Open Access

Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

  • Jason Terpolilli
  • , Yvette Hill
  • , Rui Tian
  • , John Howieson
  • , Lambert Bräu
  • , Lynne Goodwin
  • , James Han
  • , Konstantinos Liolios
  • , Marcel Huntemann
  • , Amrita Pati
  • , Tanja Woyke
  • , Konstantinos Mavromatis
  • , Victor Markowitz
  • , Natalia Ivanova
  • , Nikos Kyrpides
  • and Wayne Reeve
Corresponding author

DOI: 10.4056/sigs.4608286

Received: 15 December 2013

Accepted: 15 December 2013

Published: 20 December 2013


Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.


root-nodule bacterianitrogen fixationrhizobiaAlphaproteobacteria


An available source of nitrogen (N) is essential to life on Earth. 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 the vast majority of higher organisms. Only a subset of microbes has the necessary molecular machinery to make atmospheric N2 bioavailable by enzymatically reducing N2 to NH3. The fact that plant growth is most commonly limited by the availability of N may have provided the selective pressure for a wide range of plant genera, most of which are legumes, to evolve a symbiotic relationship with these N2-fixing microbes. These microsymbionts, collectively termed root nodule bacteria, receive a carbon source from the plant and in return supply the host with biologically fixed N. When these symbiotic interactions are optimally harnessed in agriculture, all the N-requirements of the host can be met, without the need to apply industrially synthesized N-based fertilizers, thereby increasing both the economic and environmental sustainability of the farming system [1].

Forage and fodder legumes play an integral role in sustainable farming practice, providing feed for stock while also enriching soil with bioavailable N. Worldwide, there are approximately 110 million ha of forage and fodder legumes under production [2], of which members of the Medicago genus comprise a considerable component. Two bacterial species, Ensifer meliloti and E. medicae are known to nodulate and fix N2 with Medicago spp. [3], although they differ in their symbiotic properties on some Medicago hosts. Specifically, while E. medicae can nodulate and fix N2 with M. murex, M. arabica and M. polymorpha, E. meliloti does not nodulate M. murex, does not fix with M. polymorpha and fixes N2 very poorly with M. arabica [4-6].

E. meliloti strain WSM1022 was isolated in 1987 from a nodule collected from the annual M. orbicularis growing on the Cyclades Island of Naxos in Greece. E. meliloti WSM1022 is a highly effective microsymbiont of Medicago, forming efficient N2-fixing associations with the annual species M. littoralis and M. tornata [7]. In common with E. medicae WSM419 [8], WSM1022 also fixes approximately twice as much N2 as E. meliloti 1021 on the model legume M. truncatula A17 [7]. However, unlike E. medicae WSM419, E. meliloti WSM1022 is also highly effective with the perennial M. sativa (alfalfa or lucerne) [7]. Therefore, E. meliloti WSM1022 is a broadly effective microsymbiont of Medicago spp. and as such represents a unique tool for the molecular analysis of effective N2 fixation with fully sequenced macro-and microsymbionts. Here we present a summary classification and a set of general features for E. meliloti strain WSM1022 together with a description of its genome sequence and annotation.

Classification and features

E. meliloti WSM1022 is a motile, Gram-negative rod (Figure 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 (½LA) [9], tryptone-yeast extract agar (TY) [10] or a modified yeast-mannitol agar (YMA) [11] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1Right).

Figure 1

Images of Ensifer meliloti WSM1022 using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on a solid medium (Right).

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of E. meliloti WSM1022 in a 16S rRNA sequence based tree. This strain shares 99.92% and 99.61% sequence identity (over 1290 bp) to the 16S rRNA of the fully sequenced E. meliloti 1021 [29] and E. medicae WSM419 [8] strains, respectively.

Table 1

Classification and general features of Ensifer meliloti WSM1022 according to the MIGS recommendations [12]




     Evidence code

     Current classification

      Domain Bacteria

     TAS [13]

      Phylum Proteobacteria

     TAS [14]

      Class Alphaproteobacteria

     TAS [15,16]

      Order Rhizobiales

     TAS [16,17]

      Family Rhizobiaceae

     TAS [18,19]

      Genus Ensifer

     TAS [20-22]

      Species Ensifer meliloti

     TAS [21]

     Gram stain



     Cell shape









     Temperature range



     Optimum temperature







     Oxygen requirement


     TAS [7]

     Carbon source



     Energy source





      Soil, root nodule, on host

     TAS [7]


     Biotic relationship

      Free living, symbiotic

     TAS [7]





     Biosafety level


     TAS [23]


      Root nodule

     TAS [11]


     Geographic location

      Naxos, Greece

     TAS [11]


     Soil collection date

      28 April 1987














      Not recorded

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 [24].

Figure 2

Phylogenetic tree showing the relationship of Ensifer meliloti WSM1022 (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 [25]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [26]. Bootstrap analysis [27] 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 [28]. Published genomes are indicated with an asterisk.


E. meliloti strain WSM1022 was isolated in 1987 from a nodule collected from the annual M. orbicularis growing on the Cyclades Island of Naxos in Greece. The site of collection was a gentle slope and the soil a sandy-loam texture of pH 7.5-8.0. E. meliloti forms nodules (Nod+) and fixes N2 (Fix+) on a range of annual Medicago spp. as well as the perennial M. sativa (Table 2). In common with other characterized E. meliloti strains, WSM1022 does not nodulate M. murex, does not fix N2 with M. polymorpha and M. arabica [4,5] and is a poorly effective microsymbiont of M. sphaerocarpos [11]. However, WSM1022 is broadly effective with the alkaline soil-adapted annuals M. littoralis and M. tornata as well as the widely grown perennial forage legume M. sativa. In addition, WSM1022 is also a highly effective microsymbiont for the model legume M. truncatula A17.

Table 2

Nodulation and N2 fixation properties of E. meliloti WSM1022 on selected Medicago spp. Data compiled from [7,11]

Species Name

     Cultivar or Accession

     Growth     Habit


     N2 fixation


M. truncatula





     Highly effective

M. truncatula





     Highly effective

M. truncatula





     Highly effective

M. littoralis





     Highly effective

M. tornata





     Highly effective

M. sphaerocarpos





     Poorly effective

M. arabica





     No fixation

M. polymorpha





     No fixation

M. murex





     No nodulation

M. sativa





     Highly effective

Note that ‘+’ and ‘-’ denote presence or absence, respectively, of nodulation (Nod) or N2 fixation (Fix).

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [28] and an improved-high-quality-draft genome sequence in IMG. 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 E. meliloti WSM1022.





    Finishing quality

    Improved high-quality draft


    Libraries used

    1× Illumina library


    Sequencing platforms

    Illumina HiSeq 2000


    Sequencing coverage

    Illumina: 275×



    Velvet version 1.1.04; Allpaths-LG version r42328


    Gene calling methods

    Prodigal 1.4, GenePRIMP



    NCBI project ID


    Database: IMG


    Project relevance

    Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

E. meliloti WSM1022 was cultured to mid logarithmic phase in 60 ml of TY rich medium [30] 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 [31].

Genome sequencing and assembly

The genome of Ensifer meliloti WSM1022 was sequenced at the Joint Genome Institute (JGI) using Illumina technology [32]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 12,082,430 reads totaling 1812.4 Mbp.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [31]. 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 [33] (version 1.1.04), (2) 1–3 kb simulated paired end reads were created from Velvet contigs using wgsim (Web Site), (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG [34] (version r42328). 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 –1 100 –2 100 –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 125 contigs in 121 scaffolds. The total size of the genome is 6.6 Mb and the final assembly is based on 1,812.4 Mbp of Illumina data, which provides an average 275× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [35] as part of the DOE-JGI annotation pipeline [36]. 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 [37] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [38]. 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 (Web Site). Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [39].

Genome properties

The genome is 6,649,661 nucleotides with 62.16% GC content (Table 4) and comprised of 121 scaffolds (Figure 3) of 125 contigs. From a total of 6,398 genes, 6,323 were protein encoding and 75 RNA only encoding genes. The majority of genes (80.78%) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 5.

Table 4

Genome Statistics for Ensifer meliloti WSM1022



    % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of scaffolds


Number of contigs


Total gene



RNA genes



rRNA operons



Protein-coding genes



Genes with function prediction



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


Figure 3

Graphical map of the genome of Ensifer meliloti WSM1022 showing the seven largest scaffolds. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Table 5

Number of protein coding genes of Ensifer meliloti WSM1022 associated with the general COG functional categories.



      % age

      COG Category




      Translation, ribosomal structure and biogenesis




      RNA processing and modification








      Replication, recombination and repair




      Chromatin structure and dynamics




      Cell cycle control, mitosis and meiosis




      Nuclear structure




      Defence mechanisms




      Signal transduction mechanisms




      Cell wall/membrane biogenesis




      Cell motility








      Extracellular structures




      Intracellular trafficking and secretion




      Posttranslational modification, protein turnover, chaperones




      Energy production conversion




      Carbohydrate transport and metabolism




      Amino acid transport metabolism




      Nucleotide transport and metabolism




      Coenzyme transport and metabolism




      Lipid transport and metabolism




      Inorganic ion transport and metabolism




      Secondary metabolite biosynthesis, transport and catabolism




      General function prediction only




      Function unknown




      Not in COGS







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

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


  1. Howieson JG, O’Hara GW and Carr SJ. Changing roles for legumes in Mediterranean agriculture: developments from an Australian perspective. Field Crops Res. 2000; 65:107-122 View Article
  2. Herridge DF, Peoples MB and Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil. 2008; 311:1-18 View Article
  3. 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.
  4. Garau G, Reeve WG, Brau L, Yates RJ, James D, Tiwari R, O'Hara GW and 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 View Article
  5. Rome S, Cleyet-Marel JC, Materon LA, Normand P and Brunel B. Rapid identification of Medicago nodulating strains by using two oligonucleotide probes complementary to 16S rDNA sequences. Can J Microbiol. 1997; 43:854-861 View ArticlePubMed
  6. Brunel B, Rome S, Ziani R and Cleyet-Marel JC. Comparison of nucleotide diversity and symbiotic properties of Rhizobium meliloti populations from annual Medicago species. FEMS Microbiol Ecol. 1996; 19:71-82 View Article
  7. 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
  8. 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
  9. Howieson JG, Ewing MA and D'antuono MF. Selection for acid tolerance in Rhizobium meliloti. Plant Soil. 1988; 105:179-188 View Article
  10. Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974; 84:188-198 View ArticlePubMed
  11. Terpolilli JJ. Why are the symbioses between some genotypes of Sinorhizobium and Medicago suboptimal for N2 fixation? Perth: Murdoch University; 2009. 223 p.
  12. 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
  13. 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
  14. 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.
  15. Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey's Manual of Systematic Bacteriology. Second ed: New York: Springer - Verlag; 2005, p. 1.
  16. . 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
  17. Kuykendall LD. Order VI. Rhizobiales ord. 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. 324.
  18. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  19. Conn HJ. Taxonomic relationships of certain non-sporeforming rods in soil. J Bacteriol. 1938; 36:320-321
  20. 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
  21. 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
  22. . 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 ArticlePubMed
  23. Gubler M, Hennecke H and Fix A. B. and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett. 1986; 200:186-192 View Article
  24. 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
  25. 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
  26. Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.
  27. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39:783-791 View Article
  28. 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
  29. 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
  30. 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
  31. DOE Joint Genome Institute user home. Web Site
  32. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  33. Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.
  34. Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, Sharpe T, Hall G, Shea TP and Sykes S. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci USA. 2011; 108:1513-1518 View ArticlePubMed
  35. 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
  36. Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM and Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009; 1:63-67 View ArticlePubMed
  37. 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
  38. Pruesse E, Quast C and 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 View ArticlePubMed
  39. 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