Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565
- Wayne Reeve1Email author,
- Elizabeth Drew2,
- Ross Ballard2,
- Vanessa Melino1,
- Rui Tian1,
- Sofie De Meyer1,
- Lambert Brau3,
- Mohamed Ninawi1,
- Hazuki Teshima4,
- Lynne Goodwin4,
- Patrick Chain4,
- Konstantinos Liolios5,
- Amrita Pati5,
- Konstantinos Mavromatis5,
- Natalia Ivanova5,
- Victor Markowitz6,
- Tanja Woyke5 and
- Nikos Kyrpides5
© The Author(s) 2013
Published: 20 December 2013
Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N2-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 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.
Keywordsroot-nodule bacteria nitrogen fixation rhizobia Alphaproteobacteria
Plant available nitrogen is a precious commodity in many agricultural soils and the most commonly limiting nutrient in plant growth. The supply of plant available nitrogen to nitrogen (N)-deficient farming systems is thus vital to productivity . The application of industrially fixed nitrogenous fertilizer can meet the demand for N. However, this is a costly option as the price of nitrogenous fertilizer is connected to the cost of fossil fuels required for its production. Furthermore, the use of nitrogenous fertilizer contributes to greenhouse gas emissions and pollution of the environment. A more environmentally sustainable option is to exploit the process of biological nitrogen fixation that occurs in the symbiosis between legumes and rhizobia .
In this symbiotic association, rhizobia reduce atmospheric dinitrogen (N2) into bioavailable N that can be used by the plant for growth. Pasture legumes, including the clovers that comprise the Trifolium genus, are major contributors of biologically fixed N2 to mixed farming systems throughout the world [3,4]. In Australia, soils with a history of growing Trifolium spp. have developed large and symbiotically diverse populations of Rhizobium leguminosarum bv. trifolii (R. l. trifolii) that are able to infect and form nodules on a range of clover species. The N2-fixation capacity of the symbioses established by different combinations of clover hosts (Trifolium spp.) and strains of R. l. trifolii can vary from 10 to 130% when compared to an effective host-strain combination [3–9].
R. l. trifolii strain SRDI565 (syn. N8-J ) was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum that had been inoculated with soil collected from under a mixed pasture stand from Tumet, New South Wales, Australia and grown in N deficient media for four weeks after inoculation, in the greenhouse. SRDI565 was first noted for its sub-optimal N2-fixation capacity on T. subterraneum cv. Campeda (<60% of that with strain WSM1325) and formation of white (Fix-) pseudo-nodules on T. subterraneum cv. Clare [10,11]. Here we present a preliminary description of the general features for R. leguminosarum bv. trifolii strain SRDI565 together with its genome sequence and annotation.
Classification and general features
Classification and general features of Rhizobium leguminosarum bv. trifolii SRDI565 according to the MIGS recommendations 
Species Rhizobium leguminosarum bv. trifolii
Soil, root nodule, on host
Free living, symbiotic
Soil collection date
Compatibility of SRDI565 with eleven Trifolium genotypes for nodulation (Nod) and N2-Fixation (Fix)
T. glanduliferum Boiss.
T. michelianum Savi.
T. purpureum Loisel
T. resupinatum L.
T. subterraneum L.
T. subterraneum L.
T. vesiculosum Savi.
T. fragiferum L.
T. polymorphum Poir
T. pratense L.
T. repens L.
Genome sequencing and annotation information
Genome project history
Genome sequencing project information for Rhizobium leguminosarum bv. trifolii strain SRDI565.
Improved high-quality draft
2× Illumina libraries; Std short PE & CLIP long PE
Illumina HiSeq 2000, PacBio
with Allpaths, version 39750, Velvet 1.015, phrap 4.24
Gene calling methods
Prodigal 1.4, GenePRIMP
NCBI project ID
Symbiotic N2 fixation, agriculture
Growth conditions and DNA isolation
Rhizobium leguminosarum bv. trifolii strain SRDI565 was cultured to mid logarithmic phase in 60 ml of TY rich media  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 Rhizobium leguminosarum bv. trifolii strain SRDI565 was sequenced at the Joint Genome Institute (JGI) using Illumina  data. An Illumina short-insert paired-end library with an average insert size of 243 ± 58 bp was used to generate 18,700,764 reads and an Illumina long-insert paired-end library with an average insert size of 8,446 ± 2,550 bp was used to generate 21,538,802 reads totalling 6,036 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 homepage . The initial draft assembly contained 22 contigs in 16 scaffolds. The initial draft data was assembled with Allpaths, version 39750, and the consensus was computationally shredded into 10 Kb 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 Kb 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 Kb 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 [36–38]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished), and sequencing of bridging PCR fragments with PacBio (unpublished, Cliff Han) technology. For improved high quality draft, 4 PCR PacBio consensus sequences were completed to close gaps and to raise the quality of the final sequence. The estimated total size of the genome is 7 Mb and the final assembly is based on 6,036 Mb of Illumina draft data, which provides an average 862× 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 [47,48].
Genome Statistics for Rhizobium leguminosarum bv. trifolii SRDI565
% 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 Rhizobium leguminosarum bv. trifolii SRDI565 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’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 (UMU00032). 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.
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