Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128
© The Author(s) 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.
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 . 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 , 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  which show symbiotic affinity with lucerne [3,4].
Soil acidity has long been recognized as a constraint to lucerne nodulation  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 . 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  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 .
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 . When applied correctly RRI128 has been shown to survive at more than 10,000 cells per lucerne seed at six weeks after inoculation . 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
Species Ensifer meliloti
Soil, root nodule, on host
Free living, symbiotic
Tempy, Vict., Australia
Soil collection date
Compatibility of RRI128 with various Medicago and allied genera for nodulation (Nod) and N2-fixation (Fix).
Cultivar or line
Harbinger, Herald, Angel
Hybrid disc medic
SA5045, SA32999, SA33025
Sickle fruited fenugreek
SA19917, SA35627, SA34665
Genome sequencing and annotation
Genome project history
Genome sequencing project information for Ensifer meliloti strain RRI128
1× Illumina Std library
Illumina HiSeq 2000
with Allpaths, version r39750, Velvet 1.1.04
Gene calling methods
Genbank Date of Release
September 5, 2013
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 . DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method .
Genome sequencing and assembly
The genome of Ensifer meliloti strain RRI128 was sequenced at the Joint Genome Institute (JGI) using Illumina  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 . 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 , version 1.1.04, (2) 1–3 Kb simulated paired end reads were created from Velvet contigs using wgsim , (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG  (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.
Genes were identified using Prodigal  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  RNAMMer , Rfam , TMHMM , and SignalP . Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform .
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
Genes with function prediction
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of protein coding genes of Ensifer meliloti strain RRI128 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, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
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
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.
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