Open Access

Genome sequence of Rhizobium leguminosarum bv trifolii strain WSM1689, the microsymbiont of the one flowered clover Trifolium uniflorum

  • Jason Terpolilli1,
  • Tian Rui1,
  • Ron Yates1, 2,
  • John Howieson1,
  • Philip Poole3, 4,
  • Christine Munk5,
  • Roxanne Tapia5,
  • Cliff Han5,
  • Victor Markowitz6,
  • Reddy Tatiparthi7,
  • Konstantinos Mavrommatis6,
  • Natalia Ivanova7,
  • Amrita Pati7,
  • Lynne Goodwin5,
  • Tanja Woyke7,
  • Nikos Kyrpides7 and
  • Wayne Reeve1Email author
Standards in Genomic Sciences20149:9030527

https://doi.org/10.4056/sigs.4988693

Published: 15 June 2014

Abstract

Rhizobium leguminosarum bv. trifolii is a soil-inhabiting bacterium that has the capacity to be an effective N2-fixing microsymbiont of Trifolium (clover) species. R. leguminosarum bv. trifolii strain WSM1689 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. Although WSM1689 is capable of highly effective N2-fixation with T. uniflorum, it is either unable to nodulate or unable to fix N2 with a wide range of both perennial and annual clovers originating from Europe, North America and Africa. WSM1689 therefore possesses a very narrow host range for effective N2 fixation and can thus play a valuable role in determining the geographic and phenological barriers to symbiotic performance in the genus Trifolium. Here we describe the features of R. leguminosarum bv. trifolii strain WSM1689, together with the complete genome sequence and its annotation. The 6,903,379 bp genome contains 6,709 protein-coding genes and 89 RNA-only encoding genes. This multipartite genome contains six distinct replicons; a chromosome of size 4,854,518 bp and five plasmids of size 667,306, 518,052, 341,391, 262,704 and 259,408 bp. This rhizobial genome is one of 20 sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Program.

Keywords

root-nodule bacteria nitrogen fixation lupin-nodulating rhizobia Alphaproteobacteria

Introduction

The nitrogen (N) cycle is one of the most important biogeochemical processes underpinning the existence of life on Earth. A key step in this cycle is to convert relatively inert atmospheric dinitrogen (N2) into a bioaccessible form such as ammonia (NH3) through a process referred to as biological nitrogen fixation (BNF). BNF is performed only by a specialized subset of Bacteria and Archaea that possess the necessary cellular machinery to enzymatically reduce N2 into NH3. Some of these bacteria (termed rhizobia or root nodule bacteria) have evolved non-obligatory symbiotic relationships with legumes whereby the bacteria receive a carbon source from the plant and in return supply fixed N to the host [1]. Harnessing this association can boost soil N-inputs and therefore production yields of legumes, or non-legumes grown in subsequent years, without the need for supplementation with industrially synthesized N-based fertilizers [2].

Some of the most widely cultivated pasture legumes are members of the legume genus Trifolium (clover). The natural distribution of these species spans three centers of diversity, with an estimated 28% of species in the Americas, 57% in Eurasia and 15% in sub-Saharan Africa [3]. Approximately 30 species of clover, predominately of Eurasian origin, are widely grown as annual and perennial species in pasture systems in Mediterranean and temperate climatic zones [3]. Globally-important perennial species of clover include T. repens (white clover), T. pratense (red clover), T. fragiferum (strawberry clover) and T. hybridum (alsike clover). While clovers are known to form N2-fixing symbiotic associations with Rhizobium leguminosarum bv. trifolii, there exists wide variation in symbiotic compatibility across different strains and hosts from ineffective (non-N2-fixing) nodulation to fully effective N2-fixing partnerships.

Rhizobium leguminosarum bv. trifolii strain WSM1689 was isolated in 1995 from a nodule of the perennial clover Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. T. uniflorum is one of small number of perennial Trifolium spp. found in the dry, Mediterranean basin. While WSM1689 has been shown to be either ineffective or unable to nodulate a range of annual and perennial Trifolium sp., it is a highly effective N2-fixing microsymbiont of T. uniflorum [4]. Therefore, R. leguminosarum bv. trifolii WSM1689 has a very narrow host range and thus represents a good isolate to study the genetic basis of symbiotic specificity. The availability of this sequence data also complements the already published genomes of the clover-nodulating R. leguminosarum bv. trifolii WSM1325 [5] and WSM2304 [6]. Here we present a summary classification and a set of general features for R. leguminosarum bv. trifolii strain WSM1689 together with the description of the complete genome sequence and its annotation.

Classification and features

R. leguminosarum bv. trifolii strain WSM1689 is a motile, non-sporulating, non-encapsulated, Gram-negative rod in the order Rhizobiales of the class Alphaproteobacteria. The rod-shaped form varies in size with dimensions of approximately 0.25–0.5 µm in width and 2.0 µm in length (Figure 1 Left and 1 Center). It is fast growing, forming colonies within 3–4 days when grown on half strength Lupin Agar (½LA) [7], tryptone-yeast extract agar (TY) [8] or a modified yeast-mannitol agar (YMA) [9] at 28°C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right). Minimum Information about the Genome Sequence (MIGS) is provided in Table 1.
Figure 1.

Images of Rhizobium leguminosarum bv. trifolii strain WSM1689 using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on ½LA (Right).

Table 1.

Classification and general features of Rhizobium leguminosarum bv. trifolii strain WSM1689 according to the MIGS recommendations [10,11].

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [11]

 

Phylum Proteobacteria

TAS [12]

 

Class Alphaproteobacteria

TAS [13,14]

 

Order Rhizobiales

TAS [14,15]

 

Family Rhizobiaceae

TAS [16,17]

 

Genus Rhizobium

TAS [16,1821]

 

Species Rhizobium leguminosarum bv. trifolii

TAS [16,18,21,22]

 

Strain WSM1689

TAS [4]

 

Gram stain

Negative

IDA

 

Cell shape

Rod

IDA

 

Motility

Motile

IDA

 

Sporulation

Non-sporulating

NAS

 

Temperature range

Mesophile

NAS

 

Optimum temperature

28°C

NAS

 

Salinity

Not reported

NAS

MIGS-22

Oxygen requirement

Aerobic

TAS [4]

 

Carbon source

Varied

NAS

 

Energy source

Chemoorganotroph

NAS

MIGS-6

Habitat

Soil, root nodule, host

TAS [4]

MIGS-15

Biotic relationship

Free living, symbiotic

TAS [4]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

 

Biosafety level

1

NAS [23]

 

Isolation

Root nodule

TAS [4]

MIGS-4

Geographic location

Naxos, Greece

IDA

MIGS-5

Nodule collection date

1995

IDA

MIGS-4.1

Latitude

37.128333

IDA

MIGS-4.2

Longitude

25.443333

IDA

MIGS-4.3

Depth

Not reported

 

MIGS-4.4

Altitude

Not reported

 

Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable 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). These evidence codes are from the Gene Ontology project [24].

Figure 2 shows the phylogenetic neighborhood of R. leguminosarum bv. trifolii strain WSM1689 in a 16S rRNA gene sequence based tree. This strain shares 100% (1362/1362 bp) sequence identity to the 16S rRNA gene of R. leguminosarum bv. trifolii strain WSM1325 [5] and R. leguminosarum bv. trifolii strain WSM2304 [6].
Figure 2.

Phylogenetic tree showing the relationship of Rhizobium leguminosarum bv trifolii WSM1689 (shown in bold print) to other root nodulating Rhizobium spp. in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,180 bp internal region). All positions containing gaps and missing data were eliminated. All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [25]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [26]. Bootstrap analysis [27] with 500 replicates 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 (beginning with the prefix G) for a sequencing project registered in GOLD [28]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

R. leguminosarum bv. trifolii WSM1689 is a highly effective microsymbiont of the perennial Eurasian clover Trifolium uniflorum (Table 2). In contrast, WSM1689 does not nodulate the perennial T. fragiferum and forms white ineffective (Fix) nodules with other perennial and annual clovers of Eurasian origin. Moreover, WSM1689 is either Nod or Fix on clovers of North American or African origin. Therefore, WSM1689 is unusual in having an extremely narrow clover host range for the establishment of effective N2-fixing symbiosis.
Table 2.

Compatibility of WSM1689 with both perennial and annual Trifolium genotypes for nodulation (Nod) and N2-Fixation (Fix). Data compiled from [4].

Species Name

Cultivar

Origin

Growth habit

Nod

Fix

Comment

T. uniflorum

Nil

Europe

Perennial

Nod+

Fix+

Highly effective

T. tumens

1986267

Europe

Perennial

Nod+

Fix

Ineffective

T. tumens

16758246

Europe

Perennial

Nod+

Fix

Ineffective

T. medium

21881154

Europe

Perennial

Nod+

Fix

Ineffective

T. repens

037701

Europe

Perennial

Nod+

Fix

Ineffective

T. repens

036120

Europe

Perennial

Nod+

Fix

Ineffective

T. pratense

Russian no 9

Europe

Perennial

Nod+

Fix

Ineffective

T. pratense

Redquin

Europe

Perennial

Nod+

Fix

Ineffective

T. ambiguum

Endura

Europe

Perennial

Nod+

Fix

Ineffective

T. canescens

PL4188661999

Europe

Perennial

Nod+

Fix

Ineffective

T. fragiferum

C1212

Europe

Perennial

Nod

 

No nodulation

T. polymorphum

87102

South America

Perennial

Nod+

Fix

Ineffective

T. longipes

A2436817

North America

Perennial

Nod

 

No nodulation

T. subterraneum

York

Europe

Annual

Nod+

Fix

Ineffective

T. glanduliferum

CP187182

Europe

Annual

Nod+

Fix

Ineffective

T. mulinerve

87259

Africa

Annual

Nod

 

No nodulation

T. tridentatum

CQ1263

North America

Annual

Nod+

Fix

Ineffective

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 U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [28] and a finished genome sequence in IMG/GEBA. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 3.
Table 3.

Genome sequencing project information for Rhizobium leguminosarum bv. trifolii strain WSM1689.

MIGS ID

Property

Term

MIGS-31

Finishing quality

Finished

MIGS-28

Libraries used

Illumina GAii shotgun and paired end 454 libraries

MIGS-29

Sequencing platforms

Illumina GAii and 454 GS FLX Titanium technologies

MIGS-31.2

Sequencing coverage

8.3x 454, 774.6x Illumina

MIGS-30

Assemblers

VELVET, version 1.1.05; Newbler, version 2.6; phrap, version SPS - 4.24

MIGS-32

Gene calling methods

Prodigal 1.4, GenePRIMP

 

Genbank ID

Not yet available

 

Genbank Date of Release

Not yet released

 

GOLD ID

Gi06499

 

NCBI project ID

62289

 

Database: IMG-GEBA

2510065019

 

Project relevance

Symbiotic nitrogen fixation, agriculture

Growth conditions and DNA isolation

Rhizobium leguminosarum bv. trifolii strain WSM1689 was grown to mid logarithmic phase in TY rich medium on a gyratory shaker at 28°C [29]. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [30].

Genome sequencing and assembly

The genome of Rhizobium leguminosarum bv. trifolii strain WSM1689 was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [31] and 454 technologies [32]. An Illumina GAii shotgun library which generated 73,565,648 reads totaling 5,591 Mbp, and a paired end 454 library with an average insert size of 12 Kbp which generated 376,185 reads totaling 93.4 Mbp of 454 data were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at [30]. The initial draft assembly contained 100 contigs in 4 scaffolds. The 454 paired end data was assembled with Newbler, version 2.6. The Newbler consensus sequences were computationally shredded into 2 Kbp overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.1.05 [33], and the consensus sequence computationally shredded into 1.5 Kbp 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 [3436] 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 [37], 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 93 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total genome size is 6.9 Mbp and the final assembly is based on 57.3 Mbp of 454 draft data which provides an average 8.3× coverage of the genome and 5,345 Mbp of Illumina draft data which provides an average 774.6× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [38] as part of the DOE-JGI genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [39]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant 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 [40], RNAMMer [41], Rfam [42], TMHMM [43], and SignalP [44]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [45,46].

Genome properties

The genome is 6,903,379 nucleotides with 60.94% GC content (Table 4 and Figures 3a,3b,3c,3d,3e and Figure 3f), and comprised of 6 replicons. From a total of 6,798 genes, 6,709 were protein encoding and 89 RNA only encoding genes. Within the genome, 206 pseudogenes were also identified. The majority of 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 5.
Figure 3a.

Graphical circular map of Replicon WSM1689_Rleg3_Contig1814.1 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3b.

Graphical circular map of replicon WSM1689_Rleg3_Contig1813.2 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3c.

Graphical circular map of replicon WSM1689_Rleg3_Contig1812.3 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3d.

Graphical circular map of replicon WSM1689_Rleg3_Contig1810.5 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3e.

Graphical circular map of replicon WSM1689_Rleg3_Contig1811.4 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Figure 3f.

Graphical circular map of replicon WSM1689_Rleg3_Contig1809.6 of the Rhizobium leguminosarum bv. trifolii strain WSM1689 genome. From outside to the center: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Table 4.

Genome Statistics for Rhizobium leguminosarum bv. trifolii strain WSM1689.

Attribute

Value

% of Total

Genome size (bp)

6,903,379

100.00

DNA coding region (bp)

6,004,795

86.98

DNA G+C content (bp)

4,206,909

60.94

Number of replicons

6

 

Total genes

6,798

100.00

RNA genes

89

1.31

Protein-coding genes

6,709

98.69

Genes with function prediction

5,406

79.52

Genes assigned to COGs

5,400

79.44

Genes assigned Pfam domains

5,618

82.64

Genes with signal peptides

591

8.69

Genes coding transmembrane proteins

1,524

22.42

CRISPR repeats

0

 
Table 5.

Number of protein coding genes of Rhizobium leguminosarum bv. trifolii strain WSM1689 associated with the general COG functional categories.

Code

Value

%age

COG Category

J

205

3.40

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

581

9.62

Transcription

L

153

2.53

Replication, recombination and repair

B

2

0.03

Chromatin structure and dynamics

D

39

0.65

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

66

1.09

Defense mechanisms

T

311

5.15

Signal transduction mechanisms

M

329

5.45

Cell wall/membrane biogenesis

N

81

1.34

Cell motility

Z

0

0.00

Cytoskeleton

W

0

0.00

Extracellular structures

U

82

1.36

Intracellular trafficking and secretion

O

187

3.10

Posttranslational modification, protein turnover, chaperones

C

311

5.15

Energy production conversion

G

683

11.31

Carbohydrate transport and metabolism

E

629

10.42

Amino acid transport metabolism

F

105

1.74

Nucleotide transport and metabolism

H

192

3.18

Coenzyme transport and metabolism

I

222

3.68

Lipid transport and metabolism

P

297

4.92

Inorganic ion transport and metabolism

Q

147

2.43

Secondary metabolite biosynthesis, transport and catabolism

R

795

13.17

General function prediction only

S

620

10.27

Function unknown

-

1,398

20.56

Not in COGS

-

6,037

-

Total

Declarations

Acknowledgements

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, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. We gratefully acknowledge the funding received from the Murdoch University Sir Walter Murdoch Adjunct Professor Scheme for Professor Philip Poole.

Authors’ Affiliations

(1)
Centre for Rhizobium Studies, Murdoch University
(2)
Department of Agriculture and Food
(3)
Department of Plant Sciences, University of Oxford
(4)
Sir Walter Murdoch Adjunct Professor, Murdoch University
(5)
Bioscience Division, Los Alamos National Laboratory
(6)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(7)
DOE Joint Genome Institute

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