Genome sequence of the dark pink pigmented Listia bainesii microsymbiont Methylobacterium sp. WSM2598
© Ardley et al.; licensee BioMed Central Ltd. 2014
Received: 13 June 2014
Accepted: 16 June 2014
Published: 8 December 2014
Strains of a pink-pigmented Methylobacterium sp. are effective nitrogen- (N2) fixing microsymbionts of species of the African crotalarioid genus Listia. Strain WSM2598 is an aerobic, motile, Gram-negative, non-spore-forming rod isolated in 2002 from a Listia bainesii root nodule collected at Estcourt Research Station in South Africa. Here we describe the features of Methylobacterium sp. WSM2598, together with information and annotation of a high-quality draft genome sequence. The 7,669,765 bp draft genome is arranged in 5 scaffolds of 83 contigs, contains 7,236 protein-coding genes and 18 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 G enomic E ncyclopedia for B acteria and A rchaea-R oot N odule B acteria (GEBA-RNB) project.
Nodulated legumes are important and established components of Australian agricultural systems: the value of atmospheric nitrogen (N2) fixed by rhizobia in symbiotic association with these legumes is estimated to be worth more than $2 billion annually [1, 2]. The major agricultural region of south-western Australia has a Mediterranean climate, with soils that are often acid, have a low clay content and low organic matter, and tend to be inherently infertile [3, 4]. The last forty years, however, have seen a sharp decrease in average winter rainfall by about 15–20% . This, together with the development of dryland salinity , has challenged the sustainability of using the commonly sown subterranean clover and annual medics as pasture legumes in these systems. Alternative perennial legume species (and their associated rhizobia) are therefore being sought . We have identified a suite of South African perennial, herbaceous forage legumes, including several species in the crotalarioid genus Listia (previously Lotononis) , that are potentially well-adapted to the arid climate and acid, infertile soils of the target agricultural areas.
Listia species are found in seasonally wet habitats throughout southern and tropical Africa . They produce stoloniferous roots [8, 9] and form lupinoid nodules rather than the indeterminate type found in other crotalarioid species [7, 10]. Rhizobial infection occurs by epidermal entry rather than via root hair curling . Listia-rhizobia symbioses are highly specific. The tropically distributed L. angolensis forms effective (i.e. N2-fixing) nodules with newly described species of Microvirga , while all other studied Listia species are only nodulated by strains of pigmented methylobacteria [7, 10, 12]. Unlike the methylotrophic Methylobacterium nodulans, which specifically nodulates some species of Crotalaria , the Listia methylobacteria are unable to utilize methanol as a sole carbon source . In Australia, strains of pigmented methylobacteria have been used as commercial inoculants for Listia bainesii and are able to persist in acidic, sandy, infertile soils, while remaining symbiotically and serologically stable [10, 15].
A pigmented Methylobacterium strain, WSM2598, isolated from a root nodule of L. bainesii cv “Miles” in South Africa in 2002, was found to be a highly effective nitrogen fixing microsymbiont of both L. bainesii and Listia heterophylla (previously Lotononis listii) . Here we present a set of preliminary classification and general features for Methylobacterium sp. strain WSM2598, together with the description of the genome sequence and annotation.
Species Methylobacterium sp.
Formate, succinate & glutamate
Soil, root nodule on host
Free living, symbiotic
Root nodule of Listia bainesii
Estcourt Research Station, South Africa
Sample collection date
May 27, 2002
Compatibility of Methylobacterium sp. WSM2598 with 11 host legume genotypes for nodulation (Nod) and N 2 -Fixation (Fix)
Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr.
Listia bainesii (Bak.) B.-E. van Wyk & Boatwr.
Listia heterophylla E. Mey.
Listia marlothii (Engl.) B.-E. van Wyk & Boatwr.
Listia solitudinis (Dümmer) B.-E. van Wyk & Boatwr.
Listia subulata (B.-E. van Wyk) B.-E. van Wyk & Boatwr.
Leobordea lanata (Thunb.) B.-E. van Wyk & Boatwr. (=Lotononis bolusii)
Leobordea longiflora (H. Bolus) B.-E. van Wyk & Boatwr.
Leobordea stipulosa (Bak. f.) B.-E. van Wyk & Boatwr.
Macroptilium atropurpureum (DC.) Urb. cv. Siratro
(w) indicates nodules present were white.
Genome sequencing and annotation information
Genome project history
Genome sequencing project information for Methylobacterium sp. WSM2598
Improved high quality draft
Illumina GAii standard PE and CLIP PE libraries
Illumina GAii technology
Velvet, version 1.0.05; Allpaths r39750
Gene calling method
GenBank release date
August 28, 2013
NCBI project ID
Symbiotic N2 fixation, agriculture
Growth conditions and DNA isolation
Methylobacterium sp. WSM2598 was grown to mid-logarithmic phase in TY rich media 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 draft genome of Methylobacterium sp. WSM2598 was generated at the DOE Joint Genome Institute (JGI) using Illumina technology [34, 35]. For this genome, we constructed and sequenced an Illumina short-insert paired-end library with an average insert size of 270 bp which generated 19,048,548 reads and an Illumina long-insert paired-end library with an average insert size of 6354.14 +/− 3100.07 bp which generated 18,876,864 reads totaling 5,689 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 website. The initial draft assembly contained 141 contigs in 41 scaffold(s). The initial draft data was assembled with Allpaths, version 39750, 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  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 [37–39]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished), and sequencing of bridging PCR fragments with Sanger and/or PacBio (unpublished, Cliff Han) technologies. One round of manual/wet lab finishing was also completed. 17 PCR PacBio consensus sequences were completed to close gaps and to raise the quality of the final sequence. The total (“estimated size” for the unfinished) size of the genome is 8.3 Mbp and the final assembly is based on 5,689 Mbp of Illumina draft data, which provides an average 685× 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 . Within the Integrated Microbial Genomes (IMG-ER) system , 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 , RNAMMer , Rfam , TMHMM , and SignalP . Additional gene prediction analyses and functional annotation were performed within IMG.
Genome statistics for Methylobacterium sp. WSM2598
% 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 Methylobacterium sp. WSM2598 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
WSM2598 was sequenced as part of the DOE Joint Genome Institute GEBA-RNB project. In common with other sequenced rhizobial strains, WSM2598 has a comparatively large genome of around 7.69 Mbp, with a high proportion of genes assigned to the COG functional categories associated with transcription control and signal transduction (14.69%), transport and metabolism (29.38%) and secondary metabolite biosynthesis (3.12%). These features are characteristic of soil bacteria, which inhabit oligotrophic environments with typically diverse but scarce nutrient sources. Rhizobial methylobacteria are unusual, however, in that they form symbiotic associations exclusively with African crotalarioid legume hosts, several species of which are well-adapted to arid climates and acid, infertile soils and are therefore potentially useful pasture plants in marginal agricultural systems. The molecular basis for this symbiotic specificity has yet to be determined. As WSM2598 is highly effective for N2-fixation on several of these hosts, its sequenced genome is a valuable resource for gaining an understanding of symbiotic specificity and N2-fixation in a currently understudied group of legumes and rhizobia.
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 Strategic Research Funds allocated by Murdoch University to support this project.
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