Open Access

Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128

  • Wayne Reeve
  • , Ross Ballard
  • , Elizabeth Drew
  • , Rui Tian
  • , Lambert Bräu
  • , Lynne Goodwin
  • , Marcel Huntemann
  • , James Han
  • , Reddy Tatiparthi
  • , Amy Chen
  • , Konstantinos Mavrommatis
  • , Victor Markowitz
  • , Krishna Palaniappan
  • , Natalia Ivanova
  • , Amrita Pati
  • , Tanja Woyke
  • and Nikos Kyrpides
Corresponding author

DOI: 10.4056/sigs.4929626

Received: 25 January 2014

Accepted: 25 January 2014

Published: 15 June 2014


Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with genome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contigs, contains 6,683 protein-coding genes and 87 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.


root-nodule bacterianitrogen fixationrhizobiaAlphaproteobacteria


Ensifer meliloti strain RRI128 is used in Australia to produce commercial peat cultures (referred to as Group AL inoculants) mainly for the inoculation of lucerne (Medicago sativa L.). Lucerne is sown on about 600, 000 ha annually (A. Humphries pers. com.) and is nearly always inoculated prior to sowing. RRI128 is also used for the inoculation of strand medic (Medicago littoralis Loisel) and disc medic (Medicago tornata (L.) Miller), a hybrid of the two former species, and bokhara clover (Melilotus albus Medik). RRI128 has been used commercially since 2000 when it replaced strain WSM826 [1]. Strain RRI128 was isolated from a nodule from the roots of barrel medic (Medicago truncatula Gaertn) growing in the greenhouse and inoculated with an alkaline sandy soil (pHCaCl2 7.6) collected by J. Slattery, near Tempy, Victoria.

The strain was selected for use in commercial inoculants following assessment of its nitrogen fixation capacity (effectiveness), growth on acidified agar and saprophytic competence in an in-situ soil study [2], with supporting data of satisfactory performance at ten field sites. Additional testing has shown RRI128 to be effective on 28 cultivars of lucerne (Ballard unpub. data). It also forms effective symbiosis with a range of strand and disc medics [2] which show symbiotic affinity with lucerne [3,4].

Soil acidity has long been recognized as a constraint to lucerne nodulation [5] with some evidence that strains of E. meliloti have less acidity tolerance than Ensifer medicae, possibly due to their association with Medicago species that favor neutral to alkaline soils [6]. With RRI128, constraints to lucerne nodulation are observed around pH 5. Nodulation of lucerne seedlings inoculated with RRI128 was 42% at pH 5.0 in solution culture experiments [7] and observed to decline rapidly at field sites where pHCaCl2 was below 4.7 (Ballard, unpub. data). Other strains (e.g. SRDI672) have increased lucerne nodulation in solution culture at pH 4.8 (61% cf. 12% of lucerne seedlings with nodules) but are probably approaching the limit of acidity tolerance for E. meliloti [8].

Stable colony morphology and cell survival on seed make RRI128 amenable to commercial use. RRI128 produces colonies of consistent appearance and with moderate polysaccharide when grown on yeast mannitol agar, enabling easy visual assessment of culture purity. It differs in this regard from the strain it replaced (WSM826) which produced ‘dry’ and ‘mucoid’ colony variants, in common with many of the strains that nodulate lucerne and medic [9]. When applied correctly RRI128 has been shown to survive at more than 10,000 cells per lucerne seed at six weeks after inoculation [10]. Good survival may well be characteristic of E. meliloti, since former inoculant strain WSM826 is equally competent in this regard [11,12].

Here we present a preliminary description of the general features of E. meliloti strain RRI128 together with its genome sequence and annotation.

Classification and general features

Ensifer meliloti strain RRI128 is a motile, non-sporulating, non-encapsulated, Gram-negative rod in the order Rhizobiales of the class Alphaproteobacteria. The rod-shaped form varies in size with dimensions of approximately 0.5 μm in width and 1.0-2.0 μm in length (Figure 1A). It is fast growing, forming colonies within 3-4 days when grown on TY [13] or half strength Lupin Agar (½LA) [14] at 28°C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Figure 1B).

Figure 1

Images of Ensifer meliloti strain RRI128 using (A) scanning electron microscopy and (B) light microscopy to show the colony morphology on TY plates.

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of Ensifer meliloti strain RRI128 in a 16S rRNA gene sequence based tree. This strain has 100% sequence identity (1366/1366 bp) at the 16S rRNA sequence level to the fully sequenced E. meliloti Sm1021 [30] and 99% 16S rRNA sequence (1362/1366 bp) identity to the fully sequenced E. medicae strain WSM419 [31].

Table 1

Classification and general features of Ensifer meliloti strain RRI128 according to the MIGS recommendations [15,16]




       Evidence code

     Current classification

       Domain Bacteria

       TAS [15,16]

       Phylum Proteobacteria

       TAS [17]

       Class Alphaproteobacteria

       TAS [18]

       Order Rhizobiales

       TAS [19]

       Family Rhizobiaceae

       TAS [20]

       Genus Ensifer

       TAS [21,22]

       Species Ensifer meliloti

       TAS [23,24]

       Strain RRI128

     Gram stain



     Cell shape









     Temperature range



     Optimum temperature







     Oxygen requirement



     Carbon source



     Energy source





       Soil, root nodule, on host



     Biotic relationship

       Free living, symbiotic






     Biosafety level


       TAS [25]


       Root nodule



     Geographic location

       Tempy, Vict., Australia



     Soil collection date

       Circa 1995



     Latitude     Longitude

       -35.1833       142.3833

       IDA       IDA



       0-10 cm




       Not reported

Evidence codes – IDA: Inferred from Direct Assay; 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 [26].

Figure 2

Phylogenetic tree showing the relationship of Ensifer meliloti strain RRI128 (shown in bold) with some of the root nodule bacteria in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,307 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [27], version 5.05. The tree was built using the maximum likelihood method with the General Time Reversible model. Bootstrap analysis [28] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Brackets after the strain name contain a DNA database accession number and/or a GOLD ID (beginning with the prefix G) for a sequencing project registered in GOLD [29]. Published genomes are indicated with an asterisk.


Ensifer meliloti strain RRI128 forms nodules on (Nod+) and fixes N2 (Fix+) with Medicago sativa, Melillotus albus and Trigonella balansae (Boiss. and Reuter). It also forms effective symbiosis with several species of annual medic (M. truncatula, M. littoralis and M. tornata) that happen to be closely related to each other based on their ability to be hybridized [5] and morphological and nucleotide sequence analyses of their relatedness [32]. RRI128 forms ineffective (white) nodules with Medicago polymorpha, a species that is generally recognized to have a more specific rhizobial requirement for effective symbiosis than Medicago sativa and Medicago littoralis [4,33] (Table 2).

Table 2

Compatibility of RRI128 with various Medicago and allied genera for nodulation (Nod) and N2-fixation (Fix).

Species Name

     Cultivar or line

      Common Name

      Growth Type




Medicago sativa

     *28 cultivars

      Lucerne, Alfalfa





M. littoralis

     Harbinger, Herald, Angel

      Strand medic





M. tornata

     Tornafield, Rivoli

      Disc medic





M. tornata×littoralis


      Hybrid disc medic





M. truncatula


      Barrel medic





M. polymorpha


      Burr medic





Trigonella balansae

     SA5045, SA32999, SA33025

      Sickle fruited fenugreek





Melilotus albus

     SA19917, SA35627, SA34665

      Bokhara clover





* 28 cultivars tested: Aquarius, Aurora, Cropper 9, Cuff 101, Eureka, Genesis, Hallmark, Hunterfield, Hunter River, Jinderra, ML 99, PL 55, PL 60, PL 69, Prime, SARDI Five, SARDI Seven, SARDI Ten, Sceptre, Sequel, Sequel-HR, Siriver, Trifecta, UQL1, Venus, WL525HQ, 54Q53 and 57Q75.

(w) indicates white nodules.

IDA: Inferred from Direct Assay; evidence code from the Gene Ontology project [26]

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [29] and an improved-high-quality-draft genome sequence in IMG/GEBA. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 3.

Table 3

Genome sequencing project information for Ensifer meliloti strain RRI128





     Finishing quality



     Libraries used

       1× Illumina Std library


     Sequencing platforms

       Illumina HiSeq 2000


     Sequencing coverage

       285× Illumina



       with Allpaths, version r39750, Velvet 1.1.04


     Gene calling methods

       Prodigal 1.4

     Genbank ID


     Genbank Date of Release

       September 5, 2013

     GOLD ID


     GenBank ID


     Database: IMG-GEBA


     Project relevance

       Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

Ensifer meliloti strain RRI128 was cultured to mid logarithmic phase in 60 ml of TY rich medium on a gyratory shaker at 28°C [35]. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [36].

Genome sequencing and assembly

The genome of Ensifer meliloti strain RRI128 was sequenced at the Joint Genome Institute (JGI) using Illumina [37] technology. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform, which generated 13,085,546 reads totaling 1,962 Mb of Illumina data.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI user home [36]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun, L., Copeland, A. and Han, J., unpublished). The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [38], version 1.1.04, (2) 1–3 Kb simulated paired end reads were created from Velvet contigs using wgsim [39], (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG [40] (version r39750).

Parameters for assembly steps were:

Velvet (Velvet optimizer params: --v --s 51 --e 71 --i 2 --t 1 --f "-shortPaired -fastq $FASTQ" --o "-ins_length 250 -min_contig_lgth 500")

wgsim (-e 0 -1 76 -2 76 -r 0 -R 0 -X 0,) (3) Allpaths–LG (PrepareAllpathsInputs:PHRED64=1 PLOIDY=1 FRAGCOVERAGE=125 JUMPCOVERAGE=25 LONGJUMPCOV=50, RunAllpath-sLG: THREADS=8 RUN=stdshredpairs TARGETS=standard VAPIWARNONLY=True OVERWRITE=True).

The final draft assembly contained 157 contigs in 156 scaffolds. The total size of the genome is 6.9 Mb and the final assembly is based on 1,962 Mb of Illumina data, which provides an average 285× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [41] as part of the Oak Ridge National Laboratory genome annotation pipeline. 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. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [42] RNAMMer [43], Rfam [44], TMHMM [45], and SignalP [46]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [47].

Genome properties

The genome is 6,900,273 nucleotides with 61.98% GC content (Table 4) and comprised of 156 scaffolds (Figures 3a,3b,3c,3d,3e). From a total of 6,770 genes, 6,683 were protein encoding and 87 RNA only encoding genes. The majority of genes (78.79%) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 5.

Table 4

Genome Statistics for Ensifer meliloti strain RRI128



     % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of scaffolds


Number of contigs


Total gene



RNA genes



rRNA operons


Protein-coding genes



Genes with function prediction



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


*2 copies of 5S, 1 copy of 16S and 1 copy of 23S rRNA

Figure 3a

Graphical map of YU7DRAFT_scaffold_0.1 of the genome of Ensifer meliloti strain RRI128. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3b

Graphical map of YU7DRAFT_scaffold_1.2 of the genome of Ensifer meliloti strain RRI128. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3c

Graphical map of YU7DRAFT_scaffold_2.3 of the genome of Ensifer meliloti strain RRI128. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3d

Graphical map of YU7DRAFT_scaffold_3.4 of the genome of Ensifer meliloti strain RRI128. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3e

Graphical map of YU7DRAFT_scaffold_4.5 of the genome of Ensifer meliloti strain RRI128. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Table 5

Number of protein coding genes of Ensifer meliloti strain RRI128 associated with the general COG functional categories




    COG Category




    Translation, ribosomal structure and biogenesis




    RNA processing and modification








    Replication, recombination and repair




    Chromatin structure and dynamics




    Cell cycle control, mitosis and meiosis




    Nuclear structure




    Defense mechanisms




    Signal transduction mechanisms




    Cell wall/membrane biogenesis




    Cell motility








    Extracellular structures




    Intracellular trafficking and secretion




    Posttranslational modification, protein turnover, chaperones




    Energy production conversion




    Carbohydrate transport and metabolism




    Amino acid transport metabolism




    Nucleotide transport and metabolism




    Coenzyme transport and metabolism




    Lipid transport and metabolism




    Inorganic ion transport and metabolism




    Secondary metabolite biosynthesis, transport and catabolism




    General function prediction only




    Function unknown




    Not in COGS



This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. We gratefully acknowledge the funding received from the Murdoch University Strategic Research Fund through the Crop and Plant Research Institute (CaPRI) and the Centre for Rhizobium Studies (CRS) at Murdoch University.

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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