Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128

Standards in Genomic Sciences (2014) 9:602-613 DOI:10.4056/sig s.4929626 Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128 1* 2 2 1 3 4 Wayne Reeve , Ross Ballard , Elizabeth Drew , Rui Tian , Lambert Bräu , Lynne Goodwin , 5 5 5 6 Marcel Huntemann , James Han , Reddy Tatiparthi , Amy Chen , Konstantinos 6 6 6 5 5 Mavrommatis , Victor Markowitz , Krishna Palaniappan , Natalia Ivanova , Amrita Pati , 5 5 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, nitrog en fixation, rhizobia, Alphaproteobacteria Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) g rown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with g enome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contig s, contains 6,683 protein-coding genes and 87 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. Introduction Ensifer meliloti strain RRI128 is used in Australia to produce commercial peat cultures (referred to as Group AL inoculants) mainly for the inoculation of lucerne (Medicago sativa L.). Lucerne is sown on about 600, 000 ha annually (A. Humphries pers. com.) and is nearly always inoculated prior to sowing. RRI128 is also used for the inoculation of strand medic (Medicago littoralis Loisel) and disc medic (Medicago tornata (L.) Miller), a hybrid of the two former species, and bokhara clover (Melilotus albus Medik). RRI128 has been used commercially since 2000 when it replaced strain WSM826 [1]. Strain RRI128 was isolated from a nodule from the roots of barrel medic (Medicago truncatula Gaertn) growing in the greenhouse and inoculated with an alkaline sandy soil (pHCaCl2 7.6) collected by J. Slattery, near Tempy, Victoria. The strain was selected for use in commercial inoculants following assessment of its nitrogen fixation capacity (effectiveness), growth on acidified agar and saprophytic competence in an in-situ soil study [2], with supporting data of satisfactory performance at ten field sites. Additional testing has shown RRI128 to be effective on 28 cultivars of lucerne (Ballard unpub. data). It also forms effective symbiosis with a range of strand and disc medics [2] which show symbiotic affinity with lucerne [3,4]. Soil acidity has long been recognized as a constraint to lucerne nodulation [5] with some evidence that strains of E. meliloti have less acidity The Genomic Standards Consortium Reeve et al. tolerance than Ensifer medicae, possibly due to their association with Medicago species that favor neutral to alkaline soils [6]. With RRI128, constraints to lucerne nodulation are observed around pH 5. Nodulation of lucerne seedlings inoculated with RRI128 was 42% at pH 5.0 in solution culture experiments [7] and observed to decline rapidly at field sites where pHCaCl2 was below 4.7 (Ballard, unpub. data). Other strains (e.g. SRDI672) have increased lucerne nodulation in solution culture at pH 4.8 (61% cf. 12% of lucerne seedlings with nodules) but are probably approaching the limit of acidity tolerance for E. meliloti [8]. Stable colony morphology and cell survival on seed make RRI128 amenable to commercial use. RRI128 produces colonies of consistent appearance and with moderate polysaccharide when grown on yeast mannitol agar, enabling easy visual assessment of culture purity. It differs in this regard from the strain it replaced (WSM826) which produced ‘dry’ and ‘mucoid’ colony variants, in common with many of the strains that nodulate lucerne and medic [9]. When applied correctly RRI128 has been shown to survive at more than 10,000 cells per lucerne seed at six weeks after inoculation [10]. Good survival may well be characteristic of E. meliloti, since former inoculant strain WSM826 is equally competent in this regard [11,12]. Here we present a preliminary description of the general features of E. meliloti strain RRI128 together with its genome sequence and annotation. Classification and general features Ensifer meliloti strain RRI128 is a motile, nonsporulating, non-encapsulated, Gram-negative rod A 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 1.0-2.0 μm in length (Figure 1A). It is fast growing, forming colonies within 3-4 days when grown on TY [13] or half strength Lupin Agar (½LA) [14] at 28°C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Figure 1B). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighborhood of Ensifer meliloti strain RRI128 in a 16S rRNA gene sequence based tree. This strain has 100% sequence identity (1366/1366 bp) at the 16S rRNA sequence level to the fully sequenced E. meliloti Sm1021 [30] and 99% 16S rRNA sequence (1362/1366 bp) identity to the fully sequenced E. medicae strain WSM419 [31]. Symbiotaxonomy Ensifer meliloti strain RRI128 forms nodules on (Nod+) and fixes N2 (Fix+) with Medicago sativa, Melillotus albus and Trigonella balansae (Boiss. and Reuter). It also forms effective symbiosis with several species of annual medic (M. truncatula, M. littoralis and M. tornata) that happen to be closely related to each other based on their ability to be hybridized [5] and morphological and nucleotide sequence analyses of their relatedness [32]. RRI128 forms ineffective (white) nodules with Medicago polymorpha, a species that is generally recognized to have a more specific rhizobial requirement for effective symbiosis than Medicago sativa and Medicago littoralis [4,33] (Table 2). B 1 mm 1 mm Figure 1. Images of Ensifer meliloti strain RRI 128 using (A) scanning electron microsc (...truncated)