Open Access

Genome sequence of Bradyrhizobium sp. WSM1253; a microsymbiont of Ornithopus compressus from the Greek Island of Sifnos

  • Ravi Tiwari1,
  • John Howieson1,
  • Ron Yates1, 2,
  • Rui Tian1,
  • Britanny Held3,
  • Roxanne Tapia3,
  • Cliff Han3,
  • Rekha Seshadri4,
  • T. B. K. Reddy4,
  • Marcel Huntemann4,
  • Amrita Pati4,
  • Tanja Woyke4,
  • Victor Markowitz5,
  • Natalia Ivanova4,
  • Nikos Kyrpides4, 6 and
  • Wayne Reeve1Email author
Standards in Genomic Sciences201510:113

https://doi.org/10.1186/s40793-015-0115-9

Received: 10 December 2014

Accepted: 25 November 2015

Published: 30 November 2015

Abstract

Bradyrhizobium sp. WSM1253 is a novel N2-fixing bacterium isolated from a root nodule of the herbaceous annual legume Ornithopus compressus that was growing on the Greek Island of Sifnos. WSM1253 emerged as a strain of interest in an Australian program that was selecting inoculant quality bradyrhizobial strains for inoculation of Mediterranean species of lupins (Lupinus angustifolius, L. princei, L. atlanticus, L. pilosus). In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 8,719,808 bp genome has a G + C content of 63.09 % with 71 contigs arranged into two scaffolds. The assembled genome contains 8,432 protein-coding genes, 66 RNA genes and a single rRNA operon. This improved-high-quality draft rhizobial genome is one of 20 sequenced through a DOE Joint Genome Institute 2010 Community Sequencing Project.

Keywords

root-nodule bacteria nitrogen fixation rhizobia Ornithopus

Introduction

Root nodule bacteria are soil microorganisms that can establish a symbiotic relationship with hosts from the legume plant family Leguminosae. In this intimate relationship the bacteria fix atmospheric nitrogen into ammonia for the legume, in exchange for nutrients. With the continued discovery of a large number of organisms with this capability through the last century, the slow growing, non-acid producing root nodule bacteria were separated from the fast growing acid-producing forms and designated the bradyrhizobia [1]. The initial interest in the bradyrhizobia arose from the ability of strains to nodulate agriculturally important crops such as soybean and groundnut. Today the bradyrhizobia are known to nodulate a wide variety of legumes such as Arachis hypogaea , Adenocarpus spp., Beta vulgaris , Chamaecytisus spp., Cytisus villosus , Entada koshunensis, Glycine spp., Dolichos lablab , Lespedeza spp., Lupinus spp., Ornithopus spp., Pachyrhizus erosus , Spartocytisus spp. and Teline spp. [29].

Two agriculturally important legume genera form a symbiosis with Bradyhizobium [10], the subject of this manuscript. Lupinus which is a large and diverse genus, and Ornithopus , which is a smaller forage legume genus, both nodulate and fix nitrogen with this bacterium. Lupinus angustifolius is commonly known as lupin in Europe and Australia, and lupine in North America, and its grain is widely used as an animal or human food. Lupins are either annual or perennial herbs, shrubs or trees [11]. Ornithopus is commonly known as serradella, and was originally confined to the Iberian peninsula and the Mediterranean basin, however it has become a valuable grazing plant adapted to low rainfall, acidic and infertile soils world-wide [12]. Hence, appropriate Bradyrhizobium inoculants are of particular value for the establishment of effective nitrogen-fixing symbioses with these legume genera.

In Australia, the challenge was to select inoculant strains that were optimal for N fixation in symbiosis with Lupinus angustifolius and several species of Ornithopus . These are all very important legumes in farming systems of Western Australia. They are cultivated on the same acid and sandy soils, and share microsymbionts [13]. Thus, it was important that any inoculant strain released for an individual legume species did not compromise the potential nitrogen fixation from the other legumes. Bradyrhizobium sp. WSM1253 emerged as a strain of interest in an Australian program that was selecting inoculant strains for Mediterranean species of lupins. Strain WSM1253 was isolated from a nodule of the herbaceous annual legume Ornithopus compressus in 1991 collected 2.5 km near of Kastro, towards Faros, on the Greek Island of Sifnos. This strain was found to be capable of high levels of nitrogen fixation across many species in the cross-nodulation complex of lupins and Ornithopus , being particularly effective on L. princei [14]. Here we present a preliminary description of the general features of the Ornithopus compressus microsymbiont Bradyrhizobium sp. WSM1253, together with the description of the complete genome sequence and its annotation.

Organism information

Classification and features

Bradyrhizobium sp. WSM1253 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 and dimensions of approximately 0.25 μm in width and 1.5-2.0 μm in length (Fig. 1 Left and Center). It is relatively slow growing, forming colonies after 6–7 days when grown on ½LA [15], TY [16] or YMA [17] at 28 °C. Colonies on ½LA are opaque, slightly domed and moderately mucoid with smooth margins (Fig. 1 Right).
Fig. 1

Images of Bradyrhizobium sp. WSM1253 using scanning (Left) and transmission (Center) electron microscopy as well as light microscopy to visualize colony morphology on solid media (Right)

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1 and Additional file 1: Table S1. Strain WSM1253 shares 100 % (1369/1369 bp), 99.85 % (1367/1369 bp) and 99.48 % (1362/1369 bp) 16S rRNA sequence identity with Bradyrhizobium sp. WSM1417, Bradyrhizobium sp. BTA-1T and Bradyrhizobium japonicum USDA 6T , respectively as determined using NCBI BLAST analysis [18]. Figure 2 shows the phylogenetic neighbor-hood of Bradyrhizobium sp. WSM1253 in a 16S rRNA sequence based tree.
Table 1

Classification and general features of Bradyrhizobium sp. WSM1253 [44, 45]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [45]

  

Phylum Proteobacteria

TAS [46]

  

Class Alphaproteobacteria

TAS [47, 48]

  

Order Rhizobiales

TAS [49]

  

Family Bradyrhizobiaceae

TAS [50, 51]

  

Genus Bradyrhizobium

TAS [1]

  

Species sp.

IDA

  

Strain: WSM1253

TAS [14]

 

Gram stain

Negative

IDA

 

Cell shape

Rod

IDA

 

Motility

Motile

IDA

 

Sporulation

Non-sporulating

NAS

 

Temperature range

Mesophile

NAS

 

Optimum temperature

28 °C

NAS

 

pH range; Optimum

5-9; 7

NAS

 

Carbon source

Varied

IDA

MIGS-6

Habitat

Soil, root nodule, on plant host

TAS [14]

MIGS-6.3

Salinity

Non-halophilie

NAS

MIGS-22

Oxygen requirement

Aerobic

TAS [14]

MIGS-15

Biotic relationship

free-living, symbiont

TAS [14]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

MIGS-4

Geographic location

Greek Island of Sifnos

TAS [14]

MIGS-5

Nodule collection date

1991

IDA

MIGS-4.1

Latitude

39.975

IDA

MIGS-4.2

Longitude

24.743889

IDA

MIGS-4.4

Altitude

Not reported

IDA

aEvidence 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 [52]

Fig. 2

Phylogenetic tree showing the relationship of Bradyrhizobium sp. WSM1253 (shown in bold print) to other root nodule bacteria based on aligned sequences of a 1,012 bp internal region the 16S rRNA gene. All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [41], version 5. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [42]. Bootstrap analysis [43] 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 contains a DNA database accession number and/or a GOLD ID (beginning with the prefix G) for a sequencing project registered in GOLD [22]. Published genomes are indicated with an asterisk

Symbiotaxonomy

Few of the legumes of the Mediterranean basin introduced to agriculture elsewhere are nodulated by bacteria in the genus Bradyrhizobium [19]. Amongst the notable exceptions are Lupinus and Ornithopus , which are legume genera adapted specifically to conditions of acidity and infertility [20]. Further, these two quite different legumes share a common species of Bradyrhizobium , although their modes of infection and nodule structure differ substantially [21]. WSM1253 is unusual in being a highly effective microsymbiont for many species in the two legume genera discussed, including, L. angustifolius, L. princei, L. atlanticus, L. pilosus, O. compressus, O. sativus Brot. and O. pinnatus (Table 2). WSM1253 will therefore be a valuable strain to study the genetics of nodulation and nitrogen fixation in legumes of vastly differing physiology.
Table 2

Compatibility of Bradyrhizobium sp. WSM1253 [14] with different wild and cultivated legume species

Species name

Family

Common name

Habit/Growth type

Nod

Fix

Lupinus atlanticus

Fabaceae

Atlas Lupin/Moroccan Lupin

Annual herbaceous

+

+

Lupinus pilosus

Fabaceae

Mountain blue lupin

Annual herbaceous

+

+

Lupinus princei

Fabaceae

Lupin

Annual herbaceous

+

+

Ornithopus pinnatus

Fabaceae

Sand Bird’s-foot

Annual herbaceous

+

+

Ornithopus sativus Brot.

Fabaceae

common bird’s-foot

Annual herbaceous

+

+

Ornithopus compressus

Fabaceae

Yellow serradella

Annual herbaceous

+

+

+, nodulation/fixation observed

Genome sequencing 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 for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database [22] and the improved-high-quality draft genome sequence in IMG. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 3.
Table 3

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Improved-high-quality draft

MIGS 28

Libraries used

Illumina GAii and 454 FLX libraries

MIGS 29

Sequencing platforms

Illumina and 454

MIGS 31.2

Fold coverage

659.4 × Illumina; 8.4 × 454

MIGS 30

Assemblers

Velvet 1.0.13; Newbler 2.3

MIGS 32

Gene calling methods

Prodigal 1.4

 

Locus Tag

Bra1253

GenBank ID

AHMB01000000

 

Genbank Date of Release

May 4, 2012

 

GOLD ID

Gp0007394

 

BIOPROJECT

PRJNA62341

MIGS 13

Project relevance

Symbiotic N2 fixation, agriculture

 

Source Material Identifier

WSM1253

Growth conditions and genomic DNA preparation

Bradyrhizobium sp. WSM1253 was grown on TY solid medium for 10 days, a single colony was selected and used to inoculate 5 ml TY broth medium. The culture was grown for 96 h on a gyratory shaker (200 rpm) at 28 °C [23]. Subsequently 1 ml was used to inoculate 60 ml TY broth medium and grown on a gyratory shaker (200 rpm) at 28 °C until OD 0.6 was reached. DNA was isolated from 60 ml of cells using a CTAB bacterial genomic DNA isolation method [24]. The quality of DNA was checked by 0.5 % agarose gel electrophoresis and its quantity by a NanoDrop ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, USA). A DNA concentration of 500 ng/μl and OD 260/OD 280 of 1.90 was obtained.

Genome sequencing and assembly

The draft genome of Bradyrhizobium sp. WSM1253 was generated at the DOE Joint Genome Institute using a combination of Illumina [25] and 454 technologies [26]. For this genome, we constructed and sequenced an Illumina GAii shotgun library which generated 77,541,190 reads totaling 5,893.1 Mbp, a 454 Titanium paired end library with an average insert size of 12 Kbp which generated 615,580 reads totaling 123.4 Mbp of 454 data. All general aspects of library construction and sequencing performed at the JGI [27]. The initial draft assembly contained 274 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 Kbp overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [28], and the consensus sequence was 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 [2931] 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 [32], 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 226 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 8.7 Mbp and the final assembly is based on 72.7 Mbp of 454 draft data which provides an average 8.4× coverage of the genome and 5,736.7 Mbp of Illumina draft data which provides an average 659.4× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [33], as part of the DOE-JGI genome annotation pipeline [34, 35] followed by a round of manual curation using GenePRIMP [36] for finished genomes and Draft genomes in fewer than 10 scaffolds. 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 [37] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [38]. 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 [39]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes-Expert Review system [40] developed by the Joint Genome Institute, Walnut Creek, CA, USA.

Genome properties

The genome is 8,719,808 nucleotides with 63.09 % GC content (Table 4) and comprised of 2 scaffolds (Fig. 3). From a total of 8,498 genes, 8,432 were protein encoding and 66 RNA only encoding genes. The majority of genes (66.86 %) 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 Bradyhizobium sp. WSM1253

Attribute

Value

% of Total

Genome size (bp)

8,719,808

100.00

DNA coding (bp)

7,446,464

85.40

DNA G + C (bp)

5,501,733

63.09

DNA scaffolds

2

100.00

Total genes

8,498

100.00

Protein coding genes

8,432

99.22

RNA genes

66

0.78

Pseudo genes

385

4.53

Genes in internal clusters

639

7.52

Genes with function prediction

5,682

66.89

Genes assigned to COGs

5,310

62.49

Genes with Pfam domains

6,484

76.30

Genes with signal peptides

948

11.16

Genes with transmembrane helices

1,953

22.98

CRISPR repeats

0

0.00

Fig. 3

Graphical map of the two scaffolds from the genome of Bradyhizobium sp. WSM1253. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories), 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 genes associated with general COG functional categories

Code

Value

% age

COG Category

J

235

3.83

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

430

7.01

Transcription

L

1.53

2.50

Replication, recombination and repair

B

2

0.03

Chromatin structure and dynamics

D

39

0.64

Cell cycle control, cell division, chromosome partitioning

V

170

2.77

Defense mechanisms

T

270

4.40

Signal transduction mechanisms

M

322

5.25

Cell wall/membrane/envelope biogenesis

N

105

1.71

Cell motility

U

95

1.55

Intracellular trafficking, secretion, and vesicular transport

O

246

4.01

Posttranslational modification, protein turnover, chaperones

C

441

7.29

Energy production and conversion

G

418

6.82

Carbohydrate transport and metabolism

E

643

10.49

Amino acid transport and metabolism

F

94

1.53

Nucleotide transport and metabolism

H

322

5.25

Coenzyme transport and metabolism

I

387

6.31

Lipid transport and metabolism

P

361

5.89

Inorganic ion transport and metabolism

Q

261

4.26

Secondary metabolite biosynthesis, transport and catabolism

R

667

10.88

General function prediction only

S

360

5.87

Function unknown

-

3,188

37.51

Not in COGS

Conclusions

Bradyrhizobium sp. WSM1253 was isolated from a nodule of the herbaceous annual legume Ornithopus compressus that was collected on the Greek Island of Sifnos. WSM1253 is rather unusual for a Bradyrhizobium strain in that it is highly efficient in nitrogen fixation for many species of Lupinus and Ornithopus , including L. angustifolius, L. princei, L. atlanticus, L. pilosus, O. compressus, O. sativus Brot. and O. pinnatus.

Phylogenetic analysis revealed that WSM1253 is most closely related to Bradyrhizobium sp. WSM1417. Strain WSM1417 was obtained from a Lupinus sp. nodule from Chile and differs from WSM1253 in that it cannot form an effective nitrogen-fixing symbiosis with L. angustifolius. The genomes of both of these strains have now been sequenced and this brings the total number of Bradyrhizobium genome depositions in IMG to 54; of these, strains which can symbiotically fix nitrogen have the nitrogenase-RXN MetaCyc pathway that is characterized by the multiprotein nitrogenase complex. However, strain WSM1253 is unique amongst these in that it can effectively fix nitrogen with many species of Lupinus (including L. angustifolius, L. princei, L. atlanticus, L. pilosus) and Ornithopus compressus . The genome attributes of Bradyrhizobium sp. WSM1253, in conjunction with other Bradyrhizobium genomes, will be important resources with which to build an understanding of interactions required for the successful establishment of effective symbioses with different species of Lupinus and Ornithopus .

Abbreviations

½LA: 

half strength Lupin Agar

YMA: 

Yeast Mannitol Agar

TY: 

Tryptone Yeast extract Agar

GOLD: 

Genomes OnLine Database

CTAB: 

Cetyl trimethyl ammonium bromide

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 funding received from the Australian Government for an Australia India Senior Visiting Fellowship for Ravi Tiwari.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

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

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