Open Access

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI943

  • Wayne Reeve1Email author,
  • Elizabeth Drew2,
  • Ross Ballard2,
  • Vanessa Melino1,
  • Rui Tian1,
  • Sofie De Meyer1,
  • Lambert Brau3,
  • Mohamed Ninawi1,
  • Hajnalka Daligault4, 5,
  • Karen Davenport4,
  • Tracy Erkkila4,
  • Lynne Goodwin5,
  • Wei Gu4,
  • Christine Munk4,
  • Hazuki Teshima4,
  • Yan Xu4,
  • Patrick Chain4 and
  • Nikos Kyrpides5
Standards in Genomic Sciences20139:9020232

DOI: 10.4056/sigs.4478252

Published: 20 December 2013

Abstract

Rhizobium leguminosarum bv. trifolii SRDI943 (strain syn. V2-2) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium michelianum Savi cv. Paradana that had been grown in soil collected from a mixed pasture in Victoria, Australia. This isolate was found to have a broad clover host range but was sub-optimal for nitrogen fixation with T. subterraneum (fixing 20–54% of reference inoculant strain WSM1325) and was found to be totally ineffective with the clover species T. polymorphum and T. pratense. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI943, together with genome sequence information and annotation. The 7,412,387 bp high-quality-draft genome is arranged into 5 scaffolds of 5 contigs, contains 7,317 protein-coding genes and 89 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.

Keywords

root-nodule bacteria nitrogen fixation rhizobia Alphaproteobacteria

Introduction

The availability of usable nitrogen (N) is vital for productivity in agricultural systems that are N-deficient [1]. It can be supplied exogenously in the form of industrially synthesized fertilizers. However, this practice is expensive since fertilizer manufacture depends on the availability of fossil fuels that are burnt to support the industrial process of chemical N-fixation. A far more economical practice is to supply plant-available N to farming systems by exploiting the process of biological N-fixation that occurs in a symbiotic relationship between legumes and their rhizobial microsymbionts [2]. In this specific association, atmospheric inert dinitrogen gas is converted into bioavailable N to support legume growth.

Pasture legumes, including the clovers that comprise the Trifolium genus, are major contributors of biologically fixed nitrogen (N2) to mixed farming systems throughout the world [3,4]. In Australia, soils with a history of growing Trifolium spp. have developed large and symbiotically diverse populations of Rhizobium leguminosarum bv. trifolii (R. l. trifolii) that are able to infect and nodulate a range of clover species. The N2-fixation capacity of the symbioses established by different combinations of clover hosts (Trifolium spp.) and strains of R. l. trifolii can vary from 10 to 130% when compared to an effective host-strain combination [58].

R. l. trifolii strain SRDI943 (syn. V2-2 [9]) was isolated from a nodule recovered from the roots of the annual clover Trifolium michelianum Savi cv. Paradana that had been inoculated with soil collected from under a mixed pasture at Walpeup, Victoria, Australia and grown in N deficient media for four weeks after inoculation, in the greenhouse [10]. SRDI943 forms an effective symbiosis with T. purpureum but sub-optimal N2-fixation symbiosis with T. subterraneum cv. Campeda and Clare (24 and 54% respectively of that with strain WSM1325 [9,11]). Here we present a preliminary description of the general features for R. l. trifolii strain SRDI943 together with its genome sequence and annotation.

Classification and general features

R. l. trifolii strain SRDI943 is a motile, Gram-negative rod (Figure 1 Left and Center) in the order Rhizobiales of the class Alphaproteobacteria. It is fast growing, forming colonies within 3–4 days when grown on half strength Lupin Agar (½LA) [12] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right).
Figure 1.

Images of Rhizobium leguminosarum bv. trifolii strain SRDI943 using scanning (Left) and transmission (Center) electron microscopy as well as light microscopy to show the colony morphology on solid media (Right).

Minimum information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic relationship of R. l. trifolii strain SRDI943 to root nodule bacteria in the order Rhizobiales in a 16S rRNA sequence based tree. This strain clusters closest to R. l. trifolii T24 and Rhizobium leguminosarum bv. phaseoli RRE6 with 100% and 99.8% sequence identity, respectively.
Figure 2.

Phylogenetic tree showing the relationship of Rhizobium leguminosarum bv. trifolii SRDI943 (shown in blue print) with some of the root nodule bacteria in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,307 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5.05 [31]. The tree was built using the maximum likelihood method with the General Time Reversible model. Bootstrap analysis [32] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Strains with a genome sequencing project registered in GOLD [33] are in bold print and the GOLD ID is mentioned after the accession number. Published genomes are indicated with an asterisk.

Table 1.

Classification and general features of Rhizobium leguminosarum bv. trifolii SRDI943 according to the MIGS recommendations [13]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [14]

 

Phylum Proteobacteria

TAS [15]

 

Class Alphaproteobacteria

TAS [16,17]

 

Order Rhizobiales

TAS [17,18]

 

Family Rhizobiaceae

TAS [1921]

 

Genus Rhizobium

TAS [2126]

 

Species Rhizobium leguminosarum bv. trifolii

TAS [21,23,27,28]

 

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

Non-halophile

NAS

MIGS-22

Oxygen requirement

Aerobic

TAS [11]

 

Carbon source

Varied

NAS

 

Energy source

Chemoorganotroph

NAS

MIGS-6

Habitat

Soil, root nodule, on host

TAS [9]

MIGS-15

Biotic relationship

Free living, symbiotic

TAS [9]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

 

Biosafety level

1

TAS [29]

 

Isolation

Root nodule

TAS [9]

MIGS-4

Geographic location

Victoria, Australia

TAS [9]

MIGS-5

Soil collection date

Dec, 1998

IDA

MIGS-4.1

Longitude

142.0262

IDA

MIGS-4.2

Latitude

−35.13531

IDA

MIGS-4.3

Depth

0–10cm

 

MIGS-4.4

Altitude

Not recorded

 

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 [30].

Symbiotaxonomy

R. l. trifolii SRDI943 forms nodules on (Nod+) and fixes N2 (Fix+) with a range of annual and perennial clover species of Mediterranean origin (Table 2). SRDI943 forms white, ineffective (Fix) nodules with the perennial clover T. pratense and T. polymorphum.
Table 2.

Compatibility of SRDI943 with eleven Trifolium genotypes for nodulation (Nod) and N2-Fixation (Fix)

Species Name

Cultivar

Common Name

Growth Type

Nod

Fix

Reference

T. glanduliferum Boiss.

Prima

Gland

Annual

+

+

 

T. michelianum Savi.

Bolta

Balansa

Annual

+

+

 

T. purpureum Loisel

Paratta

Purple

Annual

+

+

[11]

T. resupinatum L.

Kyambro

Persian

Annual

+

+

 

T. subterraneum L.

Campeda

Sub. clover

Annual

+

+

[9,11]

T. subterraneum L.

Clare

Sub. clover

Annual

+

+

[9,11]

T. vesiculosum Savi.

Arrotas

Arrowleaf

Annual

+

+

 

T. fragiferum L.

Palestine

Strawberry

Perennial

+

+

 

T. polymorphum Poir

Acc.#087102

Polymorphous

Perennial

+(w)

[11]

T. pratense L.

Red

Perennial

+(w)

 

T. repens L.

Haifa

White

Perennial

+

+

 

(w) indicates nodules present were white.

Genome sequencing and annotation information

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 sequence is deposited in the Genomes OnLine Database (GOLD) [33] and an improved-high-quality-draft 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 SRDI943.

MIGS ID

Property

Term

MIGS-31

Finishing quality

Improved high-quality draft

MIGS-28

Libraries used

2× Illumina libraries; Std short PE & CLIP long PE

MIGS-29

Sequencing platforms

Illumina HiSeq 2000

MIGS-31.2

Sequencing coverage

Illumina (761×)

MIGS-30

Assemblers

Velvet 1.1.05, phrap SPS-4.24, Allpaths version 39750

MIGS-32

Gene calling methods

Prodigal 1.4, GenePRIMP

 

GOLD ID

Gi08842

 

NCBI project ID

89687

 

Database: IMG

2517093000

 

Project relevance

Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

R. l. trifolii strain SRDI943 was cultured to mid logarithmic phase in 60 ml of TY rich media [34] on a gyratory shaker at 28°C. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [35].

Genome sequencing and assembly

The genome of R. l. trifolii strain SRDI943 was sequenced at the Joint Genome Institute (JGI) using an Illumina sequencing platform. An Illumina short-insert paired-end (PE) library with an average insert size of 270 bp produced 18,764,470 reads and an Illumina CLIP long-insert paired-end (PE) library with an average insert size of 9,482 bp produced 18,761,080 reads totaling 5,629 Mb of Illumina data for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at the DOE JGI user homepage [35]. The initial draft assembly contained 5 contigs in 5 scaffolds. The initial draft data was assembled with Allpaths, version 39750. The Allpaths consensus was computationally shredded into 10 Kb overlapping fake reads (shreds). Illumina sequencing data were assembled with Velvet, version 1.1.05 [36], and the consensus sequences were computationally shredded into 1.5 kb overlapping fake reads (shreds). The Allpaths consensus shreds, the Illumina VELVET consensus shreds and a sub-set of the Illumina CLIP paired-end reads were integrated using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [3739] was used in the following finishing process. The estimated genome size is 7.4 Mb and the final assembly is based on 5,629 Mb of Illumina draft data which provides an average of 761× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [40] as part of the DOE-JGI annotation pipeline [41] annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [42]. 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 ascribe a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [43], RNAMMer [44], Rfam [45], TMHMM [46], and SignalP [47]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [35,48].

Genome properties

The genome is 7,412,387 nucleotides with 60.69% GC content (Table 4) and comprised of 5 scaffolds (Figure 3) of 5 contigs. From a total of 7,406 genes, 7,317 were protein encoding and 89 RNA only encoding genes. The majority of genes (78.5%) 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 3.

Graphical map of the genome of Rhizobium leguminosarum bv. trifolii strain SRDI943. From bottom to the top of each scaffold: 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 SRDI943

Attribute

Value

% of Total

Genome size (bp)

7,412,387

100.00

DNA coding region (bp)

6,395,342

86.28

DNA G+C content (bp)

4,498,817

60.69

Number of scaffolds

5

 

Number of contigs

5

 

Total gene

7,406

100.00

RNA genes

89

1.20

rRNA operons

3

 

Protein-coding genes

7,317

98.80

Genes with function prediction

5,814

78.50

Genes assigned to COGs

5,770

77.91

Genes assigned Pfam domains

6,032

81.45

Genes with signal peptides

631

8.52

Genes with transmembrane proteins

1,618

21.85

CRISPR repeats

0

 
Table 5.

Number of protein coding genes of Rhizobium leguminosarum bv. trifolii SRDI943 associated with the general COG functional categories.

Code

Value

%age

COG Category

J

196

3.03

Translation, ribosomal structure and biogenesis

A

1

0.02

RNA processing and modification

K

652

10.06

Transcription

L

231

3.57

Replication, recombination and repair

B

2

0.03

Chromatin structure and dynamics

D

40

0.62

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

76

1.17

Defense mechanisms

T

373

5.76

Signal transduction mechanisms

M

334

5.16

Cell wall/membrane biogenesis

N

92

1.42

Cell motility

Z

1

0.02

Cytoskeleton

W

1

0.02

Extracellular structures

U

95

1.47

Intracellular trafficking and secretion

O

193

2.98

Posttranslational modification, protein turnover, chaperones

C

324

5.00

Energy production conversion

G

714

11.02

Carbohydrate transport and metabolism

E

659

10.17

Amino acid transport metabolism

F

109

1.68

Nucleotide transport and metabolism

H

192

2.96

Coenzyme transport and metabolism

I

227

3.50

Lipid transport and metabolism

P

333

5.14

Inorganic ion transport and metabolism

Q

165

2.55

Secondary metabolite biosynthesis, transport and catabolism

R

842

13.00

General function prediction only

S

627

9.68

Function unknown

-

1,636

22.09

Not in COGS

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 Strategic Research Fund through the Crop and Plant Research Institute (CaPRI), the Centre for Rhizobium Studies (CRS) at Murdoch University and the GRDC National Rhizobium Program (UMU00032). The authors would like to thank the Australia-China Joint Research Centre for Wheat Improvement (ACCWI) and SuperSeed Technologies (SST) for financially supporting Mohamed Ninawi’s PhD project.

Authors’ Affiliations

(1)
Centre for Rhizobium Studies, Murdoch University
(2)
South Australian Research and Development Institute
(3)
School of Life and Environmental Sciences, Faculty of Science & Technology, Deakin University
(4)
Bioscience Division, Los Alamos National Laboratory
(5)
DOE Joint Genome Institute

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