal, and ineffective nodules on L. burttii and L. japonicus MG-20 [12]. This polymorphic nodulation phenotype is not observed, when these hosts are inoculated with Mesorhizobium strains [12]. Rl Norway induces ineffective nodules in Pisum, and Latyrus. The nodulation and symbiotic characteristics of Rl Norway are summarized in Additional file 2: Table S1.

Genome sequencing information

Genome project history

Rl Norway was selected for sequencing, because of the striking diversity of ineffective nodulation phenotypes that it induces in Lotus , a host that belongs to a different cross-inoculation group. The complete genome sequencing was performed at the Genomics Service Unit (LMU Biocenter, Munich). The nucleotide sequences reported in this study have been deposited in the GenBank database under accession numbers CP025012.1, CP025013.1, CP025014.1, CP025015.1, CP025016.1, and CP025017.1. The data is summarized in Table 2.

Table 2 Genome sequencing project information for Rl Norway

Growth conditions and genomic DNA preparation

Rl Norway was grown at 28 °C and 180 rpm for 2 days in TY medium. Genomic DNA was isolated from 30 ml of a bacterial suspension (OD600 = 1.0) using the CTAB method [14]. The DNA quality was determined by nanodrop and gel electrophoresis.

Genome sequencing and assembly

The genome was sequenced using a combination of Illumina and MinION sequencing technologies. Library construction and sequencing were performed at the Genomics Service Unit (LMU Biocenter, Munich). For whole genome sequencing a short read DNA library was generated with the Nextera Kit (Illumina) according to manufacturer’s instructions. Sequencing (2 × 150 bp, v2 chemistry) was performed on a MiSeq sequencer (Illumina) yielding around 15 Mio paired reads and 2.3 Gb of primary sequence. A long read library was prepared with the 1D Genomic DNA Sequencing Kit (Oxford Nanopores) according to manufacturer’s instructions. MinION (Oxford Nanopores) sequencing resulted in around 180,000 sequences with a total of 670 Mb primary sequence (mean length 3.8 kb). Hybrid genome assembly with Unicycler v0.4.0 [15] using default settings resulted in six circular contigs. The average base coverage of the genome is 380x.

Genome annotation

Genome annotation was performed with RAST 2.0 [16, 17] and MicroScope [18]. Clusters of orthologous groups (COGs) of proteins were predicted using the COGNiTOR software [19], signal peptides were detected using the SignalP 4.1 server [20], and Pfam domains were predicted using the Pfam batch sequence search from EMBL-EBI [21]. Transmembrane predictions and CRISPR repeats were determined using the TMHMM Server v. 2.0 [22] and CRISPRFinder [23], respectively. All genes discussed in the text were manually inspected.

Genome properties

The genome of Rl Norway consists of 7,788,085 bp, distributed on a circular chromosome containing 63% of the genomic information and five large circular plasmids ranging from 280 to 1098 kb (Fig. 3). The complete genome and the chromosome are comparable in size to other R. leguminosarum strains [13, 24]. The chromosome contains three identical rRNA operons and 54 tRNA genes, none of which are found on any of the five plasmids (Table 3 and Fig. 3). In total 7866 protein-encoding genes were identified. BUSCO analysis [25] confirmed complete presence of the core bacteria dataset. The six replicons have a comparable mix of functional classes (Additional file 3: Figure S2A). However, all genes from the BUSCO core bacteria dataset are located on the chromosome, with only a few additional gene duplications on the plasmid replicons.

Fig. 3

The chromosome and five plasmids of Rl Norway. The plasmids are depicted to scale with the chromosome one-half of this scale. The outermost circles show protein encoding genes (blue) and rRNA and tRNA genes (red) in clockwise and counter-clockwise orientation. The inner circles indicate deviations in GC content (black) and GC skew (green/purple). Plasmid maps were generated using GCView [61]

Table 3 Genome statistics for Rl Norway

Insights from the genome sequence

Extended insights

The genomes of Rl Norway and Rlv 3841 have a very similar relative occurrence of functional protein encoding genes (Additional file 3: Figure S2B) and do not show any gross genomic alterations. Interestingly, although Rl Norway contains more protein encoding genes than Rlv 3841 (7866 vs. 7263 genes), the number of genes for which a functional annotation could be retrieved is almost identical (6106 vs. 6105 genes). Hence, the major difference lies in the number of not functionally classifiable genes (1760 vs. 1158 genes) (Table 4).

Table 4 Number of genes associated with general COG functional categories

Plasmid repertoire and genospecies classification

The five plasmids contain one set of putative repABC replication system genes each [26]. Comparative analysis of the Rep proteins from Rl Norway with those from Rlv 3841 revealed high identity between plasmids pRLN1 and pRL12, between pRLN2 and pRL11, and between pRLN5 and pRL10 (Fig. 4a). Gene content comparison and synteny analysis supported this result. Although large portions of pRLN4 and pRL9 are similar (Fig. 4b, and c), the RepABC proteins encoded in pRLN4 are more similar to their orthologs in pR132503.

Fig. 4

Genome comparison between Rl Norway and Rlv 3841. a Neighbor-joining tree of Rep proteins from both strains. Protein sequences for RepA, RepB, and RepC from the individual plasmids were aligned and the resulting alignments concatenated for analysis. Rl Norway proteins are depicted in red, Rlv3841 proteins in blue. Bootstrap values indicated on the nodes strongly support the relations between pRLN2 - pRL11, pRLN5 - pRL10, and pRLN1 - pRL12. Only bootstrap values > 70% are depicted. Branch lengths are given in terms of expected numbers of substitutions per nucleotide site. b For whole genome comparison the sequences of the chromosome and plasmids were concatenated for Rl Norway and Rlv 3841 and compared with BlastN in Easyfig 2.2.2 [62]. Levels of sequence identity are indicated by different shades of grey. c Gene contents comparison between the two strains. Depicted are the Rl Norway replicons and their respective homologous regions from the Rlv 3841 replicons. Plasmid maps were generated using BRIG [63]. Colors in the rings are the same as for the Rlv 3841 replicons in (b)

Plasmid pRLN3 is slightly different than the other replicons of Rl Norway (Additional file 3: Figure S2A). It does not exhibit significant similarity to Rlv 3841 (Fig. 4b, and c), has a slightly lower GC content and a lower proportion of protein encoding sequences (Additional file 4: Table S2), and has a higher proportion of putative encoded proteins without known homologs (Additional file 3: Figure S2A). In addition, it is the only plasmid containing potentially active transposons (2 copies) and several incomplete and therefore most likely inactivated transposon copies. The pRLN3 RepABC proteins share high similarity to their orthologs in pRL1.

For genospecies classification, we compared the Rl Norway genome to representatives of the five proposed genospecies (gsA-gsE) [13]. Typically, genomes are regarded to belong to the same species if the ANI values are above 95%. The two highest average nucleotide identity (ANI) scores (Rl CC278f: 96.34%; Rl SM51: 95.59%) were found with members of the genospecies gsD. All other comparisons resulted in ANI scores below 95% (Table 5). The ANI score between Rl Norway and Rlv 3841, which belongs to gsB, is only 93.26%. Although genospecies gsA and Rl CC278f in gsD are not yet well supported [13], the results indicate that Rl Norway belongs to genospecies gsD. This also fits well with Rl Norway having a plasmid subtype combination typical for gsD strains ([13]& personal communication Peter Young).

Table 5 Genome comparison of Rl Norway with members of the five genospecies and the respective ANI scores

Central metabolism

In terms of central metabolic genes Rl Norway resembles Rlv 3841. Both strains harbour genes encoding enzymes of the tricarboxylic acid (TCA) cycle required for aerobic respiration and energy production [27], of the pentose phosphate pathway required for the oxidation of glucose and the synthesis of nucleotides [28], and of the Entner-Doudoroff pathway for the catabolism of glucose to pyruvate [29]. Both strains lack a gene encoding the phosphofructokinase, an essential enzyme of the Embden-Meyerhof-Parnas glycolysis. These genetic similarities were reflected in a similar growth pattern in different carbon sources using Biolog GN2 MicroPlates (Additional file 1: Figure S1) [13].

A noticeable difference in the Biolog assay was the assimilation of amino acids such as D- and L-alanine, L-serine and L-proline, and nucleosides. However, no major differences were observed in the genes mediating their metabolism. The only clear exceptions were that Rl Norway lacks a putative D-serine deaminase required for the conversion of D-serine to pyruvate, but contains two putative aspartate ammonia-lyases (CUJ84_pRLN3000095, CUJ84_pRLN3000303) and two putative asparagine synthetases (CUJ84_pRLN3000485, CUJ84_pRLN3000155). In terms of amino acid transport, two ABC-type broad specificity amino-acid transporters have been characterized in Rlv 3841, Aap (AapJQMP) and Bra (BraDEFGC) [30]. The bra (CUJ84_Chr003782–3787) and aap (CUJ84_Chr001810–1813) clusters are highly conserved in Rl Norway. Another interesting difference concerned the metabolism of butanoate. In contrast to Rlv 3841, Rl Norway did not grow on γ-hydroxybutyric acid (Additional file 1: Figure S1). This is supported by the lack of a gene cluster (pRL100133–138 in Rlv 3841) associated to γ-hydroxybutyrate utilisation [13]. Furthermore, Rl Norway harbours an ortholog to the phbC1 gene (CUJ84_Chr001779), but lacks phbC2. These genes encode type I and type III poly-β-hydroxybutyrate (PHB) synthases, which are required for free-living and bacteroid PHB biosynthesis, respectively [31].

Secretion systems

Gram-negative bacteria secrete a suite of proteins via macromolecular complexes that have been classified as type 1–6 secretion systems in addition to the sec and tat transport systems [32]. A survey of the Rl Norway genome indicates that this strain contains a large repertoire of secretion systems that is distinct from the repertoire of Rlv 3841 (Table 6). Rl Norway harbours five putative type 1 secretion systems (T1SS; Table 6). T1SSa, T1SSb and T1SSc are unique to Rl Norway. Interestingly, the genes encoding the T1SSa and T1SSc systems form operons with two large genes encoding putative repeats-in-toxin (RTX) toxins. The proteins forming the T1SSd and T1SSe have orthologs with more than 90% identity in Rlv 3841. For instance, the T1SSd proteins are orthologous to the PrsD and PrsE proteins of Rlv 3841 that are required for biofilm formation [33]. Like Rlv 3841, Rl Norway lacks T2SS and T3SS, but harbours T4SS and T6SS [34].

Table 6 Secretion system repertoire in Rl Norway

Bacteria utilize T3SS, T4SS and/or T6SS to inject effector proteins directly into eukaryotic host cells or into other bacteria [35,36,37]. In rhizobia, these effectors can mediate compatibility with the host [38]. Rl Norway harbours a putative T4SS that is distinct from the T4SS from Rlv 3841. The respective T4SS encoding virB operons are not syntenic and the encoding genes share on average less than 30% identity. The T4SS of Rl Norway is encoded in the pRLN1 plasmid and is predicted to translocate proteins and not DNA, as Rl Norway lacks a VirD2 relaxase [39]. In addition, it has the peculiarity that the virB11 gene is partially duplicated and two genes are located in-between the duplication.

Rl Norway and Rlv 3841 harbour syntenic imp (tss) and hcp clusters encoding type (i) T6SS. In both cases the imp cluster is lacking orthologs to the evpJ and tssJ genes. However, a comparison to Agrobacterium tumefaciens C58 revealed that these genes are also absent in the corresponding imp and hcp operons (atu4330-atu4352). In addition, all essential genes for protein secretion are conserved [40].

T5SS are structures in which the cargo protein translocates itself across the plasma membrane. These are classified into auto-transporters (translocator and cargo encoded in the same gene) and two-partner systems (translocator and cargo are encoded by two separate genes) [41]. Rl Norway harbours two T5SS auto-transporters. However, T5SSb is split into two genes and it is probably not a bona fide T5SS. Rl Norway also has one two-partner system, in which the cargo protein is a putative filamentous hemagglutinin (Table 6). In contrast, Rlv 3841 contains three auto-transporters, but no two-partner system [34].

Symbiotic gene repertoire

Plasmid pRLN3 harbours all symbiotic genes in Rl Norway. The nod genes that are required for the synthesis and export of the nodulation factor, a key determinant in compatibility, are organised in one cluster (CUJ84_pRLN3000416–426) comprising the nodJICBADFELMN genes. They have the same organisation as the nod cluster in Rlv 3841 [24], and the encoded proteins share at least 93.6% identity with their Rlv 3841 orthologs. However, in contrast to Rlv 3841, Rl Norway lacks nodO and nodT orthologs in the proximity of the nod cluster. Interestingly, genes encoding putative transposases flank the Rl Norway nod cluster. The genes required for nitrogen fixation are located in proximity. The fixABCX (CUJ84_pRLN3000397–400) and the nifAB genes (CUJ84_pRLN3000401–402) are located almost directly downstream nodJ, whereas nifNEKDH (CUJ84_pRLN3000271–275), fixSIHG (CUJ84_pRLN3000258–261) and fixPQON (CUJ84_pRLN3000263–266) are located approximately 137.5 kb downstream of nodJ. The three subunits of the nitrogenase encoded by the nifHDK genes share 99.7, 93.5, and 96.3% identity to their respective Rlv 3841 orthologs. A noteworthy difference between both strains is that Rl Norway harbours a single fixNOQP operon encoding the essential cbb3 terminal oxidase, whereas Rlv 3841 contains two copies [24]. Furthermore, Rl Norway lacks genes encoding the FixK and FixL transcriptional regulators, which together with FnrN control the expression of the nitrogen fixation genes in other rhizobia strains [42]. Instead, Rl Norway harbours two putative fnrN genes (CUJ84_Chr002641, CUJ84_pRLN3000544) that are located in the chromosome and in the pRLN3 symbiotic plasmid. This is reminiscent of R. leguminosarum bv. viciae UPM791, in which FnrN is the global regulator of the fix genes. In this strain, FnrN is regulated by micro-aerobic conditions and binds a palindromic element called anaerobox [43, 44]. Putative anaerobox sequences were found upstream of fnrN1 (CUJ84_Chr002641) and the fixNOQP and fixGHIS operons, which suggest that FnrN might regulate their expression in Rl Norway. However, no anaerobox was found upstream of fnrN2 (CUJ84_pRLN3000544). Interestingly, fnrN2 is approximately 16.5 kb upstream of a putative uptake hydrogenase cluster comprising 18 genes (CUJ84_pRLN3000511–528). The cluster organisation resembles the hup and hyp genes from Rlv UPM791 [45]. Notably, Rlv 3841 lacks such a hydrogenase cluster.


Although detrimental in agriculture, ineffective nitrogen-fixing symbiosis remains poorly investigated. In this regard, Rl Norway is an interesting strain as it exhibits a parasitic behaviour in a wide range of hosts. Comparative genomic analyses with other R. leguminosarum strains have the potential to reveal novel factors mediating symbiotic compatibility and efficiency.



Clusters of orthologous groups


Cetyl trimethylammonium bromide


Damage associated molecular patterns



Rl :

Rhizobium leguminosarum




Type 1 secretion system


Tricarboxylic acid


  1. 1.

    Stagnari F, Maggio A, Galieni A, Pisante M. Multiple benefits of legumes for agriculture sustainability: an overview. Chem Biol Technol Agric. 2017;4(1):2.

    Article  Google Scholar 

  2. 2.

    Rubiales D, Mikic A. Introduction: legumes in sustainable agriculture. Critl Rev Plant Sci. 2015;34(1–3):2–3.

    Article  Google Scholar 

  3. 3.

    Oldroyd GE, Murray JD, Poole PS, Downie JA. The rules of engagement in the legume-rhizobial symbiosis. Annu Rev Genet. 2011;45:119–44.

    CAS  Article  Google Scholar 

  4. 4.

    Westhoek A, Field E, Rehling F, Mulley G, Webb I, Poole PS, Turnbull LA. Policing the legume-Rhizobium symbiosis: a critical test of partner choice. Sci Rep. 2017;7(1):1419.

    Article  Google Scholar 

  5. 5.

    Streeter JG. Failure of inoculant rhizobia to overcome the dominance of indigenous strains for nodule formation. Can J Microbiol. 1994;40(7):513–22.

    Article  Google Scholar 

  6. 6.

    Parker DT, Allen ON. The nodulation status of Trifolium ambiguum. Soil Sci Soc Am Pro. 1952;16(4):350–3.

    Article  Google Scholar 

  7. 7.

    Howieson JG, Yates RJ, O'Hara GW, Ryder M, Real D. The interactions of Rhizobium leguminosarum biovar trifolii in nodulation of annual and perennial Trifolium spp. from diverse centres of origin. Aust J Exp Agric. 2005;45(3):199–207.

    Article  Google Scholar 

  8. 8.

    Amesgottfred NP, Christie BR. Competition among strains of Rhizobium leguminosarum biovar trifolii and use of a diallel analysis in assessing competition. Appl Environ Microb. 1989;55(6):1599–604.

    CAS  Google Scholar 

  9. 9.

    Winarno R, Lie TA. Competition between Rhizobium strains in nodule formation - interaction between nodulating and non-nodulating strains. Plant Soil. 1979;51(1):135–42.

    Article  Google Scholar 

  10. 10.

    Bohlool BB, Ladha JK, Garrity DP, George T. Biological nitrogen fixation for sustainable agriculture - a perspective. Plant Soil. 1992;141(1–2):1–11.

    CAS  Article  Google Scholar 

  11. 11.

    Fred EB, Baldwin IL, McCoy E. Root nodule bacteria and leguminous plants; 1932.

    Google Scholar 

  12. 12.

    Gossmann JA, Markmann K, Brachmann A, Rose LE, Parniske M. Polymorphic infection and organogenesis patterns induced by a Rhizobium leguminosarum isolate from Lotus root nodules are determined by the host genotype. New Phytol. 2012;196(2):561–73.

    Article  Google Scholar 

  13. 13.

    Kumar N, Lad G, Giuntini E, Kaye ME, Udomwong P, Shamsani NJ, Young JPW, Bailly X. Bacterial genospecies that are not ecologically coherent: population genomics of Rhizobium leguminosarum. Open Biol. 2015;5(1):140133.

    Article  Google Scholar 

  14. 14.

    Murray MG, Thompson WF. Rapid isolation of high molecular-weight plant DNA. Nucleic Acids Res. 1980;8(19):4321–5.

    CAS  Article  Google Scholar 

  15. 15.

    Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595.

    Article  Google Scholar 

  16. 16.

    Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, et al. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res. 2014;42(D1):D206–14.

    CAS  Article  Google Scholar 

  17. 17.

    Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 2008;9(1):75.

    Article  Google Scholar 

  18. 18.

    Vallenet D, Calteau A, Cruveiller S, Gachet M, Lajus A, Josso A, Mercier J, Renaux A, Rollin J, Rouy Z, et al. MicroScope in 2017: an expanding and evolving integrated resource for community expertise of microbial genomes. Nucleic Acids Res. 2017;45(D1):D517–28.

    CAS  Article  Google Scholar 

  19. 19.

    Tatusov RL, Koonin EV, Lipman DJ. A genomic perspective on protein families. Science. 1997;278:631–7.

    CAS  Article  Google Scholar 

  20. 20.

    Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8:785.

    CAS  Article  Google Scholar 

  21. 21.

    Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44(D1):D279–85.

    CAS  Article  Google Scholar 

  22. 22.

    Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80.

    CAS  Article  Google Scholar 

  23. 23.

    Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nuclein Acids Res. 2007;35:W52–7.

    Article  Google Scholar 

  24. 24.

    Young JP, Crossman LC, Johnston AW, Thomson NR, Ghazoui ZF, Hull KH, Wexler M, Curson AR, Todd JD, Poole PS, et al. The genome of Rhizobium leguminosarum has recognizable core and accessory components. Genome Biol. 2006;7(4):R34.

    Article  Google Scholar 

  25. 25.

    Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31(19):3210–2.

    Article  Google Scholar 

  26. 26.

    Ramírez-Romero MA, Soberón N, Pérez-Oseguera A, Téllez-Sosa J, Cevallos MA. Structural elements required for replication and incompatibility of the Rhizobium etli symbiotic plasmid. J Bacteriol. 2000;182(11):3117–24.

    Article  Google Scholar 

  27. 27.

    Dunn MF. Tricarboxylic acid cycle and anaplerotic enzymes in rhizobia. FEMS Microbiol Rev. 1998;22(2):105–23.

    CAS  Article  Google Scholar 

  28. 28.

    Stincone A, Prigione A, Cramer T, Wamelink MMC, Campbell K, Cheung E, Olin-Sandoval V, Gruning NM, Kruger A, Alam MT, et al. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biol Rev. 2015;90(3):927–63.

    Article  Google Scholar 

  29. 29.

    Conway T. The Entner-Doudoroff pathway - history, physiology and molecular biology. FEMS Microbiol Lett. 1992;103(1):1–28.

    CAS  Article  Google Scholar 

  30. 30.

    Hosie AHF, Allaway D, Galloway CS, Dunsby HA, Poole PS. Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (bra/LIV) of the ABC family. J Bacteriol. 2002;184(15):4071–80.

    CAS  Article  Google Scholar 

  31. 31.

    Terpolilli JJ, Masakapalli SK, Karunakaran R, Webb IUC, Green R, Watmough NJ, Kruger NJ, Ratcliffe RG, Poole PS. Lipogenesis and redox balance in nitrogen-fixing pea bacteroids. J Bacteriol. 2016;198(20):2864–75.

    CAS  Article  Google Scholar 

  32. 32.

    Green E, Mecsas J. Bacterial secretion systems: an overview. In: Kudva I, Cornick N, Plummer P, Zhang Q, Nicholson T, Bannantine J, Bellaire B, editors. Virulence mechanisms of bacterial pathogens, Fifth Edition. Washington, DC: ASM Press; 2016. p. 215-39.

  33. 33.

    Russo DM, Williams A, Edwards A, Posadas DM, Finnie C, Dankert M, Downie JA, Zorreguieta A. Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum. J Bacteriol. 2006;188(12):4474–86.

    CAS  Article  Google Scholar 

  34. 34.

    Krehenbrink M, Downie JA. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae. BMC Genomics. 2008;9(1):55.

    Article  Google Scholar 

  35. 35.

    Cianfanelli FR, Monlezun L, Coulthurst SJ. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol. 2016;24(1):51–62.

    CAS  Article  Google Scholar 

  36. 36.

    Ding ZY, Atmakuri K, Christie PJ. The outs and ins of bacterial type IV secretion substrates. Trends Microbiol. 2003;11(11):527–35.

    CAS  Article  Google Scholar 

  37. 37.

    Deng WY, Marshall NC, Rowland JL, McCoy JM, Worrall LJ, Santos AS, Strynadka NCJ, Finlay BB. Assembly, structure, function and regulation of type III secretion systems. Nat Rev Microbiol. 2017;15(6):323–37.

    CAS  Article  Google Scholar 

  38. 38.

    Nelson MS, Sadowsky MJ. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. Front Plant Sci. 2015;6:491.

    Article  Google Scholar 

  39. 39.

    Alvarez-Martinez CE, Christie PJ. Biological diversity of prokaryotic type IV secretion systems. Microbiol Mol Biol Rev. 2009;73(4):775–808.

    CAS  Article  Google Scholar 

  40. 40.

    Ma LS, Hachani A, Lin JS, Filloux A, Lai EM. Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe. 2014;16(1):94–104.

    CAS  Article  Google Scholar 

  41. 41.

    Abby SS, Cury J, Guglielmini J, Neron B, Touchon M, Rocha EP. Identification of protein secretion systems in bacterial genomes. Sci Rep. 2016;6:23080.

    CAS  Article  Google Scholar 

  42. 42.

    Fischer HM. Genetic regulation of nitrogen-fixation in rhizobia. Microbiol Rev. 1994;58(3):352–86.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Gutiérrez D, Hernando Y, Palacios JM, Imperial J, Ruiz-Argüeso T. FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum bv. viciae UPM791. J Bacteriol. 1997;179(17):5264–70.

    Article  Google Scholar 

  44. 44.

    Colombo MV, Gutiérrez D, Palacios JM, Imperial J, Ruiz-Argüeso T. A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv. viciae. Mol Microbiol. 2000;36(2):477–86.

    CAS  Article  Google Scholar 

  45. 45.

    Baginsky C, Brito B, Imperial J, Palacios JM, Ruiz-Argüeso T. Diversity and evolution of hydrogenase systems in rhizobia. Appl Environ Microbiol. 2002;68(10):4915–24.

    CAS  Article  Google Scholar 

  46. 46.

    Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541–7.

    CAS  Article  Google Scholar 

  47. 47.

    Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, Gilbert J, Glockner FO, Hirschman L, Karsch-Mizrachi I, et al. The genomic standards consortium. PLoS Biol. 2011;9(6):e1001088.

    CAS  Article  Google Scholar 

  48. 48.

    Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains archaea, Bacteria, and Eucarya. PNAS. 1990;87(12):4576–9.

    CAS  Article  Google Scholar 

  49. 49.

    Garrity GM, Bell JA, Lilburn T, Phylum XIV. Proteobacteria phyl. nov., vol. 2, Part B, 2 edn. New York: Springer; 2005.

    Google Scholar 

  50. 50.

    Euzéby J. Validation list no. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006;56:1–6.

    Article  Google Scholar 

  51. 51.

    Garrity GM, Bell JA, Lilburn T, Class I. Alphaproteobacteria class. nov., vol. 2, Part C. 2nd ed. New York: Springer; 2005.

    Google Scholar 

  52. 52.

    Kuykendall LD. Order VI. Rhizobiales ord. nov. In: Bergey’s Manual of Systematic Bacteriology. Edited by Garrity GM, Brenner DJ, Kreig NR, Staley JT, vol. 2, Part C, 2 edn. New York: Springer; 2005: 324.

  53. 53.

    Kuykendall LD. Rhizobiaceae. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. New York: Springer; 2005.

    Google Scholar 

  54. 54.

    Conn HJ. Taxonomic relationships of certain non-sporeforming rods in soil. J Bacteriol. 1938;36:320–1.

    Google Scholar 

  55. 55.

    Skerman VBD, Mcgowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol. 1980;30(1):225–420.

    Article  Google Scholar 

  56. 56.

    Kuykendall LD, J.M. Y, Martínez-Romero E, Kerr A, Sawada H. Rhizobium. In: Bergey’s manual of systematic bacteriology. Edited by Garrity GM, Brenner DJ, Kreig NR, Staley JT. New York: Springer; 2005.

  57. 57.

    Frank B. Über die Pilzsymbiose der Leguminosen. Ber Dtsch Bot Ges. 1889;7:332–46.

    Google Scholar 

  58. 58.

    Young JM, Kuykendall LD, Martinez-Romero E, Kerr A, Sawada H. A revision of Rhizobium frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol 2001, 51(Pt 1):89–103.

  59. 59.

    Ramírez-Bahena MH, Garcia-Fraile P, Peix A, Valverde A, Rivas R, Igual JM, Mateos PF, Martinez-Molina E, Velazquez E. Revision of the taxonomic status of the species Rhizobium leguminosarum (frank 1879) frank 1889AL, Rhizobium phaseoli Dangeard 1926AL and Rhizobium trifolii Dangeard 1926AL. R. trifolii is a later synonym of R. leguminosarum. Reclassification of the strain R. leguminosarum DSM 30132 (=NCIMB 11478) as Rhizobium pisi sp. nov. Int J Syst Evol Microbiol. 2008;58(Pt 11):2484–90.

    Article  Google Scholar 

  60. 60.

    Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. the Gene Ontology Consortium. Nat Genet. 2000;25(1):25–9.

    CAS  Article  Google Scholar 

  61. 61.

    Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005;21(4):537–9.

    CAS  Article  Google Scholar 

  62. 62.

    Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27(7):1009–10.

    CAS  Article  Google Scholar 

  63. 63.

    Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST ring image generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12(1):1.

    Article  Google Scholar 

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We thank Martin Parniske for providing the Rl Norway strain and critical reading of the manuscript, Moritz Thomas for expert technical assistance in MinION library preparation and sequencing, and Marion Cerri for insightful discussions and reading the manuscript. We specially thank Peter Young for sharing unpublished sequences and fruitful discussions.


This work was funded by the German research foundation (DFG-grant: MA7269–1).

Author information




JL performed the imaging, the chemotaxonomic analyses, and extracted the genomic DNA. AB conducted the genome sequencing and annotation. AH conducted the genome assembly and comparisons. MM conducted the phylogenetic analysis and the manual inspection of the annotation. MM and AB conceived the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Macarena Marín.

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Additional files

Additional file 1:

Figure S1. RI Norway substrate utilization pattern determined by Biolog. In blue and yellow are indicated substrates only utilized by RI Norway and Rlv 3841, respectively. Green indicates substrates used by both strains, whereas white depicts conditions in which both strains did not grow. Rlv 3841 utilization pattern was extracted from [1]. (TIF 9702 kb)

Additional file 2:

Table S1. Nodulation phenotypes of Rl Norway on selected hosts. (DOCX 68 kb)

Additional file 3:

Figure S2. Distribution of functional classes of protein encoding genes within the RI Norway genome. (A) Functional class distribution across the six RI Norway replicons. (B) Comparison of the relative occurrence of functionally classified protein encoding genes between the RI Norway and Rlv 3841 genomes. Functional annotation (COG) was performed on WebMGA server [1]. (TIF 10046 kb)

Additional file 4:

Table S2. Genome statistics for Rl Norway. (DOCX 47 kb)

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Liang, J., Hoffrichter, A., Brachmann, A. et al. Complete genome of Rhizobium leguminosarum Norway, an ineffective Lotus micro-symbiont. Environmental Microbiome 13, 36 (2018).

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  • Symbiosis
  • Rhizobium
  • Legume
  • Ineffective nodulation
  • Genome