Genome sequence of Ensifer medicae Di28; an effective N2-fixing microsymbiont of Medicago murex and M. polymorpha

Ensifer medicae Di28 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago spp. Di28 was isolated in 1998 from a nodule recovered from the roots of M. polymorpha growing in the south east of Sardinia (Italy). Di28 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. murex and is capable of establishing a partially effective symbiotic association with the perennial M. sativa. Here we describe the features of E. medicae Di28, together with genome sequence information and its annotation. The 6,553,624 bp standard draft genome is arranged into 104 scaffolds of 104 contigs containing 6,394 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.


Introduction
Legumes are key components of sustainable agricultural systems owing to their ability to form nitrogen (N 2 )-fixing symbioses with specific soil bacteria referred to as rhizobia (or root nodule bacteria). These rhizobia are housed within legume root nodules, where they receive a source of carbon from the legume and in return supply the host with reduced nitrogen (N) in the form of ammonia [1]. The provision of this bioavailable N to the host fuels legume growth and development without the requirement for supplementation with industrially synthesized Nbased fertilizers. Furthermore, some of this biologically fixed N remains in the soil after plant harvest or senescence, resulting in an increase in soil fertility. Growing legumes in rotation with a cereal crop or as a source of forage or fodder is therefore an environmentally sustainable way of improving soil fertility and boosting agricultural productivity [2].
The legume genus Medicago is of prime importance globally as a source of forage or fodder. The perennial M. sativa (alfalfa or lucerne) is the most widely cultivated member of this genus, with over 35 million hectares grown annually. Other important species include the annuals M. polymorpha (burr medic), M. truncatula, (barrel medic) and M. murex [2][3][4]. In order to maximise the agronomic success of these forage legumes, it is imperative that they are well-matched with an effective N 2fixing microsymbiont [5,6]. While Ensifer meliloti and E. medicae are two species of rhizobia both able to nodulate and fix N 2 with Medicago spp., differences exist between these species with regard to their host range and effectiveness. Specifically, E. medicae is an effective N 2fixing symbiont of the acid tolerant annual Medicago spp. (e.g. M. polymorpha, M. murex and M. arabica), whereas E. meliloti adapted to nodulate and fix N 2 with the neutral or slightly alkaline-favoring M. truncatula, M. littoralis and M. tornata [7][8][9].
The strain E. medicae Di28 was isolated in 1998 from a nodule collected from M. polymorpha growing in the south east of Sardinia (Italy) [10]. In common with many  E. medicae strains, E. medicae Di28 is only moderately effective as a microsymbiont with M. sativa [8]. However, Di28 is capable of effective N 2 fixation with M. murex and M. polymorpha [8]. Therefore, this strain is a valuable resource in improving our understanding of the genetic determinants of highly efficient N 2 -fixing symbioses and host range, which would complement information already gained from the sequencing of the genomes of other Medicago-nodulating microsymbionts [11][12][13]. Here we present a summary classification and a set of general features for this microsymbiont together with a description of its genome sequence and annotation. . All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [27]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [28]. Bootstrap analysis [29] 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 [30]. Published genomes are indicated with an asterisk.

Organism information
Classification and general features E. medicae Di28 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) [14] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of E. medicae Di28 in a 16S rRNA sequence based tree. This strain shares 100% sequence identity (over 1,290 bp) to the 16S rRNA of the E. medicae A321 type strain and the fully sequenced E. medicae WSM419 [11].
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 [31].

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 a standard draft genome sequence in IMG. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation E. medicae Di28 was cultured to mid logarithmic phase in 60 ml of TY rich media [32] on a gyratory shaker at 28°C at 250 rpm. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [33].

Genome sequencing and assembly
The genome of Ensifer medicae Di28 was sequenced at the Joint Genome Institute (JGI) using Illumina technology [34]. An Illumina standard paired-end library was constructed and sequenced using the Illumina HiSeq  All general aspects of library construction and sequencing performed at the JGI can be found at DOE Joint Genome Institute user homepage. 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 [35]

Genome annotation
Genes were identified using Prodigal [38] as part of the DOE-JGI annotation pipeline [39]. 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 [40] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [41]. 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 INFER-NAL [42]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [43].

Genome properties
The genome is 6,553,624 nucleotides with 61.15% GC content ( Table 3) and comprised of 104 scaffolds (Figure 3) of 104 contigs. From a total of 6,469 genes, 6,394 were protein encoding and 75 RNA only encoding genes. The majority of genes (78.65%) 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 4.

Conclusions
Di28 was isolated from a nodule of M. polymorpha found in Sardinian soil of near-neutral pH. The genome size, gene count, GC content and COG profile of Di28 is comparable to that of the sequenced E. medicae strains WSM244, WSM419, WSM1115, WSM1369 and WSM4191. Of particular interest is the finding that Di28, WSM244 and WSM1369 have a relatively low pseudogene percentage (0.03-0.06%) in comparison to the other strains (4.29-6.83%). One stand-out feature from the genome of Di28 is the absence of the acid-activated lpiA gene (11,32), which is found in all other E. meliloti and E. medicae strains sequenced to date. Furthermore, the regulatory genes tcsA, tcrA and fsrR, which are required for the full acid-activated