Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece

Standards in Genomic Sciences (2014) 9:514-526 DOI:10.4056/sig s.4938652 Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece 1* 2 1 2 1 3 Wayne Reeve , Ross Ballard , John Howieson , Elizabeth Drew , Rui Tian , Lambert Bräu , 4 4 4 4 5 Christine Munk , Karen Davenport , Patrick Chain , Lynne Goodwin , Ioanna Pag ani , 5 6 5 6 Marcel Huntemann , Konstantinos Mavrommatis , Amrita Pati , Victor Markowitz , Natalia 5 5 5 Ivanova , Tanja Woyke & Nikos Kyrpides . 1 Centre for Rhizobium Studies, Murdoch University, Western Australia, Australia South Australian Research and Development Institute, Urrbrae, South Australia, Australia 3 School of Life and Environmental Sciences, Deakin University, Victoria, Australia 4 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 5 DOE Joint Genome Institute, Walnut Creek, California, USA 6 Biolog ical Data Manag ement and Technolog y Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 2 *Correspondence: Wayne Reeve () Keywords: root-nodule bacteria, nitrogen fixation, rhizobia, Alphaproteobacteria Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl ) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 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. 2 2 Introduction The genus Medicago comprises 87 species of annual and perennial legumes, including some that were formerly recognized as Trigonella and Melilotus species [1]. A small number of annual Medicago species that have been domesticated are grown extensively in the sheep-wheat zone of southern Australia, particularly where pasture regeneration after a cropping phase is desirable. Annual Medicago species are grown on more than 20 M ha [2] and are particularly valued for their contribution to farming systems, in which Medicago fix around 25 kg of N per tonne of legume dry matter produced [3]. Medicago are nodulated by two species of root nodule bacteria (Ensifer medicae and Ensifer meliloti) that are recognized as being distinct based on their different nodulation and N2 fixation phenotypes in host interaction studies and more detailed analyses of their genetics [4,5]. Ensifer medicae strain WSM1115 is used in Australia to produce commercial peat cultures (referred to as Group AM inoculants) for the inoculation of several species of annual Medicago (predominantly M. truncatula, M. polymorpha, M. scutellata, M. sphaerocarpus, M. murex, M. rugosa and M. orbicularis). WSM1115 has been used commercially since 2002 [6], when it replaced strain WSM688. WSM1115 was isolated from a nodule from the roots of burr medic (Medicago polymorpha) collected by Prof. John Howieson (Murdoch University, Australia) on the island of Samothraki, Greece. WSM1115 was selected for use in commercial inoculants having demonstrated good N2-fixation capacity with the relevant medic hosts and adequate The Genomic Standards Consortium Reeve al. saprophytic competence in moderately acidic soil (pH(CaCl2) 5). Saprophytic competence in acidic soils is a requirement of strains used to inoculate Medicago because several species (M. murex, M. sphaerocarpus and M. polymorpha) are recommended and sown into soils below pH(CaCl2) 5.5, a level that is known to limit both survival of medic rhizobia and nodulation processes [7-10]. Useful variation in saprophytic competence occurs between strains of medic rhizobia [9] and valuable insights into the mechanisms that confer acidity tolerance have been provided by studies using strain WSM419 [11], which has been recently sequenced [12]. However, the complex nature of soil adaptation means that in-situ field studies still provide the most reliable means of selecting an inoculant strain and were used to select WSM1115 for commercial use. In a cross row experiment comparing 15 strains on acidic sand (pH(CaCl2) 5.0; Dowerin, West Australia), the nodulation of plants inoculated with WSM1115 was equal to or better than that of the other strains. This translated to better plant shoot weights, which were similar to those of plants inoculated with WSM688 (the incumbent inoculant strain at time of testing) and 48% greater when compared to former inoculant strain CC169 (J. G. Howieson unpublished data). The nitrogen fixation capacity (effectiveness) of Medicago symbioses is characterized by strong interactions between the strain of rhizobia and species of Medicago [13-16]. Hence, the ability to form effective symbiosis with the species recommended for inoculation is an important consideration in inoculant strain selection. WSM1115 satisfies this requirement. In greenhouse tests it formed effective symbiosis with 16 genotypes of Medicago and overall produced 48% more shoot dry matter compared to plants inoculated with WSM688, the strain that it replaced (R.A. Ballard and N. Charman, unpublished data). A limitation of strain WSM1115 is its poor persistence in moderately saline soils (e.g. where summer salinity levels exceed 10 ECe (dS/m)). Poor nodulation of regenerating pasture was first noted in 2004 during the field evaluation and domestication of the salt tolerant annual pasture legume messina (Melilotus siculus syn. Melilotus messanensis). Subsequent studies [17] confirmed that although WSM1115 was able to nodulate and form effective symbiosis with messina, it did not persist as well as other strains (e.g. SRDI554) through the summer months when salinity levels increased. Here we present a preliminary description of the general features of Ensifer medicae strain WSM1115 together with its genome sequence and annotation. Classification and features Ensifer medicae strain WSM1115 is a motile, nonsporulating, non-encapsulated, Gram-negative rod in the order Rhizobiales of the class Alphaproteobacteria. The rod-shaped form varies in size with dimensions of approximately 0.5 μm in width and 1.0 μm in length (Figure 1A). It is fast growing, forming colonies within 3-4 days (...truncated)