Genome sequence of Ensifer medicae strain WSM1369; an effective microsymbiont of the annual legume Medicago sphaerocarpos
© The Author(s) 2013
Published: 20 December 2013
Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
One of the key nutritional constraints to plant growth and development is the availability of nitrogen (N) in nutrient deprived soils . Although the atmosphere consists of approximately 80% N, the overwhelming proportion of this is present in the form of dinitrogen (N2) which is biologically inaccessible to most plants and other higher organisms. Before the development of the Haber-Bosch process, the primary mechanism for converting atmospheric N2 into a bioaccessible form was via biological nitrogen fixation (BNF) . In BNF, N2 is made available by specialized microbes that possess the necessary molecular machinery to reduce N2 into NH3. Some plants, most of which are legumes, have harnessed BNF by evolving symbiotic relationships with specific N2-fixing microbes (termed rhizobia) whereby the host plant houses the bacteria in root nodules, supplying the microsymbiont with carbon and in return receives essential reduced N-containing products . When BNF is exploited in agriculture, some of this N2 fixed into plant tissues is ultimately released into the soil following harvest or senescence, where it can then be assimilated by subsequent crops. Compared to industrially synthesized N-based fertilizers, BNF is a low energy, low cost and low greenhouse-gas producing alternative and hence its application is crucial to increasing the environmental and economic sustainability of farming systems .
Forage and fodder legumes play vital roles in sustainable farming practice, with approximately 110 million ha under production worldwide , a significant proportion of which is made up by members of the genus Medicago. Ensifer meliloti and E. medicae are known to nodulate and fix N2 with Medicago spp , although they have differences in host specificity. While E. meliloti strains do not nodulate M. murex, nodulate but do not fix N2 with M. polymorpha and nodulate but fix very poorly with M. arabica [7,8], they are able to nodulate and fix N2 with Medicago species originating from alkaline soils including the perennial M. sativa and the annuals M. littoralis and M. tornata [9,10]. In contrast, E. medicae strains can nodulate and fix N2 with annuals well adapted to acidic soils, such as M. murex, M. arabica and M. polymorpha [7,8].
The E. medicae strain WSM1369 was isolated from a nodule collected from M. sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). This strain nodulates and fixes N2 effectively with M. polymorpha and M. sphaerocarpos . Like M. murex and M. polymorpha, M. sphaerocarpos is an annual species which is tolerant of low pH soils , with studies suggesting that it only establishes N2-fixing associations with E. medicae strains [8,9]. However, owing to a paucity of symbiotic information, it is not yet clear whether M. sphaerocarpos fixes N2 with a wide range of E. medicae strains or if this ability is restricted to a smaller set of E. medicae accessions. Therefore, genome sequences of E. medicae strains effective with M. sphaerocarpos will provide a valuable genetic resource to further investigate the symbiotaxonomy of Medicago-nodulating rhizobia and will further enhance the existing available genome data for Ensifer microsymbionts [12–15]. Here we present a summary classification and a set of general features for this microsymbiont together with a description of its genome sequence and annotation.
Classification and features
Classification and general features of Ensifer medicae WSM1369 according to the MIGS recommendations 
Species Ensifer medicae
Soil, root nodule, on host
Free living, symbiotic
Soil collection date
28 April 1993
E. medicae strain WSM1369 was isolated in 1993 from a nodule collected from the annual M. sphaerocarpos growing at San Pietro di Rudas, near Aggius, Sardinia in Italy (J. G. Howieson, pers. comm.). The site of collection was undulating grassland, with a soil derived from granite materials that had a depth of 20–40 cm and a pH of 6.0. The soil was a loamy-sand and Lathyrus and Trifolium spp. grew in association with M. sphaerocarpos. WSM1369 forms nodules (Nod+) and fixes N2 (Fix+) with M. polymorpha and M. sphaerocarpos .
Genome sequencing and annotation
Genome project history
Genome sequencing project information for E. medicae WSM1369
One Illumina fragment library
Illumina HiSeq 2000
Velvet version 1.1.04; Allpaths-LG version r39750
Gene calling methods
GenBank release date
August 28, 2013
NCBI project ID
Symbiotic N2 fixation, agriculture
Growth conditions and DNA isolation
E. medicae WSM1369 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 medicae WSM1369 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 13,712,318 reads totaling 2,057 Mbp.
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 Kbp 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: 1) Velvet (velveth: 63 -shortPaired and velvetg: -veryclean yes -exportFiltered yes -mincontiglgth 500 -scaffolding no-covcutoff 10) 2) 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 307 contigs in 307 scaffolds. The total size of the genome is 6.4 Mbp and the final assembly is based on 2,057 Mbp of Illumina data, which provides an average 321× coverage of the genome.
Genes were identified using Prodigal  as part of the DOE-JGI annotation pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA . Other non-coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform .
Genome Statistics for Ensifer medicae WSM1369
% 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 coding transmembrane proteins
Number of protein coding genes of Ensifer medicae WSM1369 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.
- O’Hara GW. The role of nitrogen fixation in crop production. J Crop Prod 1998; (2):115–138. http://dx.doi.org/10.1300/J144v01n02_06
- Olivares J, Bedmar EJ, Sanjuan J. Biological nitrogen fixation in the context of global change. Mol Plant Microbe Interact 2013; 26:486–494. PubMed http://dx.doi.org/10.1094/MPMI-12-12-0293-CRView ArticlePubMedGoogle Scholar
- Terpolilli JJ, Hood GA, Poole PS. What determines the efficiency of N2-fixing Rhizobium-Legume symbioses? Adv Microb Physiol 2012; 60:325–389. PubMed http://dx.doi.org/10.1016/B978-0-12-398264-3.00005-XView ArticlePubMedGoogle Scholar
- Howieson JG, O’Hara GW, Carr SJ. Changing roles for legumes in Mediterranean agriculture: developments from an Australian perspective. Field Crops Res 2000; 65:107–122. http://dx.doi.org/10.1016/S0378-4290(99)00081-7View ArticleGoogle Scholar
- Herridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 2008; 311:1–18. http://dx.doi.org/10.1007/s11104-008-9668-3View ArticleGoogle Scholar
- Graham P. Ecology of the root-nodule bacteria of legumes. In: Dilworth MJ, James EK, Sprent JI, Newton WE, editors. Nitrogen-Fixing Leguminous Symbioses. Dodrecht: The Netherlands: Springer; 2008. p 23–43.Google Scholar
- Rome S, Fernandez MP, Brunel B, Normand P, Cleyet-Marel JC. Sinorhizobium medicae sp. nov., isolated from annual Medicago spp. Int J Syst Bacteriol 1996; 46:972–980. PubMed http://dx.doi.org/10.1099/00207713-46-4-972View ArticlePubMedGoogle Scholar
- Garau G, Reeve WG, Brau L, Yates RJ, James D, Tiwari R, O’Hara GW, Howieson JG. The symbiotic requirements of different Medicago spp. suggest the evolution of Sinorhizobium meliloti and S. medicae with hosts differentially adapted to soil pH. Plant Soil 2005; 276:263–277. http://dx.doi.org/10.1007/s11104-005-0374-0View ArticleGoogle Scholar
- Terpolilli JJ, O’Hara GW, Tiwari RP, Dilworth MJ, Howieson JG. The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021. New Phytol 2008; 179:62–66. PubMed http://dx.doi.org/10.1111/j.1469-8137.2008.02464.xView ArticlePubMedGoogle Scholar
- Howieson JG, Nutt B, Evans P. Estimation of hoststrain compatibility for symbiotic N-fixation between Rhizobium meliloti, several annual species of Medicago and Medicago sativa. Plant Soil 2000; 219:49–55. http://dx.doi.org/10.1023/A:1004795617375View ArticleGoogle Scholar
- Initiative IOC. Climate variability and change in southwest Western Australia. 2002. p 1–34.Google Scholar
- Galibert F, Finan TM, Long SR, Puhler A, Abola P, Ampe F, Barloy-Hubler F, Barnett MJ, Becker A, Boistard P, et al. The composite genome of the legume symbiont Sinorhizobium meliloti. Science 2001; 293:668–672. PubMed http://dx.doi.org/10.1126/science.1060966View ArticlePubMedGoogle Scholar
- Reeve W, Chain P, O’Hara G, Ardley J, Nandesena K, Brau L, Tiwari R, Malfatti S, Kiss H, Lapidus A, et al. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand Genomic Sci 2010; 2:77–86. PubMed http://dx.doi.org/10.4056/sigs.43526PubMed CentralView ArticlePubMedGoogle Scholar
- Terpolilli JJ, Hill YJ, Tian R, Howieson JG, Bräu L, Goodwin L, Han J, Liolios K, Huntemann M, Pati AWT, et al. Genome sequence of Ensifer melilot strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17. Stand Genomic Sci 2013; (In press). http://dx.doi.org/10.4056/sigs.4838624
- Tak N, Gehlot HS, Kaushik M, Choudhary S, Tiwari R, Tian R, Hill YJ, Bräu L, Goodwin L, Han J, et al. Genome sequence of Ensifer sp. TW10; a Tephrosia wallichii (Biyani) microsymbiont native to the Indian Thar Desert. Stand Genomic Sci 2013; (In press). http://dx.doi.org/10.4056/sigs.4598281
- Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 1974; 84:188–198. PubMed http://dx.doi.org/10.1099/00221287-84-1-188PubMedGoogle Scholar
- Howieson JG, Ewing MA, D’antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil 1988; 105:179–188. http://dx.doi.org/10.1007/BF02376781View ArticleGoogle Scholar
- Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M, Angiuoli SV, et al. Towards a richer description of our complete collection of genomes and metagenomes “Minimum Information about a Genome Sequence” (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
- Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.View ArticleGoogle Scholar
- 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. PubMed http://dx.doi.org/10.1099/ijs.0.64188-0
- Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 1.View ArticleGoogle Scholar
- Kuykendall LD. Order VI. Rhizobiales ord. nov. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Second ed: New York: Springer-Verlag; 2005. p 324.Google Scholar
- Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225View ArticleGoogle Scholar
- Conn HJ. Taxonomic relationships of certain non-sporeforming rods in soil. J Bacteriol 1938; 36:320–321.Google Scholar
- Casida LE. Ensifer adhaerens gen. nov., sp. nov.: a bacterial predator of bacteria in soil. Int J Syst Bacteriol 1982; 32:339–345. http://dx.doi.org/10.1099/00207713-32-3-339View ArticleGoogle Scholar
- Young JM. The genus name Ensifer Casida 1982 takes priority over Sinorhizobium Chen et al. 1988, and Sinorhizobium morelense Wang et al. 2002 is a later synonym of Ensifer adhaerens Casida 1982. Is the combination Sinorhizobium adhaerens (Casida 1982) Willems et al. 2003 legitimate? Request for an Opinion. Int J Syst Evol Microbiol 2003; 53:2107–2110. PubMed http://dx.doi.org/10.1099/ijs.0.02665-0View ArticlePubMedGoogle Scholar
- Judicial Commission of the International Committee on Systematics of Prokaryotes. The genus name Sinorhizobium Chen et al. 1988 is a later synonym of Ensifer Casida 1982 and is not conserved over the latter genus name, and the species name ‘Sinorhizobium adhaerens’ is not validly published. Opinion 84. Int J Syst Evol Microbiol 2008; 58:1973. PubMed http://dx.doi.org/10.1099/ijs.0.2008/005991-0View ArticleGoogle Scholar
- Agents B. Technical rules for biological agents. TRBA (http://www.baua.de):466.
- 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; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121PubMed CentralView ArticlePubMedGoogle Scholar
- Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.Google Scholar
- Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985; 39:783–791. http://dx.doi.org/10.2307/2408678View ArticleGoogle Scholar
- Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2008; 36:D475–D479. PubMed http://dx.doi.org/10.1093/nar/gkm884PubMed CentralView ArticlePubMedGoogle Scholar
- Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR, Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology 1999; 145:1307–1316. PubMed http://dx.doi.org/10.1099/13500872-145-6-1307View ArticlePubMedGoogle Scholar
- DOE Joint Genome Institute user home. http://my.jgi.doe.gov/general/index.html.
- Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/14622418.104.22.1683View ArticlePubMedGoogle Scholar
- Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010; Chapter 11:Unit 11 5.Google Scholar
- Wgsim sequence read simulator. https://github.com/lh3/wgsim.
- Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, Sharpe T, Hall G, Shea TP, Sykes S, et al. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci USA 2011; 108:1513–1518. PubMed http://dx.doi.org/10.1073/pnas.1017351108PubMed CentralView ArticlePubMedGoogle Scholar
- Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
- Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci 2009; 1:63–67. PubMed http://dx.doi.org/10.4056/sigs.632PubMed CentralView ArticlePubMedGoogle Scholar
- Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMedPubMed CentralView ArticlePubMedGoogle Scholar
- Pruesse E, Quast C, Knittel K. Fuchs BdM, Ludwig W, Peplies J, Glöckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007; 35:7188–7196. PubMed http://dx.doi.org/10.1093/nar/gkm864PubMed CentralView ArticlePubMedGoogle Scholar
- INFERNAL. http://infernal.janelia.org
- Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar