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High-quality draft genome sequence of Ensifer meliloti Mlalz-1, a microsymbiont of Medicago laciniata (L.) miller collected in Lanzarote, Canary Islands, Spain

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Abstract

Ensifer meliloti Mlalz-1 (INSDC = ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here the features of E. meliloti Mlalz-1 are described, together with high-quality permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to Ensifer meliloti IAM 12611 T, Ensifer medicae A 321T and Ensifer numidicus ORS 1407 T, based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as E. meliloti . Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata-nodulating Ensifer strains, but ≤93% with nodC of Ensifer strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced E. meliloti strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In E. medicae strain WSM419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of E. medicae strains, which suggests genetic recombination between strain Mlalz-1 and E. medicae and the horizontal gene transfer of lpiA-acvB.

Introduction

Symbiotic nitrogen fixation by pasture legumes and their associated root nodule bacteria provides a critical contribution to sustainable animal and plant production, and the maintenance of soil fertility in agricultural systems [1,2,3]. As such, it is of direct relevance to maintaining environmentally sustainable high agricultural yields, which significantly contributes to the Sustainable Development Goals adopted in September 2015 as part of the UN’s development agenda ‘Transforming our world: the 2030 Agenda for Sustainable Development’ [4]. Medics ( Medicago spp.) are some of the most important and extensively grown pasture legumes and their specific symbiosis with strains of rhizobia belonging to either Ensifer (synonym Sinorhizobium ) meliloti or the closely related species E. medicae [5, 6] has been the subject of extensive research efforts [7].

Medicago laciniata (L.) Miller (cut leaf medic), an annual native of southern and eastern Mediterranean and Saharo-Sindian countries, is of importance because of its ability to grow in comparatively arid habitats and marginal cropping areas [8,9,10,11]. It is highly specific in its rhizobial requirements, forming a symbiosis only with a restricted subset of E. meliloti and not with strains that nodulate Medicago sativa L. (alfalfa) or Medicago truncatula Gaertn. [12, 13]. This symbiotic specificity has been linked to the rhizobial nod genes, in particular a specific nodC allele [14]. For example, van Berkum and colleagues found that most rhizobial strains isolated from Tunisian M. truncatula and M. laciniata shared chromosomal identity, but differed in their nodC alleles [15]. Based on these and other differing symbiotic traits, Villegas et al. [13] proposed two biovars within E. meliloti : bv. medicaginis for Ensifer strains that are symbiotically efficient on M. laciniata and bv. meliloti for the classical E. meliloti group that efficiently nodulates M. sativa. However, in subsequent studies the diversity observed within bv. medicaginis strains indicate that this group is certainly heterogeneous [16].

M. laciniata is native to the Canary Islands and is present on all of the islands of this archipelago, growing in environments that range from arid to subhumid. Ensifer meliloti strain Mlalz-1 was isolated from a N2-fixing nodule of M. laciniata grown in alkaline soil (pH 9.0) collected in Guatiza, in the arid Northeast of Lanzarote Island, in 2007. This strain was one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 GEBA-RNB project proposal [17, 18]. Here an analysis of the complete genome sequence of E. meliloti Mlalz-1 is provided.

Organism information

Classification and features

E. meliloti Mlalz-1 is a motile, non-sporulating, non-encapsulated, Gram-negative strain in the class Alphaproteobacteria . The rod shaped form has dimensions of approximately 0.5 μm in width and 1.0–2.0 μm in length (Fig. 1 Left and Center). It is fast growing, forming colonies after 3–5 days when grown on ½LA, TY, or a modified yeast-mannitol agar [19] at 28 °C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Fig. 1 Right). Minimum Information about the Genome Sequence (MIGS) for strain Mlalz-1 is provided in Table 1 and Additional file 1: Table S1.

Fig. 1
figure1

Images of Ensifer meliloti Mlalz-1 using scanning (Left (a)) and transmission (Center (b)) electron microscopy as well as light microscopy to visualize colony morphology on solid media (Right (c))

Table 1 Classification and general features of Ensifer meliloti Mlalz-1 in accordance with the MIGS recommendations [65] published by the Genomic Standards Consortium [66]

Symbiotaxonomy

M. laciniata is a highly specific host and its microsymbionts also appear to be highly specific since studies of Medicago isolates have shown that M. laciniata strains fail to nodulate a range of Medicago species [5, 12]. Bailly et al. [20] reported that isolates of M. laciniata nodulated and fixed nitrogen with M. truncatula, but also provided evidence that these were the progeny of horizontal transfer of the nodulation genes. Strain Mlalz-1 nodulates and is effective for nitrogen fixation with M. laciniata. We report here that strain Mlalz-1 is unable to nodulate Medicago polymorpha L., the definitive host for E. medicae strains [6].

Extended feature descriptions

Previous studies using multilocus sequence typing showed that M. laciniata rhizobia did not form a distinct chromosomal group [15]. Phylogenetic analysis of strain Mlalz-1 was performed by aligning the 16S rRNA sequence (1389 bp from scaffold 84.85) to the 16S rRNA gene sequences of Ensifer type strains (Fig. 2). Based on four variable sites within this 16S rRNA gene sequence alignment, strain Mlalz-1 is closely related to E. meliloti IAM 12611 T (= LMG 6133 T) [21], E. medicae A 321T (= LMG 19920 T) [6] and E. numidicus ORS 1407 T [22]. The available IMG 16S rRNA sequence of strain Mlalz-1 gave alignment identities of 100% to E. meliloti IAM 12611 T, 99.7% to E. medicae A 321T and 99.5% to E. numidicus ORS 1407 T. In contrast, E. meliloti IAM 12611 T and Ensifer terangae LMG 7834 T [23] were only 97.3% similar.

Fig. 2
figure2

Comparison of the 16S rRNA gene sequences of Ensifer meliloti Mlalz-1 (shown in bold) and other Ensifer spp. type strains, based on aligned 16S rRNA gene sequences of 1389 bp. Of the 1389 sites, 1279 were constant and 48 were informative. There were eight gaps overall when Mesorhizobium ciceri bv biserrulae WSM1271 was included in the analysis. Phylogenetic analysis was done using MEGA, version 6.0 [61] after manually assembling the alignment by using GeneDoc version 2.6.001 [62]. M. ciceri bv biserrulae WSM1271 was used as an outgroup and the tree was assembled using the UPGMA algorithm based on the number of nucleotide differences. This approach was used since the potential for genetic recombination among the different 16S rRNA genes as reported by van Berkum [63] cannot be ignored. Bootstrap analysis [64] with 2000 permutations of the data set was done to assess support for the branch points. Strains with a genome sequencing project registered in GOLD [24] are Ensifer adhaerens Casida AT, M. ciceri bv. biserrulae WSM1271 and Mlalz-1 and the GOLD ID is provided in place of the GenBank accession number

Genome sequencing information

Genome project history

E. meliloti Mlalz-1 was selected for sequencing at the U.S. Department of Energy funded Joint Genome Institute as part of the GEBA-RNB project [17, 18]. The root nodule bacteria in this project were selected based on environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance. In particular, strain Mlalz-1 was chosen since it has strict host specificity for M. laciniata, which is suited for cultivation in arid environments [11]. The E. meliloti Mlalz-1 genome project is deposited in the Genomes Online Database [24] and a high-quality permanent draft genome sequence (IMG Genome ID 2513237143) is deposited in IMG [25]. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 2.

Table 2 Genome sequencing project information for Ensifer meliloti Mlalz-1

Growth conditions and genomic DNA preparation

E. meliloti Mlalz-1 (= USDA 1984) was cultured on MAG solid media [26] for three days at 28 °C to obtain well grown, well separated colonies, then a single colony was selected from the plate and inoculated into 5 ml MAG broth media. The culture was grown for 48 h on a gyratory shaker (200 rpm) at 28 °C. Subsequently 1 ml was used to inoculate 50 ml of MAG and the cells were incubated on a gyratory shaker (200 rpm) at 28 °C until an OD600nm of 0.6 was reached. DNA was isolated from 50 ml of cells by Peter van Berkum according to the method described by van Berkum [26]. The final concentration of the DNA was set to 0.5 mg ml−1.

Genome sequencing and assembly

The draft genome of E. meliloti Mlalz-1 was generated at the DOE Joint genome Institute (JGI) using Illumina technology [27]. An Illumina standard PE library was constructed and sequenced using the Illumina HiSeq 2000 platform that generated 35,720,836 reads totalling 4983 Mbp. All general aspects of library construction and sequencing were done at the JGI and details can be found on the JGI website [28]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artefacts (Mingkun L, Copeland A, Han J; unpublished). The following steps for assembly were: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) [29]; (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim (version 0.3.0) [30]; (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r39750) [31]. Parameters for the assembly steps were 1) Velvet: --v --s 51 --e 71 --i 2 --t 1 --f “-shortPaired -fastq $FASTQ” --o “-ins_length 250 -min_contig_lgth 500” for Velvet and 2) wgsim: -e 0–1 76–2 76 -r 0 -R 0 -X 0. The final draft assembly contained 100 contigs in 99 scaffolds. The total size of the genome is 6.7 Mbp and the final assembly is based on 4983 Mbp of Illumina data, which provides an average of 748× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [32], as part of the DOE-JGI genome annotation pipeline [33, 34]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [35] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [36]. 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 INFERNAL [37]. Additional gene prediction analysis and manual functional annotation was done within the Integrated Microbial Genomes-Expert Review platform [38] developed by the Joint Genome Institute, Walnut Creek, CA, USA.

Genome properties

The genome is 6,664,116 bp with 62.16% GC content (Table 3) and comprised of 99 scaffolds. From a total of 6388 genes, 6314 were protein encoding and 74 RNA only encoding genes. Most genes (79.52%) 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.

Table 3 Genome statistics for Ensifer meliloti Mlalz-1
Table 4 Number of genes of Ensifer meliloti Mlalz-1 associated with the general COG functional categories

Insights from the genome sequence

E. meliloti Mlalz-1 is one of seven strains of E. meliloti that have been sequenced from the GEBA-RNB genome sequencing projects [17]. On the basis of 16S rRNA sequence identity, strain Mlalz-1 is closely related to E. meliloti IAM 12611 T (= LMG 6133 T), E. medicae A 321T (= LMG 19920 T) and E. numidicus ORS 1407 T. As the genomes of these type strains have not been sequenced or are not publically available, gANI values [39] had to be compared with other fully sequenced Ensifer strains (Table 5). E. meliloti Mlalz-1 currently forms a gANI clique with other E. meliloti strains (gANI values ≥98.14%), compared with gANI values of ≤87.9% with the finished genomes of other Ensifer strains. This supports the classification of strain Mlalz-1 as an E. meliloti strain, in accordance with the defined species affiliation cut-off value of 96.5% gANI [39]. The total genome size of strain Mlalz-1 is 6.6 Mbp, which falls within the expected size range of 6.6–8.9 Mbp for E. meliloti . The genome architecture of E. meliloti consists of a chromosome and the two symbiotic megaplasmids pSymA and pSymB [20]. Replication of a plasmid is initiated by the replication protein encoded by repC, which is present as a single copy on E. meliloti pSymA and pSymB. The E. meliloti Mlalz-1 genome carried 2 repC loci (A3CADRAFT_00120 and A3CADRAFT_01676) with highest encoded protein identity to RepC proteins of E. meliloti strains. Mlalz-1 A3CADRAFT_00120 RepC1 had highest identity (98.10%) to the RepC1 protein encoded by SMb20044 on pSymB of E. meliloti 1021. E. meliloti Mlalz-1 A3CADRAFT_01676 RepC2 had highest identity (99.00%) to the RepC2 protein encoded by SMa2391 on pSymA of E. meliloti 1021. This indicated the presence of two megaplasmids in strain Mlalz-1, and revealed that strain Mlalz-1 has a similar genome architecture to that of E. meliloti 1021.

Table 5 Pairwise gANI comparisons of selected finished genomes of sequenced Ensifer strains

Extended insights

All 29 E. meliloti strains within the gANI clique share a core set of 4948 orthologous genes, using cut off values of 1e-5 and 30% minimum protein identity. E. meliloti Mlalz-1 contains 176 unique genes, 96 (54.5%) of which encode hypothetical proteins. The unique genes include those encoding the components of a T2SS, located on scaffold A3CADRAFT_scaffold_5.6 (Fig. 3a), as well as genes that encode a DNA methyltransferase and a NitT/TauT family transport system. These T2SS components form part of a unique COG profile generated for Mlaz-1 (Table 6). The T2SS secretion system is used to translocate a wide range of proteins from the periplasm across the outer membrane [40]. Although T2SS genes are not found in other E. meliloti strains or in the Ensifer fredii strains GR64 and USDA 257, they are present in the genomes of the E. fredii strains HH103 and NGR234, in a similar gene arrangement to that observed in E. meliloti Mlalz-1 [41, 42] (Fig. 3b). Generally, the T2SS gene cluster is comprised of 12–15 genes, and strain Mlalz-1 contains the 12 required genes gspDOGLMCKEFHIJ necessary for a functional T2SS, but lacks the gspS gene found only in certain genera [43] (Fig. 3c).

Fig. 3
figure3

a Map of Ensifer meliloti Mlalz-1: A3CADRAFT_scaffold_ 5.6. b Genetic organization of the T2SS clusters in E. meliloti Mlalz-1 (Emel Mlalz-1), Ensifer fredii HH103 (Efre HH103) and Ensifer fredii NGR234 (Efre NGR234). J, gspJ; I, gspI; H, gspH; F, gspF; E, gspE; K, gspK; C, gspC; M, gspM; L, gspL; G, gspG; O, gspO; D, gspD. c Schematics of the T2SS of Gram-negative bacteria [43]. The secretin, GspD (A3CADRAFT_01569); the polytopic protein, GspF (A3CADRAFT_01561); the cytoplasmic ATPase, GspE (A3CADRAFT_01562); the major pseudopilin component, GspG (A3CADRAFT_01567); the minor pseudopilins, GspH (A3CADRAFT_01560), GspI (A3CADRAFT_01559), GspJ (A3CADRAFT_01558) and GspK (A3CADRAFT_01563); the bitopic proteins, GspL (A3CADRAFT_01566), GspC (A3CADRAFT_01564) and GspM (A3CADRAFT_01565); the peptidase, GspO (A3CADRAFT_01568)(GspS is absent from Mlalz-1); OM, outer membrane; PG, peptidoglycan; IM, inner membrane

Table 6 List of the unique COGs in Ensifer meliloti Mlalz-1

In common with some other E. meliloti strains, strain Mlalz-1 contains several genes encoding phage components. The PHASTER algorithm [44] was used to identify two resident prophages, present on scaffold A3CADRAFT_scaffold_4.5: one that was incomplete (Prophage Region 1) and one that was intact (Prophage Region 2) (Fig. 4). The proteins encoded by Prophage Region 1 (11.4 kb) and Prophage Region 2 (55 kb) were most closely related to the phage proteins of PHAGE_Mycoba_Catalina_NC031238 and PHAGE_Sinorh_phiLM21_ NC_029046, respectively.

Fig. 4
figure4

Resident prophages present in Ensifer meliloti Mlalz-1 A3CADRAFT_scaffold_4.5, imaged using PHASTER [44]. Reference locus tag for Prophage Region 1 is A3CADRAFT_01439 (phage capsid family); reference locus tag for Prophage Region 2 is A3CADRAFT_01472 (prophage tail length tape measure protein). Prophage maps not drawn to scale. Attachment site (att), coat protein (coa), fiber protein (fib), integrase (int), phage-like protein (plp), tail shaft protein (tsp), and terminase (ter). All other genes encode hypothetical proteins

The Mlaz-1 genome also contains acid-tolerance or acid-responsive genes that are orthologous to the genes identified in the comparatively acid tolerant strain E. medicae WSM419. Acid-tolerance or acid-responsive genes identified in Mlaz-1 include actA (lnt), actP, actR, actS, phrR, exoR, exoH, lpiA, acvB, degP1, mdh3, fbaB, groS, kdpB, kdpC, fixN2 and fixO2 [45,46,47,48,49,50,51,52] (Additional file 2: Table S2). It is notable that strain Mlalz-1 is unique among the sequenced Ensifer strains since it contains two versions of the highly acid-induced lpiA-acvB operon. One operon (A3CADRAFT_01189-A3CADRAFT_01190) is found on scaffold A3CADRAFT_scaffold_3.4, in a gene region that is conserved in other E. meliloti (sequence similarity >98%) and is located on the chromosome of the fully sequenced E. meliloti 1021. The second version of the lpiA-acvB operon (A3CADRAFT_05694-A3CADRAFT_05695) is located on A3CADRAFT_scaffold_47.48, in a gene region that is conserved in E. medicae genomes (sequence similarity >96%) and is located on the pSMED02 symbiotic plasmid of the fully sequenced E. medicae WSM419. The regulatory gene fsrR, required for the acid activated expression of lpiA in E. medicae WSM419 [53], is located upstream of A3CADRAFT_05694 in strain Mlalz-1. This regulatory gene is absent from the A3CADRAFT_01190 gene region, and from the lpiA-acvB gene regions of all other E. meliloti sequenced genomes. These findings suggest that E. meliloti Mlalz-1 acquired the plasmid-borne lpiA-acvB operon and associated fsrR regulatory gene by lateral transfer from an E. medicae strain.

Essential symbiotic (nod, nif and fix) genes identified in the E. meliloti Mlalz-1 genome (Additional file 2: Table S3 and S4) are located in several clusters on the following scaffolds: A3CADRAFT_scaffold_54.55 (Fig. 5a), A3CADRAFT_scaffold_61.62 (Fig. 5b), A3CADRAFT_scaffold_63.64 (Fig. 5c), A3CADRAFT_scaffold_71.72 (Fig. 5d) and A3CADRAFT_scaffold_74.75 (Fig. 5e). Nodulation of M. laciniata has been shown to require a specific nodC allele [14]. The nodC gene of strain Mlalz-1 has highest sequence identity (≥ 98%) with nodC of other M. laciniata-nodulating Ensifer strains in the NCBI database, whereas there is a lower sequence identity (≤ 93%) with nodC of Ensifer strains that nodulate other Medicago species. Nodulation of Medicago hosts requires Nod factors that are sulfated at the reducing terminus and acylated at the non-reducing terminus, with a polyunsaturated fatty acyl tail [54, 55]. The NodH sulfotransferase, together with the NodP and NodQ sulfate-activating complex, are required for Nod factor sulfation [56, 57]. Activity of NodL results in O-acetylation of the Nod factor [58], while NodE and NodF produce the specific polyunsaturated fatty acyl tail [55, 59]. Strain Mlalz-1 would appear to be typical of Ensifer strains that nodulate Medicago species since the nodEF, nodL and nodHPQ genes that are required for these specific decorations of the Nod factor are present in the genome. E. meliloti Mlalz-1 also possesses the three nodD genes that mediate host-specific activation of nodABC in the symbiotic interactions of E. meliloti with Medicago [60].

Fig. 5
figure5

Graphical map of the scaffolds; a A3CADRAFT_scaffold_54.55, b A3CADRAFT_scaffold_61.62, (c) A3CADRAFT_scaffold_63.64, d A3CADRAFT_scaffold_71.72 and e A3CADRAFT_scaffold_74.75 of Ensifer meliloti Mlalz-1 showing the location of common nodulation (nod) and fixation (nif and fix) genes within the symbiotic regions of this strain. From bottom to the top of the scaffold map: Genes on reverse strand (color by COG categories as denoted by the IMG platform), genes on forward strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew

Conclusions

E. meliloti Mlalz-1 is a rhizobial strain that is able to nodulate and fix nitrogen with the highly specific host M. laciniata. Although the 16S rRNA gene sequence divergence was insufficient to differentiate strain Mlalz-1 from E. meliloti , E. medicae or E. numidicus , a gANI value of 98.8% with the genome of E. meliloti 1021, compared with 87.9% with the genome of E. medicae WSM419 identifies strain Mlalz-1 as E. meliloti . Nodulation of M. laciniata has been shown to be dependent on the presence of a specific nodC allele, which also is present in the genome of E. meliloti Mlalz-1, based on a 98% sequence identity with the nodC of other M. laciniata-nodulating Ensifer strains [14]. However, strain Mlalz-1 is unique among sequenced E. meliloti strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. The second copy of the E. meliloti Mlalz-1 lpiA-acvB operon has highest sequence identity (>96%) with that of sequenced E. medicae strains, which infers horizontal gene transfer of this region from E. medicae .

Abbreviations

½LA:

Half strength Lupin Agar

gANI:

Genome-wide average nucleotide identity

GEBA-RNB:

Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria

IMG:

Integrated Microbial Genomes

T2SS:

Type II Secretion System

TY:

Tryptone-yeast extract

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Acknowledgements

We thank Gordon Thompson (Murdoch University) for the preparation of SEM and TEM photos. MLB thanks Alfredo Reyes-Betancort, from the Orotava Botanical Garden (Tenerife), for providing M. laciniata seeds.

Funding

This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231. We gratefully acknowledge the funding received from the Curtin University Sustainability Policy Institute and from Murdoch University’s Small Research Grants Scheme in 2016.

Author information

MLB and EV isolated the strain and provided project metadata. PvB supplied the strain, the DNA and the background information for this project and participated in drafting the manuscript. PE curated the strain and performed sequence analysis of 16S rRNA and nodC genes. RT supplied DNA to JGI and performed all imaging. JA provided symbiotic phenotype data. WAMO, JA and WR performed bioinformatics analyses and drafted the paper, MB and NB provided financial support, and MG, RS, TBKR, NI, TW, AP, VM and NK were involved in sequencing the genome and/or editing the final paper. All authors read and approved the final manuscript.

Correspondence to Wayne Reeve.

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Additional files

Additional file 1: Table S1.

Associated MIGS record for Ensifer meliloti Mlalz-1. (DOCX 52 kb)

Additional file 2: Table S2-S4.

Table S2. Acid responsive gene orthologs present in Ensifer strains. Table S3. The nodulation genes of Ensifer meliloti Mlalz-1. Table S4. The nitrogen fixation genes of Ensifer meliloti Mlalz-1. (DOCX 65 kb)

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Keywords

  • Root-nodule bacteria
  • Ensifer
  • Geba-Rnb
  • Medicago
  • lpiA-acvB operon