Open Access

Complete genome sequence of Mesorhizobium ciceri bv. biserrulae type strain (WSM1271T)

  • Kemanthi Nandasena1,
  • Ron Yates1, 5,
  • Ravi Tiwari1,
  • Graham O’Hara1,
  • John Howieson1,
  • Mohamed Ninawi1,
  • Olga Chertkov2,
  • Chris Detter2,
  • Roxanne Tapia2,
  • Shunseng Han2,
  • Tanja Woyke3,
  • Sam Pitluck3,
  • Matt Nolan3,
  • Miriam Land4,
  • Konstantinos Liolios3,
  • Amrita Pati3,
  • Alex Copeland3,
  • Nikos Kyrpides3,
  • Natalia Ivanova3,
  • Lynne Goodwin3,
  • Uma Meenakshi1 and
  • Wayne Reeve1Email author
Standards in Genomic Sciences20149:9030462

DOI: 10.4056/sigs.4458283

Published: 15 June 2014

Abstract

Mesorhizobium ciceri bv. biserrulae strain WSM1271T was isolated from root nodules of the pasture legume Biserrula pelecinus growing in the Mediterranean basin. Previous studies have shown this aerobic, motile, Gram negative, non-spore-forming rod preferably nodulates B. pelecinus — a legume with many beneficial agronomic attributes for sustainable agriculture in Australia. We describe the genome of Mesorhizobium ciceri bv. biserrulae strain WSM1271T consisting of a 6,264,489 bp chromosome and a 425,539 bp plasmid that together encode 6,470 protein-coding genes and 61 RNA-only encoding genes.

Keywords

root-nodule bacteria nitrogen fixation evolution lateral gene transfer integrative and conjugative elements symbiosis Alphaproteobacteria

Introduction

The productivity of sustainable agriculture around the world is heavily dependent on the provision of bioavailable nitrogen (N) [1]. The demand for N by non-leguminous and leguminous plants can be supplied by the application of chemically synthesized nitrogenous fertilizer onto crops and pastures. However, the production of fertilizer is costly and requires the burning of fossil fuels in the manufacturing process which increases greenhouse gas emissions. Furthermore, high application rates of fertilizer can contaminate ecosystems and waterways, and result in leaching into the environment.

In contrast, the demand for N by leguminous plants can be sustainably met through the biological process of N fixation that occurs following the successful formation of an effective symbiosis. This symbiotic nitrogen fixation (SNF) process can account for approximately 70% of the bioavailable nitrogen supplied to legumes [1].

One legume that has many beneficial agronomic attributes is Biserrula pelecinus L., which is an annual herbaceous legume native to the Mediterranean basin that was introduced into Australian soil in 1994 [2]. The beneficial agronomic attributes of this legume include drought tolerance, hard seed production, easy harvesting characteristics, insect tolerance and most importantly, a capacity to grow well in the acidic duplex soils of Australia [2,3]. This monospecific legume specifically forms an effective nitrogen fixing symbiosis with the root nodule bacterium Mesorhizobium ciceri bv. biserrulae type strain WSM1271T (= LMG23838 = HAMBI2942) [4,5]. Australian indigenous rhizobial populations were found to be incapable of nodulating B. pelecinus L [2]. However, within six years of the introduction of the inoculant into Australia, the in situ evolution of a diverse range of competitive strains capable of nodulating B. pelecinus L. compromised optimal N2-fixation with this host. This rapid emergence of less effective strains threatens the establishment of this legume species in the Australian agricultural setting. The sub-optimal strains appear to have evolved from indigenous mesorhizobia that acquired the island of genes associated with symbiosis from the original inoculant, WSM1271T, following a horizontal gene transfer event [6].

In this report, a summary classification and a set of general features for M. ciceri bv. biserrulae strain WSM1271T are presented together with the description of the complete genome sequence and its annotation.

Classification and features

M. ciceri strain WSM1271T is a motile, Gram-negative, non-spore-forming rod (Figure 1 and Figure 2) in the order Rhizobiales of the class Alphaproteobacteria. They are moderately fast growing, forming 2–4 mm diameter colonies within 3–4 days, and have a mean generation time of 4–6 h when grown in half Lupin Agar (½LA) broth [7] at 28 °C. Colonies on ½LA are white-opaque, slightly domed, moderately mucoid with smooth margins (Figure 3).
Figure 1.

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using scanning electron microscopy.

Figure 2.

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using transmission electron microscopy.

Figure 3.

Image of Mesorhizobium ciceri bv. biserrulae strain WSM1271T using the appearance of colony morphology on solid media.

The organism tolerates a pH range between 5.5 and 9.0. Carbon source utilization and fatty acid profiles have been described before [6]. Minimum Information about the Genome Sequence (MIGS) is provided in Table 1
Table 1.

Classification and features of Mesorhizobium ciceri bv. biserrulae strain WSM1271T according to the MIGS recommendations [8,9].

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [9]

 

Phylum Proteobacteria

TAS [10]

 

Class Alphaproteobacteria

TAS [11,12]

 

Order Rhizobiales

TAS [11,13]

 

Family Phyllobacteriaceae

TAS [11,14]

 

Genus Mesorhizobium

TAS [15]

 

Species Mesorhizobium ciceri bv biserrulae

TAS [15]

 

Gram stain

Negative

TAS [6]

 

Cell shape

Rod

TAS [6]

 

Motility

Motile

TAS [6]

 

Sporulation

Non-sporulating

TAS [16]

 

Temperature range

Mesophile

TAS [16]

 

Optimum temperature

28°C

TAS [6]

 

Salinity

Unknown

NAS

MIGS-22

Oxygen requirement

Aerobic

TAS [16]

 

Carbon source

Arabinose, β-gentibiose, glucose, mannitol & melibiose

TAS [6]

 

Energy source

Chemoorganotroph

TAS [16]

MIGS-6

Habitat

Soil, root nodule, host

TAS [6]

MIGS-15

Biotic relationship

Free living, Symbiotic

TAS [6]

MIGS-14

Pathogenicity

None

NAS

 

Biosafety level

1

TAS [17]

 

Isolation

Root nodule

TAS [5,6]

MIGS-4

Geographic location

5 km before Bottida, Sardinia

TAS [2,5]

MIGS-5

Nodule collection date

April 1993

TAS [4]

MIGS-4.1

Longitude

9.012008

NAS

MIGS-4.2

Latitude

40.382709

NAS

MIGS-4.3

Depth

10 cm

NAS

MIGS-4.4

Altitude

295 m

TAS [5]

Evidence codes - 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). Evidence codes are from the Gene Ontology project [18].

Figure 4 shows the phylogenetic neighborhood of M. ciceri bv. biserrulae strain WSM1271T in a 16S rRNA sequence based tree. This strain clustered in a tight group, which included M. australicum, M. ciceri, M. loti and M. shangrilense and had >99% sequence identity with all four type strains. Our polyphasic taxonomic study indicates that WSM1271T is a new biovar of nodulating bacteria [5].
Figure 4.

Phylogenetic tree showing the relationships of Mesorhizobium ciceri bv. biserrulae WSM1271T (shown in bold print) with root nodule bacteria in the order Rhizobiales based on aligned sequences of the 16S rRNA gene (1,290 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA [19]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model. Bootstrap analysis [20] was performed with 500 replicates 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 [21]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

M. ciceri bv. biserrulae strain WSM1271T has an extremely narrow legume host range for symbiosis only forming highly effective nitrogen-fixing root nodules on Biserrula pelecinus. L. This strain also nodulates the closely related species Astragalus membranaceus but does not nodulate 21 other legume species nodulated by Mesorhizobium spp [5]. The high degree of specificity in the symbiotic relationships of this strain is representative of root nodule bacteria isolated from B. pelecinus L. growing in undisturbed landscapes in the Mediterranean basin, and is an important example of a highly specific legume host-root nodule bacteria relationship in an annual herbaceous legume used as a forage species in agriculture.

Genome sequencing and annotation

Genome project history

The Joint Genome Institute (JGI) operated by US Department of Energy (DOE) sequenced, finished and annotated WSM1271 as part of the Community Sequencing Program (CSP). The genome project is deposited in the Genomes OnLine Database [21]. The finished genome sequence is in GenBank. The CSP selects projects on the basis of environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance. Table 2 summarizes the project information.
Table 2.

Genome sequencing project information for Mesorhizobium ciceri bv. biserrulae strain WSM1271T

MIGS ID

Property

Term

MIGS-31

Finishing quality

Finished

MIGS-28

Libraries used

Illumina GAii shotgun library, 454 Titanium standard library and paired end 454 libraries

MIGS-29

Sequencing platforms

Illumina and 454 technologies

MIGS-31.2

Sequencing coverage

454 (26.8x) and Illumina (124x)

MIGS-30

Assemblers

Newbler, version 2.3 and Velvet version 0.7.63, PHRAP and CONSED

MIGS-32

Gene calling method

Prodigal, GenePrimp

 

Genbank ID

CP002447, CP002448

 

Genbank Date of Release

November 10, 2012

 

GOLD ID

Gc01578

 

NCBI project ID

48991

 

Database: IMG

649633066

 

Project relevance

Symbiotic nitrogen fixation, agriculture

Growth conditions and DNA isolation

M. ciceri bv. biserrulae strain WSM1271T was grown to mid logarithmic phase in TY rich medium [22] on a gyratory shaker at 28 °C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [23].

Genome sequencing and assembly

The Joint Genome Institute (JGI) generated the draft genome of M. ciceri bv. biserrulae WSM1271T using a combination of Illumina [24] and 454 technologies [25]. The sequencing of an Illumina GAii shotgun library generated 23,461,369 reads totaling 844.6 Mb, a 454 Titanium standard library which generated 277,881 reads and a paired end 454 libraries with average insert size of 1.137 +/− 2.842 Kb and 4.378 +/− 1.094 kb which generated 40,653 and 130,843 reads totaling 244.0 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [23]. The initial draft assembly contained 32 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 0.7.63 [26], and the consensus sequences were computationally shredded into 1.5 Kb 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 [2729] 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 [30], 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 49 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total size of the genome is 6,890,027 bp and the final assembly is based on 112.0 Mb of 454 draft data which provides an average 26.8× coverage of the genome and 832.1 Mb of Illumina draft data which provides an average 124× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePrimp pipeline [32]. 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 assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [33], RNAMMer [34], Rfam [35], TMHMM [36], and SignalP [37]. Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform [38].

Genome properties

The genome is 6,690,028 bp long with a 62.56% GC content (Table 3) and comprises a single chromosome and a single plasmid. From a total of 6,531 genes, 6,470 were protein encoding and 61 RNA only encoding genes. Within the genome, 206 pseudogenes were also identified. The majority of genes (70.74%) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4, and Figures 5, 6 and 7.
Figure 5.

Graphical circular map of the chromosome. 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 6.

Graphical circular map of the plasmid of Mesorhizobium ciceri bv. biserrulae WSM1271T. 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 7.

Color code for Figure 5 and 6.

Table 3.

Genome Statistics for Mesorhizobium ciceri bv. biserrulae strain WSM1271T.

Attribute

Value

% of Total

Genome size (bp)

6,690,028

100.00

DNA coding region (bp)

5,791,860

86.57

DNA G+C content (bp)

4,185,397

62.56

Number of replicons

2

 

Extrachromosomal elements

1

 

Total genes

6,531

100.00

RNA genes

61

0.93

Protein-coding genes

6,470

99.07

Genes with function prediction

4,620

70.74

Genes assigned to COGs

5174

79.22

Genes assigned Pfam domains

5398

82.65

Genes with signal peptides

597

9.14

Genes with transmembrane helices

1528

23.40

Table 4.

Number of protein coding genes of Mesorhizobium ciceri bv. biserrulae WSM1271T associated with the general COG functional categories.

Code

Value

%age

COG Category

J

193

3.35

Translation, ribosomal structure and biogenesis

A

1

0.02

RNA processing and modification

K

492

8.53

Transcription

L

156

2.71

Replication, recombination and repair

B

6

0.10

Chromatin structure and dynamics

D

35

0.61

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

63

1.09

Defense mechanisms

T

238

4.13

Signal transduction mechanisms

M

290

5.03

Cell wall/membrane biogenesis

N

62

1.08

Cell motility

Z

0

0.00

Cytoskeleton

W

2

0.03

Extracellular structures

U

124

2.15

Intracellular trafficking and secretion

O

185

3.21

Posttranslational modification, protein turnover, chaperones

C

356

6.17

Energy production conversion

G

535

9.28

Carbohydrate transport and metabolism

E

732

12.70

Amino acid transport metabolism

F

92

1.60

Nucleotide transport and metabolism

H

204

3.54

Coenzyme transport and metabolism

I

235

4.08

Lipid transport and metabolism

P

274

4.75

Inorganic ion transport and metabolism

Q

175

3.04

Secondary metabolite biosynthesis, transport and catabolism

R

731

12.68

General function prediction only

S

585

10.15

Function unknown

-

1,357

20.78

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 Australian Research Council Discovery grant (DP0880896), Murdoch University Strategic Research Fund through the Crop and Plant Research Institute (CaPRI) and the Centre for Rhizobium Studies (CRS) at Murdoch University. 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)
Bioscience Division, Los Alamos National Laboratory
(3)
DOE Joint Genome Institute
(4)
Oak Ridge National Laborator
(5)
Department of Agriculture and Food

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Copyright

© The Author(s) 2014