Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 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
An available source of nitrogen (N) is essential to life on Earth. Although the atmosphere consists of approximately 80% N, the overwhelming proportion of this is present in the form of dinitrogen (N2) which is biologically inaccessible to the vast majority of higher organisms. Only a subset of microbes has the necessary molecular machinery to make atmospheric N2 bioavailable by enzymatically reducing N2 to NH3. The fact that plant growth is most commonly limited by the availability of N may have provided the selective pressure for a wide range of plant genera, most of which are legumes, to evolve a symbiotic relationship with these N2-fixing microbes. These microsymbionts, collectively termed root nodule bacteria, receive a carbon source from the plant and in return supply the host with biologically fixed N. When these symbiotic interactions are optimally harnessed in agriculture, all the N-requirements of the host can be met, without the need to apply industrially synthesized N-based fertilizers, thereby increasing both the economic and environmental sustainability of the farming system [1]. Forage and fodder legumes play an integral role in sustainable farming practice, providing feed for stock while also enriching soil with bioavailable N. Worldwide, there are approximately 110 million ha of forage and fodder legumes under production [2], of which members of the Medicago genus comprise a considerable component. Two bacterial species, Ensifer meliloti and E. medicae are known to nodulate and fix N2 with Medicago spp.  [4][5][6]. E. meliloti strain WSM1022 was isolated in 1987 from a nodule collected from the annual M. orbicularis growing on the Cyclades Island of Naxos in Greece. E. meliloti WSM1022 is a highly effective microsymbiont of Medicago, forming efficient N2fixing associations with the annual species M. littoralis and M. tornata [7]. In common with E. medicae WSM419 [8], WSM1022 also fixes approximately twice as much N2 as E. meliloti 1021 on the model legume M. truncatula A17 [7]. However, unlike E. medicae WSM419, E. meliloti WSM1022 is also highly effective with the perennial M. sativa (alfalfa or lucerne) [7]. Therefore, E. meliloti WSM1022 is a broadly effective microsymbiont of Medicago spp. and as such represents a unique tool for the molecular analysis of effective N2 fixation with fully sequenced macroand microsymbionts. Here we present a summary classification and a set of general features for E. meliloti strain WSM1022 together with a description of its genome sequence and annotation.

Classification and features
E. meliloti WSM1022 is a motile, Gram-negative 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) [9], tryptone-yeast extract agar (TY) [10] or a modified yeast-mannitol agar (YMA) [11] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins ( Figure 1Right). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of E. meliloti WSM1022 in a 16S rRNA sequence based tree. This strain shares 99.92% and 99.61% sequence identity (over 1290 bp) to the 16S rRNA of the fully sequenced E. meliloti 1021 [29] and E. medicae WSM419 [8] strains, respectively. Symbiotaxonomy E. meliloti strain WSM1022 was isolated in 1987 from a nodule collected from the annual M. orbicularis growing on the Cyclades Island of Naxos in Greece. The site of collection was a gentle slope and the soil a sandy-loam texture of pH 7.5-8.0. E. meliloti forms nodules (Nod + ) and fixes N2 (Fix + ) on a range of annual Medicago spp. as well as the perennial M. sativa (Table 2). In common with other characterized E. meliloti strains, WSM1022 does not nodulate M. murex, does not fix N2 with M. polymorpha and M. arabica [4,5] and is a poorly effective microsymbiont of M. sphaerocarpos [11]. However, WSM1022 is broadly effective with the alkaline soil-adapted annuals M. littoralis and M. tornata as well as the widely grown perennial forage legume M. sativa. In addition, WSM1022 is also a highly effective microsymbiont for the model legume M. truncatula A17.  Phylum Proteobacteria TAS [14] Class Alphaproteobacteria TAS [15,16] Order Rhizob iales TAS [16,17] Family Rhizob iaceae TAS [18,19] Genus Ensifer TAS [20][21][22] Species Ensifer meliloti TAS [21] Gram stain Negative IDA 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 generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [24]. Standards in Genomic Sciences All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [25]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [26]. Bootstrap analysis [27] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Brackets after the strain name contain a DNA database accession number and/or a GOLD ID (beg inning with the prefix G) for a sequencing project registered in GOLD [28]. Published g enomes are indicated with an asterisk.

Genome sequencing and annotation 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 [28] and an improved-high-quality-draft genome sequence in IMG. Sequencing, finishing and annota-tion were performed by the JGI. A summary of the project information is shown in Table 3.

Genome annotation
Genes were identified using Prodigal [35] as part of the DOE-JGI annotation pipeline [36]. 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. The tRNAScanSE tool [37] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [38]. 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 (http://infernal.janelia.org). Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [39].

Genome properties
The genome is 6,649,661 nucleotides with 62.16% GC content ( Table 4) and comprised of 121 scaffolds ( Figure 3) of 125 contigs. From a total of 6,398 genes, 6,323 were protein encoding and 75 RNA only encoding genes. The majority of genes (80.78%) 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.