Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park, Australia
© The Author(s) 2014
Published: 15 June 2014
Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
Keywordsroot-nodule bacteria nitrogen fixation rhizobia Betaproteobacteria
Burkholderia spp. are ubiquitous in the environment and are found in nearly all terrestrial and some marine ecosystems. They have adapted to occupy numerous niches and may have saprophytic, parasitic, pathogenic or symbiotic lifestyles . Emerging evidence suggests an ancient and stable symbiosis between Burkholderia and Mimosa genera within South America  and between Burkholderia and legumes from the Papilionoideae subfamily in South Africa [3,4]. Despite this, there is very little data regarding the symbiosis between Burkholderia and endemic legumes outside of South America and South Africa.
In Australia, legumes are predominately nodulated by species from the genera Bradyrhizobium, Ensifer, and Rhizobium [5,6]. There are no published genomes or species descriptions of symbiotic Burkholderia spp. isolated in Australia and there is a paucity of information on the interaction between Burkholderia and endemic Australia legumes. Burkholderia sp. WSM2230 was isolated from an effective nitrogen fixing nodule on Kennedia coccinea grown in an acidic soil (pH(CaCl2) 4.8) collected from Karijini National Park, Western Australia. Its symbiotic phenotype was authenticated in glasshouse experiments (Watkin, unpublished). Recently this isolate was revived from long-term storage from frozen glycerol stocks but failed to form nodules on K. coccinea in axenic glasshouse trials (Walker, unpublished). In this regard, it is interesting that the South African microsymbiont B. tuberum STM678T only infrequently forms effective nodules on Macroptilium atropurpureum (Siratro). A recent study  revealed that B. tuberum forms effective nodules on Siratro when water levels are reduced and temperature is increased. Unlike B. tuberum STM678T, the annotation of the genome sequence of the laboratory cultured strain of WSM2230 failed to identify nodulation genes and this offers an explanation for the lack of a nodulation phenotype.
Establishing the genomic sequences of Australian Burkholderia will be beneficial to understand the mutualistic interactions occurring between plant and rhizosphere organisms in low-pH soil. WSM2230 was only isolated from Karijini National Park acidic soil (pH(CaCl2) 4.8) and other sites where the soil pH was higher (pH(CaCl2) >7) did not contain any Burkholderia symbionts. In these more alkaline soils, numerous Bradyrhizobium and Rhizobium spp. were instead trapped (Watkin, unpublished). Soil pH is an edaphic variable that controls microbial biogeography  and the acid tolerance of Burkholderia has been shown to account for the biogeographical distribution of this genus .
The genome of WSM2230 is one of two Australian Burkholderia genomes (the other being that of WSM2232 (GOLD ID Gi08832)) that have now been sequenced through the Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) program. Here we present a preliminary description of the general features of the Burkholderia sp. WSM2230 together with its genome sequence and annotation. The genomes of WSM2232 and WSM2230 will be an important resource to identify the processes enabling such isolates to adapt to the infertile, highly acidic soils that dominate the Australian landscape.
Classification and features
Classification and general features of Burkholderia sp. strain WSM2230 according to the MIGS recommendations 
Species Burkholderia sp.
Soil, root nodule, on host
Free living, symbiotic
Root nodule of Kennedia coccinea
Karijini National Park, Australia
Soil collection date
Compatibility of WSM2230 with nine legume species for nodulation (Nod) and N2-Fixation (Fix)
Sturts Desert Pea
Genome sequencing and annotation
Genome project history
Genome sequencing project information for Burkholderia sp. WSM2230
Improved high-quality draft
1× Illumina library
Illumina HiSeq 2000
Velvet version 1.1.04; Allpaths-LG version r39750
Gene calling methods
NCBI project ID
Symbiotic N2 fixation, agriculture
Growth conditions and DNA isolation
Burkholderia sp. strain WSM2230 was cultured to mid logarithmic phase in 60 ml of TY rich medium on a gyratory shaker at 28°C . DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method .
Genome sequencing and assembly
The genome of Burkholderia sp. strain WSM2230 was sequenced at the Joint Genome Institute (JGI) using Illumina technology . An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 15,498,652 reads totaling 2,324 Mbp.
All general aspects of library construction and sequencing performed at the JGI can be found at the JGI user home . All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun, L., Copeland, A. and Han, J., unpublished). The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet  (version 1.1.04), (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim (https://github.com/lh3/wgsim), (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG  (version r39750). Parameters for assembly steps were: 1) Velvet —v —s 51 —e 71 —i 2 —t 1 —f “-shortPaired -fastq $FASTQ” —o “-ins_length 250 -min_contig_lgth 500”), 2) wgsim (-e 0 -1 76 -2 76 -r 0 -R 0 -X 0), 3) Allpaths-LG (STD_1,project,assembly,fragment,1,200,35,,,inward,0,0 SIMREADS,project,assembly,jumping,1,,,3000,300,inward,0,0).
The final draft assembly contained 33 contigs in 33 scaffolds. The total size of the genome is 6.3 Mbp and the final assembly is based on 2,324 Mbp of Illumina data, which provides an average 368× 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. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA . Other non-coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [40,41].
Genome Statistics for Burkholderia sp. strain WSM2230
% 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 Burkholderia sp. strain WSM2230 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, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
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
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.
- Compant S, Nowak J, Coenye T, Clement C, Barka EA. Diversity and occurrence of Burkholderia spp. in the natural environment. FEMS Microbiol Rev 2008; 32:607–626. PubMed http://dx.doi.org/10.1111/j.1574-6976.2008.00113.xView ArticlePubMedGoogle Scholar
- Bontemps C, Elliott GN, Simon MF. Dos Reis Júnior FB, Gross E, Lawton RC, Neto NE, De Fatima Loureiro M, De Faria SM, Sprent JI and others. Burkholderia species are ancient symbionts of legumes. Mol Ecol 2010; 19:44–52. PubMed http://dx.doi.org/10.111365-294X.2009.04458.XView ArticlePubMedGoogle Scholar
- Garau G, Yates RJ, Deiana P, Howieson JG. Novel strains of nodulating Burkholderia have a role in nitrogen fixation with Papilionoid herbaceous legumes adapted to acid, infertile soils. Soil Biol Biochem 2009; 41:125–134. http://dx.doi.org/10.1016/j.soilbio.2008.10.011View ArticleGoogle Scholar
- Angus AA, Hirsch AM. Insights into the history of the legume-betaproteobacterial symbiosis. Mol Ecol 2010; 19:28–30. PubMed http://dx.doi.org/10.111365-294X.2009.04459.XView ArticlePubMedGoogle Scholar
- Thrall PH, Laine A, Broadhurst LM, Bagnall DJ, Brockwell J. Symbiotic effectiveness of rhizobial mutualists varies in interactions with native Australian legume genera. PLoS ONE 2011; 6:e23545. PubMed http://dx.doi.org/10.1371/journal.pone.0023545PubMed CentralView ArticlePubMedGoogle Scholar
- Hoque MS, Broadhurst LM, Thrall PH. Genetic characterisation of root nodule bacteria associated with Acacia salicina and A. stenophylla (Mimosaceae) across south-eastern Australia. Int J Syst Evol Microbiol 2011; 61:299–309. PubMed http://dx.doi.org/10.1099/ijs.0.021014-0View ArticlePubMedGoogle Scholar
- Angus AA, Lee A, Lum MR, Shehayeb M, Hessabi R, Fujishige NA, Yerrapragada S, Kano S, Song N, Yang P, et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant Soil 2013; 369:543–562. http://dx.doi.org/10.1007/s11104-013-1590-7View ArticleGoogle Scholar
- Fierer N, Jackson RB. The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci USA 2006; 103:626–631. PubMed http://dx.doi.org/10.1073/pnas.0507535103PubMed CentralView ArticlePubMedGoogle Scholar
- Stopnisek N, Bodenhausen N, Frey B, Fierer N, Eberl L, Weisskopf L. Genus-wide acid tolerance accounts for the biogeographical distribution of soil Burkholderia populations. Environ Microbiol 2013. PubMed http://dx.doi.org/10.1111/14622920.12211
- Miller JH. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; 1972.Google 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
- Beringer JE. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 1974; 84:188–198. PubMed http://dx.doi.org/10.1099/00221287-84-1-188PubMedGoogle Scholar
- Terpolilli JJ. Why are the symbioses between some genotypes of Sinorhizobium and Medicago suboptimal for N2 fixation? Perth: Murdoch University; 2009. 223 p.Google 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 II. Betaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 575.View ArticleGoogle Scholar
- Validation List No. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2006; 56:1–6. PubMed http://dx.doi.org/10.1099/ijs.0.64188-0
- Garrity GM, Bell JA, Lilburn T. Order I. Burkholderiales 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. 575.View ArticleGoogle Scholar
- Garrity GM, Bell JA, Lilburn T. Family I. Burkholderiaceae fam. 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. 575.View ArticleGoogle Scholar
- Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List No. 45. Int J Syst Bacteriol 1993; 43:398–399. http://dx.doi.org/10.1099/00207713-43-2-398
- Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, Hashimoto Y, Ezaki T, Arakawa M. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol 1992; 36:1251–1275. PubMed http://dx.doi.org/10.11348-0421.1992.tb02129.xView ArticlePubMedGoogle Scholar
- Gillis M, Van TV, Bardin R, Goor M, Hebbar P, Willems A, Segers P, Kersters K, Heulin T, Fernandez MP. Polyphasic taxonomy in the genus Burkholderia leading to an emended description of the genus and proposition of Burkholderia vietnamiensis sp. nov. for N2-fixing isolates from rice in Vietnam. Int J Syst Bacteriol 1995; 45:274–289. http://dx.doi.org/10.1099/00207713-45-2-274View ArticleGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121PubMed CentralView ArticlePubMedGoogle Scholar
- Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York: Oxford University Press; 2000.Google Scholar
- Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985; 39:783–791. http://dx.doi.org/10.2307/2408678View ArticleGoogle Scholar
- Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2008; 36:D475–D479. PubMed http://dx.doi.org/10.1093/nar/gkm884PubMed CentralView ArticlePubMedGoogle Scholar
- Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR, Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology 1999; 145:1307–1316. PubMed http://dx.doi.org/10.1099/13500872-145-6-1307View ArticlePubMedGoogle Scholar
- DOI Joint Genome Institute user home. http://my.jgi.doe.gov/general/index.html.
- Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/146224126.96.36.1993View ArticlePubMedGoogle Scholar
- Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.
- Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, Sharpe T, Hall G, Shea TP, Sykes S, et al. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci USA 2011; 108:1513–1518. PubMed http://dx.doi.org/10.1073/pnas.1017351108PubMed CentralView ArticlePubMedGoogle Scholar
- Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
- Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci 2009; 1:63–67. PubMed http://dx.doi.org/10.4056/sigs.632PubMed CentralView ArticlePubMedGoogle Scholar
- 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. PubMedPubMed CentralView ArticlePubMedGoogle Scholar
- Pruesse E, Quast C, Knittel K. Fuchs BdM, Ludwig W, Peplies J, Glöckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007; 35:7188–7196. PubMed http://dx.doi.org/10.1093/nar/gkm864PubMed CentralView ArticlePubMedGoogle Scholar
- INFERNAL. http://infernal.janelia.org
- Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar
- Integrated Microbial Genomes (IMG-ER) platform. http://img.jgi.doe.gov/er