Complete genome sequence of Mesorhizobium ciceri bv. biserrulae type strain (WSM1271T)

Mesorhizobium ciceri bv. biserrulae strain WSM1271T was isolated from root nodules of the pasture legume Biserrula pelecinus growing in the Mediterranean basin. Previous studies have shown this aerobic, motile, Gram negative, non-spore-forming rod preferably nodulates B. pelecinus – a legume with many beneficial agronomic attributes for sustainable agriculture in Australia. We describe the genome of Mesorhizobium ciceri bv. biserrulae strain WSM1271T consisting of a 6,264,489 bp chromosome and a 425,539 bp plasmid that together encode 6,470 protein-coding genes and 61 RNA-only encoding genes.


Introduction
The productivity of sustainable agriculture around the world is heavily dependent on the provision of bioavailable nitrogen (N) [1]. The demand for N by non-leguminous and leguminous plants can be supplied by the application of chemically synthesized nitrogenous fertilizer onto crops and pastures. However, the production of fertilizer is costly and requires the burning of fossil fuels in the manufacturing process which increases greenhouse gas emissions. Furthermore, high application rates of fertilizer can contaminate ecosystems and waterways, and result in leaching into the environment. In contrast, the demand for N by leguminous plants can be sustainably met through the biological process of N fixation that occurs following the successful formation of an effective symbiosis. This symbiotic nitrogen fixation (SNF) process can account for approximately 70% of the bioavailable nitrogen supplied to legumes [1]. One legume that has many beneficial agronomic attributes is Biserrula pelecinus L., which is an annual herbaceous legume native to the Mediterranean basin that was introduced into Australian soil in 1994 [2]. The beneficial agronomic attributes of this legume include drought tolerance, hard seed production, easy harvesting characteristics, insect tolerance and most importantly, a capacity to grow well in the acidic duplex soils of Australia [2,3]. This monospecific legume specifically forms an effective nitrogen fixing symbiosis with the root nodule bacterium Mesorhizobium ciceri bv. biserrulae type strain WSM1271 T (= LMG23838 = HAMBI2942) [4,5]. Australian indigenous rhizobial populations were found to be incapable of nodulating B. pelecinus L [2]. However, within six years of the introduction of the inoculant into Australia, the in situ evolution of a diverse range of competitive strains capable of nodulating B. pelecinus L. compromised optimal N2-fixation with this host. This rapid emergence of less effective strains threatens the establishment of this legume species in the Australian agricultural setting. The sub-optimal strains appear to have evolved from indigenous mesorhizobia that acquired the island of genes associated with symbiosis from the original inoculant, WSM1271 T , following a horizontal gene transfer event [6].
In this report, a summary classification and a set of general features for M. ciceri bv. biserrulae strain WSM1271 T are presented together with the description of the complete genome sequence and its annotation.

Classification and features
M. ciceri strain WSM1271 T is a motile, Gramnegative, non-spore-forming rod ( Figure 1 and Figure 2) in the order Rhizobiales of the class Alphaproteobacteria. They are moderately fast growing, forming 2-4 mm diameter colonies within 3-4 days, and have a mean generation time of 4-6 h when grown in half Lupin Agar (½LA) broth [7] at 28 °C. Colonies on ½LA are white-opaque, slightly domed, moderately mucoid with smooth margins (Figure 3).
The organism tolerates a pH range between 5.5 and 9.0. Carbon source utilization and fatty acid profiles have been described before [6]. Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 4 shows the phylogenetic neighborhood of M. ciceri bv. biserrulae strain WSM1271 T in a 16S rRNA sequence based tree. This strain clustered in a tight group, which included M. australicum, M. ciceri, M. loti and M. shangrilense and had >99% sequence identity with all four type strains. Our polyphasic taxonomic study indicates that WSM1271 T is a new biovar of nodulating bacteria [5].

Symbiotaxonomy
M. ciceri bv. biserrulae strain WSM1271 T has an extremely narrow legume host range for symbiosis only forming highly effective nitrogen-fixing root nodules on Biserrula pelecinus. L. This strain also nodulates the closely related species Astragalus membranaceus but does not nodulate 21 other legume species nodulated by Mesorhizobium spp [5]. The high degree of specificity in the symbiotic relationships of this strain is representative of root nodule bacteria isolated from B. pelecinus L. growing in undisturbed landscapes in the Mediterranean basin, and is an important example of a highly specific legume hostroot nodule bacteria relationship in an annual herbaceous legume used as a forage species in agriculture.     [8,9].

MIGS ID Property Term Evidence code
Domain Bacteria TAS [9] Phylum Proteobacteria TAS [10] Class Alphaproteobacteria TAS [11,12] Current classification Order Rhizob iales TAS [11,13] Family Phyllobacteriaceae TAS [11,14] Genus Mesorhizob ium TAS [15] Species Mesorhizob ium c iceri bv biserrulae TAS [15] Gram stain Negative TAS [6] Cell Evidence codes -TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Nontraceable 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). Evidence codes are from the Gene Ontolog y project [18]. . All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [19]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model. Bootstrap analysis [20] was performed with 500 replicates 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 [21]. Published genomes are indicated with an asterisk.

Genome sequencing and annotation Genome project history
The Joint Genome Institute (JGI) operated by US Department of Energy (DOE) sequenced, finished and annotated WSM1271 as part of the Community Sequencing Program (CSP). The genome project is deposited in the Genomes OnLine Database [21]. The finished genome sequence is in GenBank. The CSP selects projects on the basis of environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and bi-ogeochemical importance. Table 2 summarizes the project information.

Growth conditions and DNA isolation
M. ciceri bv. biserrulae strain WSM1271 T was grown to mid logarithmic phase in TY rich medium [22] on a gyratory shaker at 28 °C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [23].

Genome sequencing and assembly
The Joint Genome Institute (JGI) generated the draft genome of M. ciceri bv. biserrulae WSM1271 T using a combination of Illumina [24] and 454 technologies [25].  [26], and the consensus sequences were computationally shredded into 1.5 Kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS -4.24 (High Performance Software, LLC). The software Consed [27][28][29] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [30], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 49 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total size of the genome is 6,890,027 bp and the final assembly is based on 112.0 Mb of 454 draft data which provides an average 26.8× coverage of the genome and 832.1 Mb of Illumina draft data which provides an average 124× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePrimp pipeline [32]. 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 [33], RNAMMer [34], Rfam [35], TMHMM [36], and SignalP [37]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [38].

Genome properties
The genome is 6,690,028 bp long with a 62.56% GC content (Table 3) and comprises a single chromosome and a single plasmid. From a total of 6,531 genes, 6,470 were protein encoding and 61 RNA only encoding genes. Within the genome, 206 pseudogenes were also identified. The majority of genes (70.74%) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4, and Figures 5,6 and 7.