Complete genome sequence of Mesorhizobium australicum type strain (WSM2073T)

Mesorhizobium australicum strain WSM2073T was isolated from root nodules on the pasture legume Biserrula pelecinus growing in Australia in 2000. This aerobic, motile, gram negative, non-spore-forming rod is poorly effective in N2 fixation on B. pelecinus and has gained the ability to nodulate B. pelecinus following in situ lateral transfer of a symbiosis island from the original inoculant strain for this legume, Mesorhizobium ciceri bv. biserrulae WSM1271. We describe that the genome size of M. australicum strain WSM2073T is 6,200,534 bp encoding 6,013 protein-coding genes and 67 RNA-only encoding genes. This genome does not contain any plasmids but has a 455.7 kb genomic island from Mesorhizobium ciceri bv. biserrulae WSM1271 that has been integrated into a phenylalanine-tRNA gene.


Introduction
Biological nitrogen fixation (BNF) contributes substantially to the productivity of sustainable agriculture around the world and approximately 80% of biologically fixed nitrogen (N) is estimated to be contributed by the symbiotic association between root nodule bacteria (RNB) and leguminous plants [1]. This process of symbiotic nitrogen fixation (SNF) enables 175 million tons of atmospheric nitrogen (N2) to be fixed each year into a plant available form. SNF therefore reduces the need to apply fertilizer to provide bioavailable nitrogen, decreases greenhouse gas emissions derived from fertilizer manufacture, alleviates chemical leaching into the environment from the over application of fertilizer, and substantially enhances soil nitrogen for crop and animal production [2][3][4]. Because of substantial SNF benefits, considerable effort has been devoted to sourcing legumes from different geographical locations to improve legume productivity in different agricultural settings [3].
The Mediterranean legume Biserrula pelecinus L. is one of only three deep rooted annual legume species widely used in commerce with the potential to reduce the development of dryland salinity in Australia and was therefore introduced into Australia in 1994. Native RNB in Australian soil were not capable of nodulating B. pelecinus and therefore this host was inoculated with the inoculant strain Mesorhizobium ciceri bv. biserrulae WSM1271 [5] to obtain an effective symbiosis. Six years after the introduction of this legume into Western Australia, isolates were recovered from root nodules on B. pelecinus growing in Northam, Western Australia that were compromised in their nitrogen fixation capacity. The gradual replacement of the inoculant by established strains of RNB that are competitive for nodulation but suboptimal in N2 fixation threatens the successful establishment of this new legume in agriculture [6].
One of these poorly effective but competitive strains that was isolated from a nodule of B. pelecinus grown in the wheat belt of Western Australia can only fix <40% N2 compared to the original inoculant M. ciceri bv. biserrulae WSM1271. This strain has been designated as WSM2073 T (= LMG 24608 = HAMBI 3006) and is now the recognized type strain for the species Mesorhizobium australicum [7]. The species name au.stra.li'cum. N.L. neut. adj. australicum is in reference to where this isolate originated from [7] and represents a dominant chromosomal type strain surviving as a soil saprophyte in the Western Australian wheat belt [6,8] that appears to have the capacity to acquire symbiotic genes through horizontal transfer [9].
In this report we present a summary classification and a set of general features for M. australicum strain WSM2073 T together with the description of the complete genome sequence and its annotation. Here we reveal that a 455.7 Kb genomic island from the inoculant Mesorhizobium ciceri bv. biserrulae WSM1271 has been horizontally transferred into M. australicum strain WSM2073 T and integrated into the phenylalanine-tRNA gene.

Classification and features
M. australicum strain WSM2073 T is a motile, gram negative, non-spore-forming rod ( Figure 1 Left and Center) 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 [10] at 28 °C. Colonies on ½LA are whiteopaque, slightly domed, moderately mucoid with smooth margins (Figure 1 Right). Strains of this organism are able to tolerate a pH range between 5.5 and 9.0. More information on the carbon source utilization and fatty acid profiles were described before [7]. Minimum Information about a Genome Sequence (MIGS) is given in Table 1. Figure 2 shows the phylogenetic neighborhood of M. australicum strain WSM2073 T in a 16S rRNA sequence based tree. This strain clustered in a tight group which included M. shangrilense, M. loti and M. ciceri and had >99% sequence similarity with all four type strains. However, based on a polyphasic taxonomic study we have identified this strain to belong to a new species [7].

Symbiotaxonomy
M. australicum strain WSM2073 T has an extremely narrow legume host range for symbiosis only forming partially effective nitrogen-fixing root nodules on Biserrula pelecinus L [6]. This strain also nodulates the closely related species Astragalus membranaceus but does not nodulate 21 other legume species nodulated by Mesorhizobium spp. [6]. Strain WSM2073 T has similar highly specific symbiotic nodulation capabilities to M. ciceri bv. biserrulae WSM1271, but is a poor N-fixer on B. pelecinus L.  Phylum Proteobacteria TAS [13] Class Alphaproteobacteria TAS [14,15] Order Rhizob iales TAS [15,16] Family Phyllobacteriaceae TAS [15,17] Genus Mesorhizob ium TAS [18] Species Mesorhizob ium australicum TAS [7] Gram stain Negative TAS [7] Cell , 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 Ontolog y project [21]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements. . All sites were informative and there were no g ap-containing sites. Phylog enetic analyses were performed using MEGA [22]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model. Bootstrap analysis [23] 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 [24] Published genomes are indicated with an asterisk. Standards in Genomic Sciences

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 US Department of Energy Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [24] and the complete genome sequence in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Growth conditions and DNA isolation
M. australicum strain WSM2073 T was grown to mid logarithmic phase in TY medium (a rich medium) [25] on a gyratory shaker at 28°C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethylammonium bromide) bacterial genomic DNA isolation method.

Genome sequencing and assembly
The draft genome of M. australicum strain WSM2073 T was generated at the DOE Joint genome Institute (JGI) using a combination of Illumina [26] and 454 technologies [27]. Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 0.7.63 [28], 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 [29][30][31] 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 [32], 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 59 additional reactions were necessary to close gaps and to raise the quality of the finished sequence.
The total size of the genome is 6,200,534 bp and the final assembly is based on 257 Mb of 454 draft data which provides an average 28× coverage of the genome and 13,385 Mb of Illumina draft data which provides an average 2159× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [33] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePrimp pipeline [34]. 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 [35], RNAMMer [36], Rfam [37], TMHMM [38], and SignalP [39]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [40].

Genome properties
The genome is 6,200,534 bp long with a 62.84% GC content (Table 3, Figure 3) and comprised of a single chromosome. From all the genes present in the genome, 6,013 were protein coding genes and 67 RNA only encoding genes. Two hundred and twenty one pseudogenes were also identified. The majority of protein coding genes (4,875; 80.18%) were assigned a putative function whilst the remaining protein coding genes were annotated as encoding hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.