Open Access

Permanent draft genome sequences of the symbiotic nitrogen fixing Ensifer meliloti strains BO21CC and AK58

  • Marco Galardini
  • , Marco Bazzicalupo
  • , Emanuele Biondi
  • , Eveline Brambilla
  • , Matteo Brilli
  • , David Bruce
  • , Patrick Chain
  • , Amy Chen
  • , Hajnalka Daligault
  • , Karen Walston Davenport
  • , Shweta Deshpande
  • , John C. Detter
  • , Lynne A. Goodwin
  • , Cliff Han
  • , James Han
  • , Marcel Huntemann
  • , Natalia Ivanova
  • , Hans-Peter Klenk
  • , Nikos C. Kyrpides
  • , Victor Markowitz
  • , Kostas Mavrommatis
  • , Stefano Mocali
  • , Matt Nolan
  • , Ioanna Pagani
  • , Amrita Pati
  • , Francesco Pini
  • , Sam Pitluck
  • , Giulia Spini
  • , Ernest Szeto
  • , Hazuki Teshima
  • , Tanja Woyke
  • and Alessio Mengoni
Corresponding author

DOI: 10.4056/sigs.3797438

Received: 15 December 2013

Accepted: 15 December 2013

Published: 20 December 2013


Ensifer (syn. Sinorhizobium) meliloti is an important symbiotic bacterial species that fixes nitrogen. Strains BO21CC and AK58 were previously investigated for their substrate utilization and their plant-growth promoting abilities showing interesting features. Here, we describe the complete genome sequence and annotation of these strains. BO21CC and AK58 genomes are 6,985,065 and 6,974,333 bp long with 6,746 and 6,992 genes predicted, respectively.


AerobicmotileGram-negativemesophilicchemoorganotrophicchemoautotrophicsoilplant symbiontbiological nitrogen fixationEnsifer (Sinorhizobium) melilotilegume yield


Strains AK58 and BO21CC belong to the species Ensifer (syn. Sinorhizobium) meliloti (Alphaproteobacteria, Rhizobiales, Rhizobiaceae, Sinorhizobium/Ensifer group) [1,2], an important symbiotic nitrogen fixing bacterial species that associates with roots of leguminous plants of several genera, mainly from Melilotus, Medicago and Trigonella [3]. These strains have been originally isolated from Medicago spp. during a long course experiment (BO21CC) and from plants collected in the north Aral sea region (Kazakhstan) (AK58). Previous analyses conducted by comparative genomic hybridization (CGH), nodulation tests and Phenotype Microarray™(Biolog, Inc.) showed that AK58 (= DSM 23808) and BO21CC (= DSM 23809) are highly diverse in both genomic and phenotypic properties. In particular, they show different symbiotic phenotypes with respect to the crop legume Medicago sativa L [4,5]. In a previous collaboration with DOE-JGI, the genomes of strains AK83 (= DSM 23913) and BL225C (= DSM 23914) were also sequenced, allowing the identification of putative genetic determinants for their different symbiotic phenotypes [6]. Consequently, interest in strains AK58 and BO21CC arose, since genomic analysis of these strains would foster a greater understanding of the E. meliloti pangenome [7], and facilitate deeper investigation of the genomic determinants responsible for differences in symbiotic performances between E. meliloti strains found in nature. These research goals may lead to improved strain selection and better inoculants of the legume crop M. sativa.

Classification and features

Representative genomic 16S rRNA sequences of strains AK58 and BO21CC were compared with those present in the Ribosomal Database by using Match Sequence module of Ribosomal Database Project [8]. Representative genomic 16S rRNA sequences of closer phylogenetic relatives of the genus Ensifer/Sinorhizobium and of Rhizobiales family (as outgroup) were then selected from IMG-ER database [Table 1] [16]. All strains from the genus Ensifer/Sinorhizobium form a close cluster, including strains AK58 and BO21CC, thus confirming the affiliation of these two strains within the species. Figure 1 shows the phylogenetic neighborhood of E. meliloti AK58 and BO21CC in a 16S rRNA based tree.

Table 1

Classification and general features of E. meliloti AK58 and BO21CC according to the MIGS recommendations [9] and the Names for Life database [10]




     Evidence code

      Current classification

     Domain Bacteria

     TAS [11]

     Phylum Proteobacteria

     TAS [12]

     Class Alphaproteobacteria

     TAS [12]

     Order Rhizobiales

     TAS [12]

     Family Rhizobiaceae

     TAS [12]

     Genus Ensifer

     TAS [2,12]

     Species Ensifer meliloti

     TAS [13]

     Strain BO21CC     Strain AK58

     TAS [4,5]

      Gram stain


     TAS [12]

      Cell shape


     TAS [12]



     TAS [12]



     TAS [12]

      Temperature range

     mesophile, 20-37°C

     TAS [12]

      Optimum temperature


     TAS [12]


     Tolerate 1.0% NaCl

     TAS [12]


      Oxygen requirement


     TAS [12]

      Carbon source

     carbohydrates and salts of organic acids

     TAS [12]

      Energy metabolism


     TAS [12]



     Soil, root nodules of legumes

     TAS [3,12]


      Biotic relationship

     free living, symbiont

     TAS [12]



     not reported

      Biosafety level


     TAS [14]



     BO21CC: root nodules of Medicago sativa cv. ‘Oneida’     AK58: root nodules of Medicago falcata

     TAS [4]


      Geographic location

     BO21CC: Lodi, Italy     AK58: Kazakhstan,

     TAS [4]


      Sample collection time

     BO21CC: 1997     AK58: 2001




     BO21CC: 45.31     AK58: 58.75




     BO21CC: 9.50     AK58: 48.98




     not reported



     BO21CC: 70 m     AK58: 305 m


Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [15].

Figure 1

Phylogenetic consensus tree showing the position of E. meliloti AK58 and BO21CC strains in the Ensifer/Sinorhizobium genus. The phylogenetic tree was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [17], chosen as model with the lowest BIC scores (Bayesian Information Criterion) after running a Maximum Likelihood fits of 24 different nucleotide substitution models (Model Test). The bootstrap consensus tree inferred from 500 replicates [18] is taken to represent the phylogenetic pattern of the taxa analyzed [18]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree with the highest log likelihood (-3411.7124) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (G, parameter = 0.3439). A total of 1,284 nt positions were present in the final dataset. Model test and Maximum Likelihood inference were conducted in MEGA5 [19]. In bold E. meliloti AK58 and BO21CC strains.

E. meliloti AK58 and BO21CC show different symbiotic phenotypes with respect to the host plant Medicago sativa, as well as differences in substrates utilization [5]. Moreover E. meliloti AK58 and BO21CC present differences in cell morphology also, with AK58 being smaller than BO21CC and the other E. meliloti strains for which genome sequencing is available (Figure 2). Interestingly, BO21CC is also showing cells with a ratio between cell axes nearer 1 (more rounded cells), when compared with AK58 and with the other E. meliloti strains (Figure 2).

Figure 2

Cell morphology and cell size analysis of E. meliloti strains. Cell size analysis with Pixcavator IA software [20] of logarithmically grown cultures (OD600=0.6) in TY medium of AK58, BO21CC, plus other completely sequenced E. meliloti strains is reported. Cell size is expressed as cell area in µm2, while roundness is the ratio between the two main axes of the cell. Standard errors after more than 300 individual observations are reported. Different letters indicate significant differences (P<0.05) after 1-way ANOVA.

Genome sequencing information

Genome project history

AK58 and BO21CC strains were selected for sequencing on the basis of the Community Sequencing Program 2010 of DOE Joint Genome Institute (JGI) in relation to the project entitled “Complete genome sequencing of Sinorhizobium meliloti AK58 and BO21CC strains: Improving alfalfa performances through the exploitation of Sinorhizobium genomic data”. The overall rationale for their genome sequencing was related to the identification of genomic determinants of different symbiotic performances between S. meliloti strains. The genome project is deposited in the Genomes On Line Database [21] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE-JGI. A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information





     Finishing quality

     High-Quality Draft


     Libraries used

     Two genomic libraries: one 454 PE library (9 kb insert size),one Illumina library


     Sequencing platforms

     Illumina GAii, 454 GS FLX Titanium


     Sequencing coverage

     60 × (AK58) 910 × (BO21CC) Illumina; 8.8 × pyrosequence



     Newbler version 2.3, Velvet version 1.0.13, phrap version, 1.080812, Allpaths version 39750,


     Gene calling method


     GenBank Date of Release

BO21CC: AXBL01000000AK58: AXBM00000000

     GOLD ID

     BO21CC: Gi07569     AK58: Gi07577

     NCBI project ID

     BO21CC: 375171     AK58: 928722

     Database: IMG

     BO21CC: 9144     AK58: 7327


     Source material identifier

     BO21CC: DSM23809     AK58: DSM23808

     Project relevance

     CSP2010, biotechnological, biodiversity

Growth conditions and DNA isolation

E. meliloti strains AK58 and BO21CC (DSM23808 and DSM23809, respectively) were grown in DSMZ medium 98 (Rhizobium medium) [22] at 28°C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer with modification st/LALMP [23] for strain AK58 and additional 5 µl proteinase K incubation at 58° for 1 hour for strain BO21CC, respectively. DNA will be available on request through the DNA Bank Network [24].

Genome sequencing and assembly

The draft genomes were generated at the DOE Joint Genome Institute (JGI) using Illumina data [25]. For BO21CC genome, we constructed and sequenced an Illumina short-insert paired-end library with an average insert size of 270 bp which generated 76,033,356 reads and an Illumina long-insert paired-end library with an average insert size of 9,141.74 ± 1,934.63 bp, which generated 4,563,348 reads totaling 6,463 Mbp of Illumina data. For AK58, a combination of Illumina [25] and 454 technologies [26] was used. For the AK58 genome we constructed and sequenced an Illumina GAii shotgun library which generated 80,296,956 reads totaling 6,102.6 Mb, a 454 Titanium standard library which generated 0 reads and 1 paired end 454 library with an average insert size of 10 kb, which generated 326,569 reads totaling 96 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [27]. The initial draft assemblies contained 194 contigs in 16 scaffold(s) for BO21CC, and 311 contigs in 5 scaffolds for AK58.

For BO21CC the initial draft data was assembled with Allpaths and the consensus was computationally shredded into 10 Kbp overlapping fake reads (shreds). The Illumina draft data was also assembled with Velvet, version 1.1.05 [28], and the consensus sequences were computationally shredded into 1.5 Kbp overlapping fake reads (shreds). The Illumina draft data was assembled again with Velvet using the shreds from the first Velvet assembly to guide the next assembly. The consensus from the second Velvet assembly was shredded into 1.5 Kbp overlapping fake reads. The fake reads from the Allpaths assembly and both Velvet assemblies and a subset of the Illumina CLIP paired-end reads were assembled using parallel phrap, version 4.24 (High Performance Software, LLC). Possible mis-assemblies were corrected with manual editing in Consed [29-31].

Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished), and sequencing of bridging PCR fragments with Sanger and/or PacBio (unpublished, Cliff Han) technologies. For improved high quality draft and noncontiguous finished projects, one round of manual/wet lab finishing may have been completed. Primer walks, shatter libraries, and/or subsequent PCR reads may also be included for a finished project. A total of 128 additional sequencing reactions and 126 PCR PacBio consensus sequences were completed to close gaps and to raise the quality of the final sequence. The total ("estimated size" for unfinished) size of the BO21CC genome is 7.1 Mb and the final assembly is based on 6,463 Mbp of Illumina draft data, which provides an average 910 × coverage of the genome.

For AK58, the 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.6 (20110517_1502). The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with Velvet, version 1.1.05 [28], and the consensus sequence was computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [29-31] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [32], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 0 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size of AK58 is 7 Mb and the final assembly is based on 61.5 Mb of 454 draft data which provides an average 8.8 × coverage of the genome and 420 Mb of Illumina draft data which provides an average 60 × coverage of the genome.

Genome annotation

Genes were identified using Prodigal [33] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [34]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [16].

Genome properties

The High-Quality draft assemblies of the genomes consist of 41 scaffolds for BO21CC and 9 scaffolds for AK58 representing overall 6,985,065 and 6,974,333 bp, respectively. The overall G+C content was 62.12% and 62.04% for BO21CC and AK58, respectively (Table 3a and Table 3b). Of the 6,746 and 6,992 genes predicted, 5,357 and 5,549 were protein-coding genes, and 105 and 79 RNAs were present in BO21CC and AK58, respectively. The large majority of the protein-coding genes (79.32% and 78.03%, BO21CC and AK58, respectively) were assigned a putative function as COGs. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3a

Genome Statistics for strain BO21CC



    % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of scaffolds


Total genes



RNA genes



rRNA operons


tRNA genes



Protein-coding genes

Genes with function prediction (proteins)



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



Table 3b

Genome statistics for strain AK58




Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of scaffolds


Total genes



RNA genes



rRNA operons


tRNA genes



Protein-coding genes



Genes with function prediction (proteins)



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides


     20.42% %

Genes with transmembrane helices



*only one rRNA operon appears to be complete.

Table 4

Number of genes associated with the general COG functional categories














    Translation, ribosomal structure and biogenesis






    RNA processing and modification












    Replication, recombination and repair






    Chromatin structure and dynamics






    Cell cycle control, cell division, chromosome partitioning






    Nuclear structure






    Defense mechanisms






    Signal transduction mechanisms






    Cell wall/membrane biogenesis






    Cell motility












    Extracellular structures






    Intracellular trafficking and secretion, and vesicular transport






    Posttranslational modification, protein turnover, chaperones






    Energy production and conversion






    Carbohydrate transport and metabolism






    Amino acid transport and metabolism






    Nucleotide transport and metabolism






    Coenzyme transport and metabolism






    Lipid transport and metabolism






    Inorganic ion transport and metabolism






    Secondary metabolites biosynthesis, transport and catabolism






    General function prediction only






    Function unknown



We are grateful to Dr. M.L. Roumiantseva and Dr. B. Simarov (Research Institute for Agricultural Microbiology, St-Petersburg-Puskin, Russia) for original isolation and the permission to use strain AK58 in this work. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy Under Contract No. DE-AC02-05CH11231.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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