Genome sequence of the Ornithopus/Lupinus-nodulating Bradyrhizobium sp. strain WSM471
- Wayne Reeve1Email author,
- Sofie De Meyer1,
- Jason Terpolilli1,
- Vanessa Melino1,
- Julie Ardley1,
- Rui Tian1,
- Ravi Tiwari1,
- John Howieson1,
- Ronald Yates1, 2,
- Graham O’Hara1,
- Mohamed Ninawi1,
- Megan Lu3,
- David Bruce3,
- Chris Detter3,
- Roxanne Tapia3,
- Cliff Han3,
- Chia-Lin Wei3,
- Marcel Huntemann3,
- James Han3,
- I-Min Chen5,
- Konstantinos Mavromatis3,
- Victor Markowitz5,
- Natalia Ivanova3,
- Ioanna Pagani3,
- Amrita Pati3,
- Lynne Goodwin4,
- Tanja Woyke3 and
- Nikos Kyrpides3
© The Author(s) 2013
Published: 20 December 2013
Bradyrhizobium sp. strain WSM471 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen- (N2) fixing root nodule formed on the annual legume Ornithopus pinnatus (Miller) Druce growing at Oyster Harbour, Albany district, Western Australia in 1982. This strain is in commercial production as an inoculant for Lupinus and Ornithopus. Here we describe the features of Bradyrhizobium sp. strain WSM471, together with genome sequence information and annotation. The 7,784,016 bp high-quality-draft genome is arranged in 1 scaffold of 2 contigs, contains 7,372 protein-coding genes and 58 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.
Keywordsroot-nodule bacteria nitrogen fixation rhizobia Alphaproteobacteria
The most abundant form of nitrogen (N) occurs in the atmosphere as a chemically inert dinitrogen (N2) gas. However, N2 needs to be converted first into a biologically useable form through the unique process of N2 fixation . The incorporation of fixed N into biologically essential macromolecules provides the basis for the continuance of life on Earth. Bioavailable N can be chemically synthesized (primarily through the products obtained from the Haber-Bosch process) or biologically fixed by N2-fixing diazotrophs. The highest contribution to biological fixation occurs from the process of symbiotic nitrogen fixation (SNF). The estimated total annual input from SNF ranges from 139–175 million tons  which provides ∼70% of the N currently utilized in agriculture. However, various constraints from edaphic conditions can limit SNF capacity in certain agricultural areas. To extend productive crops and pastures into these regions, considerable efforts have been devoted to sourcing legume hosts and their compatible microsymbionts from different geographical locations that are edaphically and climatically suited to the challenging areas into which they are to be introduced .
These selection programs have enabled the domestication of new Mediterranean legume species that have overcome the deficiencies of the use of traditional species . Seven species new to Australian agriculture have been commercialized since 1993 including the Papilionoid legume Ornithopus sativus (serradella) . This hard-seeded deep-rooted and acid tolerant pasture legume has shown particular promise in acidic sandy soils exposed to low rainfall , with the potential to be established in four million hectares of sandy soils for which no other suitable legume pasture exists . The hard seeded nature of this legume makes it well adapted to crop rotation systems . Currently, serradella is the most widely sown pasture in Western Australia and has proven to be a highly productive legume with high nutritive value .
The strains of lupin-nodulating Bradyrhizobium that also nodulate seradella are unusual since they have the capacity to establish symbioses with Mediterranean derived herbaceous and crop legumes endemic to the cool climatic regions of the world. Before the 1990s, the commercial inoculant for serradella (Ornithopus spp.) in Australia was Bradyrhizobium sp. strain WU425, however during the breeding and evaluation of well adapted cultivars of O. sativus, it was revealed that WSM471 produced 15% more biomass with this legume than did WU425 . Strain WSM471 was isolated from nodules of O. pinnatus collected in Western Australia, in 1982, although it was almost certainly accidentally introduced to Australia . Because of its superior capacity to fix nitrogen with O. sativus relative to other strains of Bradyrhizobium, strain WSM471 was released as a commercial inoculant for this legume in Australia in 1996  and remains in current usage. This strain is also the commercial “back-up” for inoculation of lupins in Australia. Here we present a summary classification and a set of general features for Bradyrhizobium sp. strain WSM471 together with the description of the complete genome sequence and its annotation.
Classification and general features
Classification and general features of Bradyrhizobium sp. strain WSM471 according to the MIGS recommendations .
Species Bradyrhizobium sp.
Soil, root nodule on host
Free living, symbiotic
Albany, Western Australia
Nodule collection date
Bradyrhizobium sp. strain WSM471 was isolated from nodules of Ornithopus pinnatus collected from Oyster Harbour, near Albany, Western Australia (34.98 lat; 117.96 long), in 1982. The purpose of the collection of the nodules that gave rise to WSM471 was to seek strains of nodulating bacteria that might improve the winter nitrogen fixation capacity of the symbiosis with Lupinus angustifolius. This symbiosis seemed to be limited by low winter temperatures, which was later confirmed by Peltzer et al. . Strain WSM471 is highly effective for nitrogen fixation with the grain legumes L. pilosus, L. angustifolius and L. atlanticus, and also the forage legumes O. pinnatus, O. sativus and O. compressus [5,23]. Because WSM471 has a broad range for symbiotic nitrogen fixation across both pulse and forage legumes, and is in commercial usage, it was chosen as a candidate strain for sequencing.
Genome sequencing and annotation information
Genome project history
Genome sequencing project information for Bradyrhizobium sp. strain WSM471.
Illumina GAii shotgun and paired end 454 libraries
Illumina GAii and 454 GS FLX Titanium technologies
6.9× 454 paired end, Illumina 625.6
Velvet1.0.13, Newbler 2.3, phrap 4.24
Gene calling methods
Prodigal 1.4, GenePRIMP
Genbank Date of Release
February 2, 2012
NCBI project ID
Symbiotic N2-fixation, agriculture
Growth conditions and DNA isolation
Bradyrhizobium sp. strain WSM471 was grown to mid logarithmic phase in TY rich medium  on a gyratory shaker at 28°C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method .
Genome sequencing and assembly
The genome of Bradyrhizobium sp. WSM471 was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina  and 454 technologies . An Illumina GAii shotgun library which generated 67,039,982 reads totaling 5,095 Mb and 1 paired end 454 library with an average insert size of 5 Kb which generated 397,976 reads totaling 83.7 Mb of 454 were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website . The initial draft assembly contained 236 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with Velvet, version 1.0.13 , and the consensus sequence were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [29–31] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 327 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 7.8 Mb and the final assembly is based on 53.8 Mb of 454 draft data which provides an average 6.9× coverage of the genome and 4,879.9 Mb of Illumina draft data which provides an average 625.6× coverage of the genome.
Genes were identified using Prodigal  as part of the DOE-JGI Annotation pipeline  followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , RNAMMer , Rfam , TMHMM , and SignalP . Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform .
Genome Statistics for Bradyrhizobium sp. strain WSM471.
% of Total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of scaffolds
Number of contigs
Genes with function prediction
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of protein coding genes of Bradyrhizobium sp. strain WSM471 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 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. The authors would like to thank the Australia-China Joint Research Centre for Wheat Improvement (ACCWI) and SuperSeed Technologies (SST) for financially supporting Mohamed Ninawi’s PhD project.
- O’Hara GW. The role of nitrogen fixation in crop production. J Crop Prod 1998; 1:115–138. http://dx.doi.org/10.1300/1144v01n02_06View 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
- 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
- Nichols P, Loi A, Nutt B, Snowball R Revell C. Domestication of new Mediterranean annual pasture legumes. In: Huyghe C, editor. Sustainable Use of Genetic Diversity in Forage and Turf Breeding: Springer; 2010. p 137–142.
- Ballard R. Assessment of strains of Bradyrhizobium sp. (Lupinus) for serradellas (Ornithopus spp.). Aust J Exp Agric 1996; 36:63–70. http://dx.doi.org/10.1071/EA9960063View ArticleGoogle Scholar
- Stepkowski T, Moulin L, Krzyzanska A, McInnes A, Law IJ, Howieson J. European origin of Bradyrhizobium populations infecting lupins and serradella in soils of Western Australia and South Africa. Appl Environ Microbiol 2005; 71:7041–7052. PubMed http://dx.doi.org/10.1128/AEM.71.11.7041-7052.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Bullard GK, Roughley RJ, Pulsford DJ. The legume inoculant industry and inoculant quality control in Australia: 1953–2003. Aust J Exp Agric 2005; 45:127–140. http://dx.doi.org/10.1071/EA03159View ArticleGoogle 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
- 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.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
- 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
- Garrity GM, Bell JA, Lilburn TG. Family VII. Bradyrhizobiaceae. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Volume 2. New York: Springer-Verlag; 2005. p 438.Google Scholar
- Jordan DC. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int J Syst Bacteriol 1982; 32:136–139. http://dx.doi.org/10.1099/00207713-32-1-136View 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
- 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(Database issue):D475–D479. PubMed http://dx.doi.org/10.1093/nar/gkm884PubMed CentralView ArticlePubMedGoogle Scholar
- Peltzer SC, Abbott LK, Atkins CA. Effect of low root-zone temperature on nodule initiation in narrow-leafed lupin (Lupinus angustifolius L.). Aust J Agric Res 2002; 53:355–365. http://dx.doi.org/10.1071/AR01092View ArticleGoogle Scholar
- Howieson JG, Reeve N, Yates RJ. The selection of effective Brady rhizobium sp. (Lupinus) for new lupin and serradella species. In: Dracup M, Palta J, editors. Proceedings of the First Australian Lupin Technical Symposium. Perth, Western Australia: Department of Agriculture; 1994. p 270–273.Google 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. http://my.jgi.doe.gov/general/index.html
- Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/14622422.214.171.1243View ArticlePubMedGoogle Scholar
- Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005; 437:376–380. PubMedPubMed CentralPubMedGoogle Scholar
- Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010; Chapter 11:Unit 11 5.
- Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998; 8:186–194. PubMed http://dx.doi.org/10.1101/gr.83.175View ArticlePubMedGoogle Scholar
- Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 1998; 8:175–185. PubMed http://dx.doi.org/10.1101/gr.8.3.175View ArticlePubMedGoogle Scholar
- Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998; 8:195–202. PubMed http://dx.doi.org/10.1101/gr.8.3.195View ArticlePubMedGoogle Scholar
- Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Valafar HRAH, editor. Proceeding of the 2006 international conference on bioinformatics & computational biology: CSREA Press; 2006. p 141–146.
- 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
- Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010; 7:455–457. PubMed http://dx.doi.org/10.1038/nmeth.1457View 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. PubMePubMed CentralView ArticlePubMedGoogle Scholar
- Lagesen K Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007; 35:3100–3108. PubMed http://dx.doi.org/10.1093/nar/gkm160PubMed CentralView ArticlePubMedGoogle Scholar
- Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR. Rfam: an RNA family database. Nucleic Acids Res 2003; 31:439–441. PubMed http://dx.doi.org/10.1093/nar/gkg006PubMed CentralView ArticlePubMedGoogle Scholar
- Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001; 305:567–580. PubMed http://dx.doi.org/10.1006/jmbi.2000.4315View ArticlePubMedGoogle Scholar
- Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795. PubMed http://dx.doi.org/10.1016/j.jmb.2004.05.028View ArticlePubMedGoogle Scholar
- 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