Open Access

Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece

  • Wayne Reeve
  • , Ross Ballard
  • , John Howieson
  • , Elizabeth Drew
  • , Rui Tian
  • , Lambert Bräu
  • , Christine Munk
  • , Karen Davenport
  • , Patrick Chain
  • , Lynne Goodwin
  • , Ioanna Pagani
  • , Marcel Huntemann
  • , Konstantinos Mavrommatis
  • , Amrita Pati
  • , Victor Markowitz
  • , Natalia Ivanova
  • , Tanja Woyke
  • and Nikos Kyrpides
Corresponding author

DOI: 10.4056/sigs.4938652

Received: 31 December 2013

Accepted: 31 December 2013

Published: 15 June 2014

Abstract

Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N2 fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl2) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 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.

Keywords:

root-nodule bacterianitrogen fixationrhizobiaAlphaproteobacteria

Introduction

The genus Medicago comprises 87 species of annual and perennial legumes, including some that were formerly recognized as Trigonella and Melilotus species [1]. A small number of annual Medicago species that have been domesticated are grown extensively in the sheep-wheat zone of southern Australia, particularly where pasture regeneration after a cropping phase is desirable. Annual Medicago species are grown on more than 20 M ha [2] and are particularly valued for their contribution to farming systems, in which Medicago fix around 25 kg of N per tonne of legume dry matter produced [3].

Medicago are nodulated by two species of root nodule bacteria (Ensifer medicae and Ensifer meliloti) that are recognized as being distinct based on their different nodulation and N2 fixation phenotypes in host interaction studies and more detailed analyses of their genetics [4,5].

Ensifer medicae strain WSM1115 is used in Australia to produce commercial peat cultures (referred to as Group AM inoculants) for the inoculation of several species of annual Medicago (predominantly M. truncatula, M. polymorpha, M. scutellata, M. sphaerocarpus, M. murex, M. rugosa and M. orbicularis). WSM1115 has been used commercially since 2002 [6], when it replaced strain WSM688. WSM1115 was isolated from a nodule from the roots of burr medic (Medicago polymorpha) collected by Prof. John Howieson (Murdoch University, Australia) on the island of Samothraki, Greece.

WSM1115 was selected for use in commercial inoculants having demonstrated good N2-fixation capacity with the relevant medic hosts and adequate saprophytic competence in moderately acidic soil (pH(CaCl2) 5).

Saprophytic competence in acidic soils is a requirement of strains used to inoculate Medicago because several species (M. murex, M. sphaerocarpus and M. polymorpha) are recommended and sown into soils below pH(CaCl2) 5.5, a level that is known to limit both survival of medic rhizobia and nodulation processes [7-10]. Useful variation in saprophytic competence occurs between strains of medic rhizobia [9] and valuable insights into the mechanisms that confer acidity tolerance have been provided by studies using strain WSM419 [11], which has been recently sequenced [12]. However, the complex nature of soil adaptation means that in-situ field studies still provide the most reliable means of selecting an inoculant strain and were used to select WSM1115 for commercial use. In a cross row experiment comparing 15 strains on acidic sand (pH(CaCl2) 5.0; Dowerin, West Australia), the nodulation of plants inoculated with WSM1115 was equal to or better than that of the other strains. This translated to better plant shoot weights, which were similar to those of plants inoculated with WSM688 (the incumbent inoculant strain at time of testing) and 48% greater when compared to former inoculant strain CC169 (J. G. Howieson unpublished data).

The nitrogen fixation capacity (effectiveness) of Medicago symbioses is characterized by strong interactions between the strain of rhizobia and species of Medicago [13-16]. Hence, the ability to form effective symbiosis with the species recommended for inoculation is an important consideration in inoculant strain selection. WSM1115 satisfies this requirement. In greenhouse tests it formed effective symbiosis with 16 genotypes of Medicago and overall produced 48% more shoot dry matter compared to plants inoculated with WSM688, the strain that it replaced (R.A. Ballard and N. Charman, unpublished data).

A limitation of strain WSM1115 is its poor persistence in moderately saline soils (e.g. where summer salinity levels exceed 10 ECe (dS/m)). Poor nodulation of regenerating pasture was first noted in 2004 during the field evaluation and domestication of the salt tolerant annual pasture legume messina (Melilotus siculus syn. Melilotus messanensis). Subsequent studies [17] confirmed that although WSM1115 was able to nodulate and form effective symbiosis with messina, it did not persist as well as other strains (e.g. SRDI554) through the summer months when salinity levels increased.

Here we present a preliminary description of the general features of Ensifer medicae strain WSM1115 together with its genome sequence and annotation.

Classification and features

Ensifer medicae strain WSM1115 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 μm in length (Figure 1A). It is fast growing, forming colonies within 3-4 days when grown on TY [18] or half strength Lupin Agar (½LA) [19] at 28°C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Figure 1B).

Figure 1

Images of Ensifer medicae strain WSM1115 using (A) scanning electron microscopy and (B) light microscopy to show the colony morphology on a solid medium.

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of Ensifer medicae strain WSM1115 in a 16S rRNA gene sequence based tree. This strain has 100% sequence identity (1,366/1,366 bp) at the 16S rRNA sequence level to the fully sequenced Ensifer medicae strain WSM419 [12] and 99% 16S rRNA sequence (1362/1366 bp) identity to the fully sequenced E. meliloti Sm1021 [36].

Table 1

Classification and general features of Ensifer medicae strain WSM1115 according to the MIGS recommendations [20]

MIGS ID

     Property

    Term

    Evidence code

     Current classification

    Domain Bacteria

    TAS [21]

    Phylum Proteobacteria

    TAS [22]

    Class Alphaproteobacteria

    TAS [23,24]

    Order Rhizobiales

    TAS [22,25]

    Family Rhizobiaceae

    TAS [26,27]

    Genus Ensifer

    TAS [28-30]

    Species Ensifer medicae

    TAS [29]

    Strain WSM1115

     Gram stain

    Negative

    IDA

     Cell shape

    Rod

    IDA

     Motility

    Motile

    IDA

     Sporulation

    Non-sporulating

    NAS

     Temperature range

    Mesophile

    NAS

     Optimum temperature

    28°C

    NAS

     Salinity

    Non-halophile

    NAS

MIGS-22

     Oxygen requirement

    Aerobic

    IDA

     Carbon source

    Varied

    NAS

     Energy source

    Chemoorganotroph

    NAS

MIGS-6

     Habitat

    Soil, root nodule, on host

    IDA

MIGS-15

     Biotic relationship

    Free living, symbiotic

    IDA

MIGS-14

     Pathogenicity

    Non-pathogenic

    IDA

     Biosafety level

    1

    TAS [31]

     Isolation

    Root nodule

    IDA

MIGS-4

     Geographic location

    Samothraki, Greece

    IDA

MIGS-5

     Time of sample collection

    May, 1987

    IDA

MIGS-4.1

     Latitude

    40.4900

    IDA

MIGS-4.2

     Longitude

    25.6500

    IDA

MIGS-4.3

     Depth

    <10 cm

    IDA

MIGS-4.4

     Altitude

    325 m

    IDA

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 [32].

Figure 2

Phylogenetic tree showing the relationship of Ensifer medicae WSM1115 (shown in bold print) to other Ensifer spp. in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,290 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [33]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [34]. Bootstrap analysis [35] 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 [32]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

Ensifer medicae strain WSM1115 forms nodules (Nod+) and fixes N2 (Fix+) with a range of annual and perennial Medicago species and Melilotus species (Table 2). Levels of N2 fixation in combination with Medicago littoralis is suboptimal, that species generally forming more effective associations with strains of Ensifer meliloti including strain RRI128 [38]. The level of N2 fixation with Melilotus albus is also noted as positive, but has been observed to vary markedly with different plant accessions.

Table 2

Compatibility of Ensifer medicae WSM1115 with various Medicago and allied genera for nodulation (Nod) and N2-fixation (Fix)

Species Name

    Cultivar or line

    Common Name

    Growth Type

   Nod

    Fix

    Reference

M. polymorpha

    Santiago/Cavalier/Scimitar

    Burr

    Annual

   +

    +

    IDA

M. truncatula.

    Caliph/Jester

    Barrel

    Annual

   +

    +

    IDA

M. murex

    Zodiac

    Murex

    Annual

   +

    +

    IDA

M. sphaerocarpus

    Orion

    Sphere

    Annual

   +

    +

    IDA

M. scutellata

    Sava/Silver/Essex

    Snail

    Annual

   +

    +

    IDA

M. rugosa

    Paraponto

    Gama

    Annual

   +

    +

    IDA

M. littoralis

    Herald/Harbinger

    Strand

    Annual

   +

    Poor

    IDA

M. orbicularis

    Estes

    Button

    Annual

   +

    +

     [15]

M. rigiduloides

    Accession PI 227850

    Rigid

    Annual

   +(w)

    -

     [15]

M. rigidula

    Accession PI 495552

    Tifton

    Annual

   +(w)

    -

     [15]

M. arabica

    Local ecotype

    Spotted

    Annual

   +

    +

     [15]

M. minima

    Devine

    Woolly burr

    Annual

   +

    +

     [15]

M. sativa

    SARDI Ten

    Lucerne

    Perennial

   +

    +

    IDA

M. lupulina

    ‘BEBLK’

    Black

    Perennial

   +

    +

     [15]

Melilotus siculus

    Accessions SA40006 & 39909

    Messina

    Annual

   +

    +

     [17]

Melilotus albus

    various accessions

    Bokhara clover

    Biennial

   +

    +

    IDA

(w) indicates nodules present were white.

IDA: Inferred from Direct Assay from the Gene Ontology project [37].

Genome sequencing and annotation 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 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 [32] and a 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 medicae strain WSM1115

MIGS ID

     Property

     Term

MIGS-31

     Finishing quality

     Permanent high quality draft

MIGS-28

     Libraries used

     2× Illumina libraries; Std short PE & CLIP long PE

MIGS-29

     Sequencing platforms

     Illumina HiSeq 2000

MIGS-31.2

     Sequencing coverage

     530× Illumina

MIGS-30

     Assemblers

     with Allpaths, version 38445, Velvet 1.1.05, phrap 4.24

MIGS-32

     Gene calling methods

     Prodigal 1.4, GenePRIMP

     Genbank ID

     AQZC01000000

     Genbank Date of Release

     April 22, 2013

     GOLD ID

     Gi08906

     NCBI project ID

     74391

     Database: IMG-GEBA

     2512875026

     Project relevance

     Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

Ensifer medicae strain WSM1115 was cultured to mid logarithmic phase in 60 ml of TY rich medium on a gyratory shaker at 28°C [39]. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [40].

Genome sequencing and assembly

The genome of Ensifer medicae strain WSM1115 was sequenced at the Joint Genome Institute (JGI) using Illumina [41] data. An Illumina standard paired-end library with a minimum insert size of 270 bp was used to generate 23,080,558 reads totaling 3,462 Mbp and an Illumina CLIP paired-end library with an average insert size of 9,584 + 2,493 bp was used to generate 2,163,668 reads totaling 324 Mbp of Illumina data (unpublished, Feng Chen).

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI user home [40]. The initial draft assembly contained 57 contigs in 11 scaffolds. The initial draft data was assembled with Allpaths, version 38445, 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 [42], 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 [43-45]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished), and sequencing of bridging PCR fragments. The estimated total size of the genome is 6.9 Mbp and the final assembly is based on 3,654 Mbp of Illumina draft data, which provides an average 530× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [46] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [47]. 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. 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 [48], RNAMMer [49], Rfam [50], TMHMM [51], and SignalP [52]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [53].

Genome properties

The genome is 6,861,065 nucleotides with 61.16% GC content (Table 4) and comprised of 7 scaffolds (Figures 3a,3b,3c,3d,3e,3f and Figure 3g) From a total of 6,872 genes, 6,789 were protein encoding and 83 RNA only encoding genes. The majority of genes (76.25%) 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 medicae strain WSM1115

Attribute

      Value

       % of Total

Genome size (bp)

      6,861,065

       100.00

DNA coding region (bp)

      5,918,651

       86.26

DNA G+C content (bp)

      4,196,062

       61.16

Number of scaffolds

      7

Number of contigs

      28

Total gene

      6,872

       100.00

RNA genes

      83

       1.21

rRNA operons

      3

       0.04

Protein-coding genes

      6,789

       98.79

Genes with function prediction

      5,240

       76.25

Genes assigned to COGs

      5,168

       75.20

Genes assigned Pfam domains

      5,424

       78.93

Genes with signal peptides

      571

       8.31

Genes coding membrane proteins

      1,483

       21.58

CRISPR repeats

      0

Figure 3a

Graphical maps of SinmedDRAFT_Scaffold1.2 of the Ensifer medicae strain WSM1115 genome sequence. 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 maps of SinmedDRAFT_Scaffold2.1 of the Ensifer medicae strain WSM1115 genome sequence. 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 maps of SinmedDRAFT_Scaffold5.3 of the Ensifer medicae strain WSM1115 genome sequence. 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 maps of SinmedDRAFT_Scaffold3.7 of the Ensifer medicae strain WSM1115 genome sequence. 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 maps of SinmedDRAFT_Scaffold6.5 of the Ensifer medicae strain WSM1115 genome sequence. 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 3f

Graphical maps of SinmedDRAFT_Scaffold4.6 of the Ensifer medicae strain WSM1115 genome sequence. 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 3g

Graphical maps of SinmedDRAFT_Scaffold7.4 of the Ensifer medicae strain WSM1115 genome sequence. 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 medicae strain WSM1115 associated with the general COG functional categories.

Code

       Value

        %age

        COG Category

J

       186

        3.23

        Translation, ribosomal structure and biogenesis

A

       0

        0.00

        RNA processing and modification

K

       527

        9.16

        Transcription

L

       269

        4.68

        Replication, recombination and repair

B

       3

        0.05

        Chromatin structure and dynamics

D

       43

        0.75

        Cell cycle control, mitosis and meiosis

Y

       0

        0.00

        Nuclear structure

V

       55

        0.96

        Defense mechanisms

T

       244

        4.24

        Signal transduction mechanisms

M

       272

        4.73

        Cell wall/membrane biogenesis

N

       68

        1.18

        Cell motility

Z

       0

        0.00

        Cytoskeleton

W

       1

        0.02

        Extracellular structures

U

       112

        1.95

        Intracellular trafficking and secretion

O

       195

        3.39

        Posttranslational modification, protein turnover, chaperones

C

       335

        5.82

        Energy production conversion

G

       575

        10.00

        Carbohydrate transport and metabolism

E

       609

        10.59

        Amino acid transport metabolism

F

       106

        1.84

        Nucleotide transport and metabolism

H

       194

        3.37

        Coenzyme transport and metabolism

I

       205

        3.56

        Lipid transport and metabolism

P

       286

        4.97

        Inorganic ion transport and metabolism

Q

       164

        2.85

        Secondary metabolite biosynthesis, transport and catabolism

R

       726

        12.62

        General function prediction only

S

       577

        10.03

        Function unknown

-

       1,704

        24.80

        Not in COGS

-

       5,752

        Total

Declarations

Acknowledgements

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 and the GRDC National Rhizobium Program (Project UMU63).


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.

References

  1. Small E. Alfalfa and Relatives: Evolution and Classification of Medicago. Ottawa, Canada: NRC Reserach Press; 2011.
  2. Hill MJ and Donald GE. ; 1998
  3. Peoples MB and Baldock JA. Nitrogen dynamics of pastures: nitrogen fixation inputs, the impact of legumes on soil nitrogen fertility, and the contributions of fixed nitrogen to Australian farming systems. Aust J Exp Agric. 2001; 41:327-346 View Article
  4. Rome S, Brunel B, Normand P, Fernandez M and Cleyet-Marel JC. Evidence that two genomic species of Rhizobium are associated with Medicago truncatula. Arch Microbiol. 1996; 165:285-288 View ArticlePubMed
  5. Rome S, Fernandez MP, Brunel B, Normand P and Cleyet-Marel JC. Sinorhizobium medicae sp. nov., isolated from annual Medicago spp. Int J Syst Bacteriol. 1996; 46:972-980 View ArticlePubMed
  6. Bullard GK, Roughley RJ and Pulsford DJ. The legume inoculant industry and inoculant quality control in Australia: 1953–2003. Aust J Exp Agric. 2005; 45:127-140 View Article
  7. Brockwell J, Pilka A and Holliday RA. Soil pH is a major determinant of the numbers of naturally occurring Rhizobium meliloti in non-cultivated soils in central New South Wales. Aust J Exp Agric. 1991; 31:211-219 View Article
  8. Denton MD, Hill CR, Bellotti WD and Coventry DR. Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations. Appl Soil Ecol. 2007; 35:441-448 View Article
  9. Howieson JG and Ewing MA. Acid tolerance in the Rhizobium meliloti-Medicago symbiosis. Aust J Agric Res. 1986; 37:55-64 View Article
  10. Howieson JG, Robson AD and Abbott LK. Acid-tolerant species of Medicago produce root exudates at low pH which induce the expression of nodulation genes in Rhizobium meliloti. Aust J Plant Physiol. 1992; 19:287-296 View Article
  11. Reeve WG, Brau L, Castelli J, Garau G, Sohlenkamp C, Geiger O, Dilworth MJ, Glenn AR, Howieson JG and Tiwari RP. The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions. Microbiology. 2006; 152:3049-3059 View ArticlePubMed
  12. Reeve W, Chain P, O'Hara G, Ardley J, Nandesena K, Brau L, Tiwari R, Malfatti S, Kiss H and Lapidus A. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand Genomic Sci. 2010; 2:77-86 View ArticlePubMed
  13. Brockwell J and Hely FW. Symbiotic characteristics of Rhizobium meliloti: an appraisal of the systematic treatment of nodulation and nitrogen fixation interactions between hosts and rhizobia of diverse origins. Australian Journal of Agricultural Economics. 1966; 17:885-889
  14. Howieson JG, Nutt B and 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 Article
  15. Interrante SM, Singh R, Islam MA, Stein JD, Young CA and Butler TJ. Effectiveness of Sinorhizobium inoculants on annual medics. Crop Sci. 2011; 51:2249-2255 View Article
  16. Terpolilli JJ, O'Hara GW, Tiwari RP, Dilworth MJ and 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 View ArticlePubMed
  17. Bonython AL, Ballard RA, Charman N, Nichols PGH and Craig AD. New strains of rhizobia that nodulate regenerating messina (Melilotus siculus) plants in saline soils. Crop Pasture Sci. 2011; 62:427-436 View Article
  18. Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974; 84:188-198 View ArticlePubMed
  19. Howieson JG, Ewing MA and D'antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil. 1988; 105:179-188 View Article
  20. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M and Angiuoli SV. 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 View ArticlePubMed
  21. Woese CR, Kandler O and 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 View ArticlePubMed
  22. 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.
  23. Garrity GM, Bell JA, Lilburn TG. Class I. Alphaproteobacteria In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey's Manual of Systematic Bacteriology. Second ed. Volume 2: New York: Springer - Verlag; 2005, p. 1.
  24. 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 View ArticlePubMed
  25. 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.
  26. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  27. Conn HJ. Taxonomic relationships of certain non-sporeforming rods in soil. J Bacteriol. 1938; 36:320-321
  28. Judicial Commission of the International Committee on Systematics of P. 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. International Journal of Systematic and Evolutionary Microbiology 2008;58(Pt 8):1973.
  29. 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 View ArticlePubMed
  30. Casida LE. Ensifer adhaerens gen. nov., sp. nov.: a bacterial predator of bacteria in soil. Int J Syst Bacteriol. 1982; 32:339-345 View Article
  31. Agents B. Technical rules for biological agents. TRBA ():466.Web Site
  32. Liolios K, Mavromatis K, Tavernarakis N and 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 View ArticlePubMed
  33. Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2011; 28:2731-2739 View ArticlePubMed
  34. Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.
  35. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39:783-791 View Article
  36. Galibert F, Finan TM, Long SR, Puhler A, Abola P, Ampe F, Barloy-Hubler F, Barnett MJ, Becker A and Boistard P. The composite genome of the legume symbiont Sinorhizobium meliloti. Science. 2001; 293:668-672 View ArticlePubMed
  37. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  38. Ballard RA, Slattery JF and Charman N. Host range and saprophytic competence of Sinorhizobium meliloti - a comparison of strains for the inoculation of lucerne, strand and disc medics. Aust J Exp Agric. 2005; 45:209-216 View Article
  39. Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR and Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology. 1999; 145:1307-1316 View ArticlePubMed
  40. . Web Site
  41. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  42. Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.
  43. Ewing B and Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8:175-185 View ArticlePubMed
  44. Ewing B, Hillier L, Wendl MC and Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998; 8:175-185 View ArticlePubMed
  45. Gordon D, Abajian C and Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998; 8:195-202 View ArticlePubMed
  46. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  47. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  48. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964PubMed
  49. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T and Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007; 35:3100-3108 View ArticlePubMed
  50. Griffiths-Jones S, Bateman A, Marshall M, Khanna A and Eddy SR. Rfam: an RNA family database. Nucleic Acids Res. 2003; 31:439-441 View ArticlePubMed
  51. Krogh A, Larsson B, von Heijne G and Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001; 305:567-580 View ArticlePubMed
  52. Bendtsen JD, Nielsen H, von Heijne G and Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340:783-795 View ArticlePubMed
  53. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed