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. [3], although they differ in their symbiotic properties on some Medicago hosts. Specifically, while E. medicae can nodulate and fix N2 with M. murex, M. arabica and M. polymorpha, E. meliloti does not nodulate M. murex, does not fix with M. polymorpha and fixes N2 very poorly with M. arabica [4–6].

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 N2-fixing 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 macro-and 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).

Figure 1.
figure 1

Images of Ensifer meliloti WSM1022 using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on a solid medium (Right).

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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.

Figure 2.
figure 2

Phylogenetic tree showing the relationship of Ensifer meliloti WSM1022 (shown in bold print) to other Ensifer spp. in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,290 bp internal region). 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 (beginning with the prefix G) for a sequencing project registered in GOLD [28]. Published genomes are indicated with an asterisk.

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Table 1. Classification and general features of Ensifer meliloti WSM1022 according to the MIGS recommendations [12]
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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.

Table 2. Nodulation and N2 fixation properties of E. meliloti WSM1022 on selected Medicago spp. Data compiled from [7,11]†
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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 annotation were performed by the JGI. A summary of the project information is shown in Table 3.

Table 3. Genome sequencing project information for E. meliloti WSM1022.
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Growth conditions and DNA isolation

E. meliloti WSM1022 was cultured to mid logarithmic phase in 60 ml of TY rich medium [30] 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 [31].

Genome sequencing and assembly

The genome of Ensifer meliloti WSM1022 was sequenced at the Joint Genome Institute (JGI) using Illumina technology [32]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 12,082,430 reads totaling 1812.4 Mbp.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [31]. 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 [33] (version 1.1.04), (2) 1–3 kb 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 [34] (version r42328). Parameters for assembly steps were: 1) Velvet (velveth: 63 -shortPaired and velvetg: -veryclean yes -exportFiltered yes -mincontiglgth 500 -scaffolding no-covcutoff 10) 2) wgsim (-e 0 -1 100 -2 100 -r 0 -R 0 -X 0) 3) Allpaths-LG (PrepareAllpathsInputs:PHRED64=1 PLOIDY=1 FRAGCOVERAGE=125 JUMPCOVERAGE=25 LONGJUMPCOV=50, RunAllpath-sLG: THREADS=8 RUN=stdshredpairs TARGETS=standard VAPIWARNONLY=True OVERWRITE=True). The final draft assembly contained 125 contigs in 121 scaffolds. The total size of the genome is 6.6 Mb and the final assembly is based on 1,812.4 Mbp of Illumina data, which provides an average 275× coverage of the genome.

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.

Figure 3.
figure 3

Graphical map of the genome of Ensifer meliloti WSM1022 showing the seven largest scaffolds. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

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Table 4. Genome Statistics for Ensifer meliloti WSM1022
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Table 5. Number of protein coding genes of Ensifer meliloti WSM1022 associated with the general COG functional categories.
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