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High-quality permanent draft genome sequence of Ensifer medicae strain WSM244, a microsymbiont isolated from Medicago polymorpha growing in alkaline soil

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Standards in Genomic Sciences201510:126

  • Received: 4 September 2015
  • Accepted: 3 November 2015
  • Published:


Ensifer medicae WSM244 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago species. WSM244 was isolated in 1979 from a nodule recovered from the roots of the annual Medicago polymorpha L. growing in alkaline soil (pH 8.0) in Tel Afer, Iraq. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced to date. It is effective at fixing nitrogen with M. polymorpha L., as well as with more alkaline-adapted Medicago spp. such as M. littoralis Loisel., M. scutellata (L.) Mill., M. tornata (L.) Mill. and M. truncatula Gaertn. This strain is also effective with the perennial M. sativa L. Here we describe the features of E. medicae WSM244, together with genome sequence information and its annotation. The 6,650,282 bp high-quality permanent draft genome is arranged into 91 scaffolds of 91 contigs containing 6,427 protein-coding genes and 68 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.


  • Root-nodule bacteria
  • Nitrogen fixation
  • Symbiosis
  • Alphaproteobacteria
  • Ensifer


Root nodule bacteria that fix atmospheric nitrogen in association with annual and perennial pasture legumes have important roles in agriculture. Some of the most important associations in temperate and Mediterranean regions are the Ensifer ( Sinorhizobium 1) - Medicago symbioses that produce nutritious feed for animals. Medicago is a genus within tribe Trifolieae, which is included in the “temperate herbaceous papilionoid” Inverted Repeat Lacking Clade (IRLC) legumes [1, 2]. Species of Medicago are amongst the most extensively grown forage and pasture plants and have been cultivated ever since Medicago sativa L. (alfalfa, or lucerne) was first domesticated in the Near East and/or Central Asia in about 5000 BC. In addition to perennial M. sativa L., annual medic species used widely in agriculture include M. tornata (L.) Mill. (disc medic), the model legume M. truncatula Gaertn. (barrel medic) and M. littoralis Loisel. (strand medic), together with more recently commercialised species such as M. polymorpha L. (burr medic) and M. murex Willd. (murex medic) [3]. Medicago spp. are symbiotically specific: nearly all studied species are nodulated by strains of rhizobia belonging to either Ensifer medicae or the closely related species E. meliloti [4, 5]. E. medicae can be distinguished from E. meliloti by its ability to nodulate and fix nitrogen with M. polymorpha L. [5].

Ensifer medicae WSM244 was isolated in 1979 from a root nodule of M. polymorpha L. growing on alkaline soil (pH 8.0) near Tel Afer, Iraq [6]. This strain was superior in N2-fixation on a range of medics (M sativa L., M truncatula Gaertn., M. tornata L., M. polymorpha L., M. littoralis Loisel., M scutellata (L.) Mill.) in glasshouse tests in Australia and field trials in Iraq in 1980, and was recommended for development as an inoculant in Iraq (D. Chatel, pers com.). WSM244 has also been used in trials aimed at developing acid-tolerant inoculant strains for pasture medics, as the acid-sensitive nature of the microsymbiont is a constraint to the growth and persistence of Medicago spp. in agricultural regions with moderately acidic soils [7]. When field tested in an acidic soil (pH 5.0 CaCl2) in Western Australia, WSM244 survived at the site of inoculation for two years, but unlike several more acid tolerant strains it did not demonstrate saprophytic competence and was unable to colonize the soil [8]. This characteristic of WSM244 as an acid-soil sensitive strain correlates with its acid sensitive profile for growth in laboratory media and an inability to maintain a neutral intracellular pH when exposed to pH 6.0 or less [9]. This is in contrast to other E. medicae strains, which typically are the dominant microsymbiont partners of annual medics growing on acid soils, in contrast to the more acid-sensitive E. meliloti , which preferentially associates with alkaline-soil-adapted Medicago spp. [10]. The pH response phenotype of WSM244 is in marked contrast to the sequenced acid tolerant E. medicae strain WSM419 [11]. Sequencing the genome of WSM244 and comparing its attributes with an acid-tolerant strain such as WSM419 would provide a means of establishing the molecular determinants required for adaptation to acid soils. This strain was therefore selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project [12]. Here we present a summary classification and a set of general features for E. medicae strain WSM244, together with a description of its genome sequence and annotation.

Organism information

Classification and features

E. medicae WSM244 is a motile, Gram-negative rod (Fig. 1 Left and Center) in the order Rhizobiales of the class Alphaproteobacteria . It is fast growing, forming colonies within 3–4 days when grown on half strength Lupin Agar [13], tryptone-yeast extract agar [14] or a modified yeast-mannitol agar [15] at 28 °C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Fig. 1 Right).
Fig. 1
Fig. 1

Images of Ensifer medicae WSM244 using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on solid media (Right)

Figure 2 shows the phylogenetic relationship of E. medicae WSM244 in a 16S rRNA sequence based tree. This strain is the most phylogenetically related to Ensifer medicae WSM419 and Ensifer meliloti LMG 6133T based on the 16S rRNA gene alignment, with sequence identities of 100 % and 99.71 %, respectively, as determined using the EzTaxon-e database, which contains the sequences of validly published type strains [16]. Minimum Information about the Genome Sequence for WSM244 is provided in Table 1 and Additional file 1: Table S1.
Fig. 2
Fig. 2

Phylogenetic tree showing the relationship of Ensifer medicae WSM244 (shown in bold blue print) to other type and non-type strains in the Ensifer genus and to other root nodule bacteria species in the order Rhizobiales, based on aligned sequences of the 16S rRNA gene (1,283 bp internal region). (The species name “Sinorhizobium chiapanecum” has not been validly published.) Azorhizobium caulinodans ORS 571T was used as an outgroup. All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 6 [37]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [38]. Bootstrap analysis [39] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Strains with a genome sequencing project registered in GOLD [17] are in bold font and the GOLD ID is provided after the GenBank accession number. Finished genomes are indicated with an asterisk

Table 1

Classification and general features of Ensifer medicae WSM244 in accordance with the MIGS recommendations [40] published by the Genome Standards Consortium [41]




Evidence codea



Domain Bacteria

TAS [42]


Phylum Proteobacteria

TAS [43, 44]


Class Alphaproteobacteria

TAS [45, 46]


Order Rhizobiales

TAS [47]


Family Rhizobiaceae

TAS [48]


Genus Ensifer

TAS [4951]


Species Ensifer medicae

TAS [5]


Strain: WSM244

TAS [6]


Gram stain




Cell shape












Temperature range

10–40 °C



Optimum temperature

25–30 °C



pH range; Optimum

6–10; 6.5–8

TAS [9]


Carbon source

Arabinose, galactose, mannitol, tryptone

TAS [9]



Soil; root nodule on host (Medicago polymorpha L.)

TAS [8]



0.89–2.0 % (w/v)



Oxygen requirement


TAS [8]


Biotic relationship

Free living, symbiotic

TAS [8]



Biosafety level 1

TAS [52]


Geographic location

Tel Afer, Iraq

TAS [6]


Sample collection


TAS [6]




TAS [6]




TAS [6]



0–10 cm




400 m

TAS [6]

aEvidence 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 [53] (


WSM244 nodulates and is effective for nitrogen fixation with M. littoralis Loisel., M sativa L., M. tornata (L.) Mill. [3], M. murex Willd., M. polymorpha L., M truncatula Gaertn. [8] and M scutellata (L.) Mill. (D. Chatel per com). WSM244 nodulates and is partially effective for nitrogen fixation with M. rotata Boiss. and M. rugosa Desr., but does not nodulate M. blancheana Boiss. (D. Chatel per com). The symbiotic characteristics of E. medicae WSM244 on a range of selected hosts are summarised in Additional file 2: Table S2.

Genome sequencing information

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 Genomic Encyclopedia of Bacteria and Archaea, The Root Nodulating Bacteria chapter project at the U.S. Department of Energy, Joint Genome Institute. The genome project is deposited in the Genomes OnLine Database [17] and a high-quality permanent draft genome sequence is deposited in IMG [18]. Sequencing, finishing and annotation were performed by the JGI [19]. A summary of the project information is shown in Table 2.
Table 2

Genome sequencing project information for E. medicae WSM244





Finishing quality

High-quality draft


Libraries used

Illumina Standard shotgun library


Sequencing platforms

Illumina HiSeq 2000


Fold coverage

677x Illumina



Velvet version 1.1.04; ALLPATHS v. r41043


Gene calling methods

Prodigal 1.4


Locus Tag

A3C7 (


Genbank ID



Genbank Date of Release

July 9 2013



Gp0010265 (





Source Material Identifier



Project relevance

Symbiotic N2 fixation, agriculture

Growth conditions and genomic DNA preparation

E. medicae WSM244 was grown on TY solid medium [14] for three days, then a single colony was selected and used to inoculate 5 ml TY broth medium. The culture was grown for 48 h on a gyratory shaker (200 rpm) at 28 °C. Subsequently 1 ml was used to inoculate 60 ml TY broth medium and grown on a gyratory shaker (200 rpm) at 28 °C until OD 0.6 was reached. DNA was isolated from 60 ml of cells using a CTAB bacterial genomic DNA isolation method ( Final concentration of the DNA was 0.5 mg ml−1.

Genome sequencing and assembly

The draft genome of E. medicae WSM244 was generated at the DOE Joint genome Institute (JGI) using the Illumina technology [20]. An Illumina Std shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 22,576,268 reads totaling 3,386.4 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website. 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, Han J. unpublished) . The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) [21], (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 r41043) [22]. Parameters for assembly steps were: 1) Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –export- Filtered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) 2) wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0) 3) Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True) . The final draft assembly contained 91 contigs in 91 scaffolds. The total size of the genome is 6.7 Mbp and the final assembly is based on 789.1 Mbp of Illumina data, which provides an average 118.7x coverage of the genome.

Genome annotation

Genes were identified using Prodigal [23], as part of the DOE-JGI genome annotation pipeline [24, 25]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [26] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [27]. 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 [28]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform [29] developed by the Joint Genome Institute, Walnut Creek, CA, USA [30].

Genome properties

The genome is 6,650,282 nucleotides with 61.21 % GC content (Table 3) and comprised of 91 scaffolds of 91 contigs. From a total of 6,495 genes, 6,427 were protein encoding and 68 RNA only encoding genes. The majority of protein-coding genes (79.34 %) 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 4.
Table 3

Genome statistics for Ensifer medicae WSM244



% of Total

Genome size (bp)



DNA coding (bp)



DNA G + C (bp)



DNA scaffolds



Total genes



Protein coding genes



RNA genes



Pseudo genes



Genes in internal clusters



Genes with function prediction



Genes assigned to COGs



Genes with Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats



Table 4

Number of genes of Ensifer medicae WSM244 associated with general COG functional categories



% age of total (4,567)





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/envelope biogenesis




Cell motility








Extracellular structures




Intracellular trafficking, 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 metabolite biosynthesis, transport and catabolism




General function prediction only




Function unknown




Mobilome: prophages, transposons




Not in COGS

Insights from the genome sequence

WSM244 is one of six strains of E. medicae and one of 30 E. medicae or E. meliloti Medicago -nodulating strains that have been sequenced and whose genomes have been deposited in the IMG database. The genome of WSM244 falls within the expected size range of 6.4–7.2 Mbp for E. medicae . As observed in other E. medicae genomes, WSM244 possesses a large number of genes assigned to COG functional categories for: transport and metabolism of amino acids (12.15 %), carbohydrates (11.17 %), inorganic ions (5.3 %), lipids (3.91 %) and coenzymes (3.32 %), transcription (8.63 %) and signal transduction (3.66 %). The WSM244 genome contains only four pseudo genes, the numbers of which are highly variable in sequenced E. medicae strains and can be as high as 485 ( E. medicae WSM4191). All six E. medicae strains share high ANI values of 99.18–99.67 %, which is consistent with the low levels of genetic diversity found in E. medicae populations [31]. The six E. medicae strains share 5,425 core orthologous genes. WSM244 contains 202 unique genes, including those found in clusters encoding a putative polyketide synthase, phage proteins and a sulfonate transport system. Around 72 % of these unique genes encode hypothetical proteins. Strain WSM244 is particularly interesting, as it lacks the acid tolerance of other E. medicae strains. The genome of this strain does contain orthologs of acid response or acid tolerance genes that were initially discovered in E. medicae WSM419. These genes include actA (lnt), actP, actR, actS, phrR, lpiA and acvB [3235]. WSM244 also contains the tcsA-tcrA-fsrR- regulatory gene cluster which is required for the low-pH-activation of lpiA and acvB in E. medicae WSM419 [36]. This finding is in direct contrast to the absence of fsrR, tcsA and tcrA in the the acid-sensitive strain E. meliloti 1021. This suggests that either there may be differences in pH responsive gene expression in the WSM244 background, or that acid tolerant E. medicae strains possess other candidate genes that are required for low pH adaptation and have not yet been identified.


WSM244 is of particular interest as it was isolated from M. polymorpha growing in alkaline soil and it lacks the acid tolerance of E. medicae strains isolated from medics growing in acid Sardinian and Greek soils [9]. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced to date. Analysis of its sequenced genome and comparison with other sequenced E. medicae and E. meliloti genomes will yield new insights into the molecular basis of acid tolerance in rhizobia and into the ecology and biogeography of the Ensifer-Medicago symbiosis.


Editorial note—Readers are advised that in Opinion 84 the Judicial Commission of the International Committee on Systematics of Prokaryotes ruled that the genus name Ensifer Casida 1982 has priority over Sinorhizobium Chen et al. 1988 and the names are synonyms [1]. It was further concluded that the transfer of members of the genus Sinorhizobium to the genus Ensifer, as proposed by Young [2] would not cause confusion.




Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria


Inverted Repeat Lacking Clade



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. We thank Gordon Thompson (Murdoch University) for the preparation of SEM and TEM photos.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

Centre for Rhizobium Studies, Murdoch University, Murdoch, Australia
DOE Joint Genome Institute, Walnut Creek, CA, USA
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia


  1. Steele KP, Ickert-Bond SM, Zarre S, Wojciechowski MF. Phylogeny and character evolution in Medicago (Leguminosae): Evidence from analyses of plastid trnK/matK and nuclear GA3ox1 sequences. Am J Bot. 2010;97:1142–55.View ArticlePubMedGoogle Scholar
  2. Steele KP, Wojciechowski MF. Phylogenetic systematics of tribes Trifolieae and Vicieae (Fabaceae), based on sequences of the plastid gene matK (Papilionoideae: Leguminosae). In: Klitgaard BB, Bruneau A, editors. Advances in Legume Systematics, part 10. Kew: Royal Botanic Garden; 2003. p. 355–70.Google Scholar
  3. Howieson JG, Nutt B, Evans P. Estimation of host-strain compatibility for symbiotic N-fixation between Rhizobium meliloti, several annual species of Medicago and Medicago sativa. Plant Soil. 2000;219:49–55.View ArticleGoogle Scholar
  4. Béna G, Lyet A, Huguet T, Olivieri I. Medicago – Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago. J Evol Biol. 2005;18:1547–58.View ArticlePubMedGoogle Scholar
  5. 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;46:972–80.View ArticlePubMedGoogle Scholar
  6. Materon LA, Danso SKA. Nitrogen fixation in two annual Medicago legumes, as affected by inoculation and seed density. Field Crops Res. 1991;26:253–62.View ArticleGoogle Scholar
  7. Howieson JG, Ewing MA, Thorn CW, Revell CK. Increased yield in annual species of Medicago grown in acidic soil in response to inoculation with acid tolerant Rhizobium meliloti. In: Wright RJ, Baligar VC, Murrmann RP, editors. Plant-Soil Interactions at Low pH, vol. 45. Dordrecht: Springer Netherlands; 1991. p. 589–95. Developments in Plant and Soil Sciences].View ArticleGoogle Scholar
  8. Howieson JG, Ewing MA. Acid tolerance in the Rhizobium meliloti-Medicago symbiosis. Aust J Agric Res. 1986;37:55–64.View ArticleGoogle Scholar
  9. O’Hara GW, Goss TJ, Dilworth MJ, Glenn AR. Maintenance of intracellular pH and acid tolerance in Rhizobium meliloti. Appl Environ Microb. 1989;55:1870–6.Google Scholar
  10. Garau G, Reeve WG, Brau L, Deiana P, Yates RJ, James D, et al. 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;276:263–77.View ArticleGoogle Scholar
  11. Reeve W, Chain P, O’Hara G, Ardley J, Nandesena K, Brau L, et al. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand Genomic Sci. 2010;2:77–86.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Reeve WG, Ardley JK, Tian R, Eshragi L, Yoon JW, Ngamwisetkun P, et al. A genomic encyclopedia of the root nodule bacteria: Assessing genetic diversity through a systematic biogeographic survey. Stand Genomic Sci. 2014;9:39.Google Scholar
  13. Howieson JG, Ewing MA, D’antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil. 1988;105:179–88.View ArticleGoogle Scholar
  14. Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974;84:188–98.PubMedGoogle Scholar
  15. Vincent JM. A manual for the practical study of the root-nodule bacteria. International Biological Programme. Oxford: Blackwell Scientific Publications; 1970.Google Scholar
  16. Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012;62:716–21.View ArticlePubMedGoogle Scholar
  17. Reddy TBK, Thomas AD, Stamatis D, Bertsch J, Isbandi M, Jansson J, et al. The Genomes OnLine Database (GOLD) v. 5: a metadata management system based on a four level (meta)genome project classification. Nucleic Acids Res. 2015;43:D1099–D1106.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Pillay M, et al. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res. 2014;42:D560–7.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, et al. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS One. 2012;7:e48837.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5:433–8.View ArticlePubMedGoogle Scholar
  21. Zerbino DR, Birney E. Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Research. 2008;18:821–9.PubMed CentralView ArticlePubMedGoogle Scholar
  22. Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, et al. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci U S A. 2011;108:1513–8.PubMed CentralView ArticlePubMedGoogle Scholar
  23. 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 CentralView ArticlePubMedGoogle Scholar
  24. Mavromatis K, Ivanova NN, Chen IMA, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009;1:63–7.PubMed CentralView ArticlePubMedGoogle Scholar
  25. Chen IM, Markowitz VM, Chu K, Anderson I, Mavromatis K, Kyrpides NC, et al. Improving microbial genome annotations in an integrated database context. PLoS One. 2013;8:e54859.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Lowe TM, Eddy SR. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research. 1997;25:955–64.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007;35:7188–96.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Kamphuis LG, Williams AH, Küster H, Trengove RD, Singh KB, Oliver RP, et al. Phoma medicaginis stimulates the induction of the octadecanoid and phenylpropanoid pathways in Medicago truncatula. Molecular Plant Pathology. 2012. doi:10.1111/j.1364-3703.2011.00767.x.PubMedGoogle Scholar
  29. The Integrated Microbial Genomes (IMG) platform.
  30. 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;25:2271–8.View ArticlePubMedGoogle Scholar
  31. Epstein B, Branca A, Mudge J, Bharti AK, Briskine R, Farmer AD, et al. Population genomics of the facultatively mutualistic bacteria Sinorhizobium meliloti and S. medicae. PLoS Genet. 2012;8:e1002868.PubMed CentralView ArticlePubMedGoogle Scholar
  32. Tiwari RP, Reeve WG, Dilworth MJ, Glenn AR. Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system. Microbiology. 1996;142(Pt 7):1693–704.View ArticlePubMedGoogle Scholar
  33. Tiwari RP, Reeve WG, Dilworth MJ, Glenn AR. An essential role for actA in acid tolerance of Rhizobium meliloti. Microbiology. 1996;142(Pt 3):601–10.View ArticlePubMedGoogle Scholar
  34. Reeve WG, Tiwari RP, Kale NB, Dilworth MJ, Glenn AR. ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH-induced copper toxicity. Mol Microbiol. 2002;43:981–91.View ArticlePubMedGoogle Scholar
  35. Poole PS, Hynes MF, Johnston AWB, Tiwari RP, Reeve WG, Downie JA. Physiology of root-nodule bacteria. In: Dilworth MJ, James EK, Sprent JI, Newton WE, editors. Nitrogen-fixing Leguminous Symbioses, vol. 7. Dordrecht: Springer Netherlands; 2008. p. 241–92. Nitrogen Fixation: Origins, Applications, and Research Progress].View ArticleGoogle Scholar
  36. Reeve WG, Brau L, Castelli J, Garau G, Sohlenkamp C, Geiger O, et al. The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions. Microbiology. 2006;152:3049–59.View ArticlePubMedGoogle Scholar
  37. 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;28:2731–9.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.Google Scholar
  39. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–91.View ArticleGoogle Scholar
  40. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, 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;26:541–7.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, et al. The Genomic Standards Consortium. PLoS Biology. 2011;9:e1001088.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Evol Microbiol 2005, 55:2235–2238Google Scholar
  44. Chen WX, Wang ET, Kuykendall LD. The Proteobacteria. New York: Springer - Verlag; 2005.Google Scholar
  45. List of new names and new combinations previously effectively, but not validly, published. International Journal of Systematic and Evolutionary Microbiology 2006, 56:1–6.Google Scholar
  46. Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Secondth ed. New York: Springer; 2005.Google Scholar
  47. Kuykendall LD. Order VI. Rhizobiales ord. nov. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Secondth ed. New York: Springer; 2005. p. 324.Google Scholar
  48. Kuykendall LD. Family I. Rhizobiaceae. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. New York: Springer; 2005.Google Scholar
  49. Kuykendall LD, Hashem FM, Wang ET. Genus VII. Sinorhizobium. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology, vol. 2. New York: Springer; 2005. p. 358–61.View ArticleGoogle Scholar
  50. 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.View ArticleGoogle Scholar
  51. Casida LE. Ensifer adhaerens gen. nov., sp. nov.: a bacterial predator of bacteria in soil. Int J Syst Bacteriol. 1982;32:339–45.View ArticleGoogle Scholar
  52. Biological Agents: Technical rules for biological agents. []
  53. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.PubMed CentralView ArticlePubMedGoogle Scholar


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