Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565.

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.


Introduction
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 [1]. 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 [2].
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][4][5][6][7][8][9]. R. l. trifolii strain SRDI565 (syn. N8-J [10]) 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
R. l. trifolii strain SRDI565 is a motile, Gramnegative 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) [12] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right).

Symbiotaxonomy
R. l. trifolii SRDI565 forms nodules on (Nod + ), and fixes N2 (Fix + ) with, a range of annual and perennial clover species of Mediterranean origin ( Table  2). SRDI565 forms white, ineffective (Fix -) nodules with annual clovers T. glanduliferum and T. subterraneum cv. Clare, and with the perennial clovers T. pratense and T. polymorphum. SRDI565 does not form nodules on T. vesiculosum.

Genome sequencing and annotation information 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 [30] 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.
Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of R. l. trifolii strain SRDI565 in a 16S rRNA sequence based tree. This strain clusters closest to R. l. trifolii T24 and Rhizobium leguminosarum bv. phaseoli RRE6 with 99.8% and 99.6% sequence identity, respectively.

Growth conditions and DNA isolation
Rhizobium leguminosarum bv. trifolii strain SRDI565 was cultured to mid logarithmic phase in 60 ml of TY rich media [31] 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 [32].   [13] MIGS ID Property Term Evidence code

Current classification
Domain Bacteria TAS [13,14] Phylum Proteobacteria TAS [15] Class Alphaproteobacteria TAS [16] Order Rhizob iales TAS [17,18] Family Rhizob iaceae TAS [19,20] Genus 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 g enerally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [27]. (shown in blue print) with some of the root nodule bacteria in the orde r Rhizob iales based on alig ned sequences of the 16S rRNA g ene (1,307 bp internal reg ion). All sites were informative and there were no g apcontaining sites. Phylogenetic analyses were performed using MEGA, version 5.05 [28]. The tree was built using the maximum likelihood method with the General Time Reversible model. Bootstrap analysis [29] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Strains with a g enome sequencing project reg istered in GOLD [30] are in bold print and the GOLD ID is shown after the accession number. Published g enomes are indicated with an asterisk.

Genome sequencing and assembly
The genome of Rhizobium leguminosarum bv. trifolii strain SRDI565 was sequenced at the Joint Genome Institute (JGI) using Illumina [33] 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 longinsert 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 [34]. 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 [35], 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 pairedend reads were assembled using parallel phrap, version 4.24 (High Performance Software, LLC). Possible mis-assemblies were corrected with manual editing in Consed [36][37][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.

Genome annotation
Genes were identified using Prodigal [39] as part of the DOE-JGI annotation pipeline [40], followed by a round of manual curation using the JGI GenePRIMP pipeline [41]. 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. These data sources were combined to assert a product description for each predicted protein. Noncoding genes and miscellaneous features were predicted using tRNAscan-SE [42], RNAMMer [43], Rfam [44], TMHMM [45], and SignalP [46]. Addi-tional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [47,48].

Genome properties
The genome is 6,905,599 nucleotides with 60.67% GC content ( Table 4) and comprised of 7 scaffolds (Figures 3,4,5,6,7,8,and 9) of 7 contigs. From a total of 6,836 genes, 6,750 were protein encoding and 86 RNA-only encoding genes. The majority of genes (77.98%) 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.          . From bottom to the top of each scaffold: Genes on forwa rd stra nd (color by COG categ ories as denoted by the IMG platform), Genes on reverse strand (color by COG categ ories), RNA g enes (tRNAs g reen, sRNAs red, other RNAs black), GC content, GC skew.