Open Access

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

  • Wayne Reeve
  • , Elizabeth Drew
  • , Ross Ballard
  • , Vanessa Melino
  • , Rui Tian
  • , Sofie De Meyer
  • , Lambert Brau
  • , Mohamed Ninawi
  • , Hajnalka Daligault,
  • , Karen Davenport
  • , Tracy Erkkila
  • , Lynne Goodwin
  • , Wei Gu
  • , Christine Munk
  • , Hazuki Teshima
  • , Yan Xu
  • , Patrick Chain
  • and Nikos Kyrpides
Corresponding author

DOI: 10.4056/sigs.4478252

Received: 15 December 2013

Accepted: 15 December 2013

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 bacterianitrogen fixationrhizobiaAlphaproteobacteria

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 [5-8].

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.

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 [19-21]

    Genus Rhizobium

    TAS [21-26]

    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 MIGS-4.2

    Longitude    Latitude

    142.0262    -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].

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.

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 [37-39] 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.

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

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 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.


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