Open Access

Genome sequence of Ensifer sp. TW10; a Tephrosia wallichii (Biyani) microsymbiont native to the Indian Thar Desert

  • Nisha Tak1,
  • Hukam S. Gehlot1,
  • Muskan Kaushik1,
  • Sunil Choudhary1,
  • Ravi Tiwari2,
  • Rui Tian2,
  • Yvette Hill2,
  • Lambert Bräu3,
  • Lynne Goodwin4,
  • James Han5,
  • Konstantinos Liolios5,
  • Marcel Huntemann5,
  • Krishna Palaniappan6,
  • Amrita Pati5,
  • Konstantinos Mavromatis5,
  • Natalia Ivanova5,
  • Victor Markowitz6,
  • Tanja Woyke5,
  • Nikos Kyrpides5 and
  • Wayne Reeve2Email author
Standards in Genomic Sciences20139:9020304

DOI: 10.4056/sigs.4598281

Published: 20 December 2013

Abstract

Ensifer sp. TW10 is a novel N2-fixing bacterium isolated from a root nodule of the perennial legume Tephrosia wallichii Graham (known locally as Biyani) found in the Great Indian (or Thar) desert, a large arid region in the northwestern part of the Indian subcontinent. Strain TW10 is a Gram-negative, rod shaped, aerobic, motile, non-spore forming, species of root nodule bacteria (RNB) that promiscuously nodulates legumes in Thar Desert alkaline soil. It is fast growing, acid-producing, and tolerates up to 2% NaCl and capable of growth at 40oC. In this report we describe for the first time the primary features of this Thar Desert soil saprophyte together with genome sequence information and annotation. The 6,802,256 bp genome has a GC content of 62% and is arranged into 57 scaffolds containing 6,470 protein-coding genes, 73 RNA genes and a single rRNA operon. This genome is one of 100 RNB 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 Great Indian (or Thar) Desert is a large, hot, arid region in the northwestern part of the Indian subcontinent. It is the 18th largest desert in the world covering 200,000 square km with 61% of its landmass occupying Western Rajasthan. The landscape occurs at low altitude (<1500 m above sea level) and extends from India into the neighboring country of Pakistan [1]. The Thar Desert region is characterized by low annual precipitation (50 to 300 mm), high thermal load and alkaline soils that are poor in texture and fertility [2]. Despite these harsh conditions, the Thar Desert has very rich plant diversity in comparison to other desert landscapes [3]. Approximately a quarter of the plants in the Thar Desert are used to provide animal fodder or food, fuel, medicine or shelter for local inhabitants [4].

The Indian Thar desert harbors several native and exotic plants of the Leguminoseae family [2] including native legume members of the sub-families Caesalpinioideae, Mimosoideae and Papilionoideae that have adapted to the harsh Thar desert environment [5]. The Papilionoid genus Tephrosia can be found throughout this semi-arid to arid environment and these plants are among the first to grow after monsoonal rains. The generic name is derived from the Greek word “tephros” meaning “ash-gray” since dense trichomes on the leaves provide a greyish tint to the plant. Many species within this genus produce the potent toxin rotenone, which historically has been used to poison fish. It is a perennial shrub that has adapted to the harsh desert conditions by producing a long tap root system and dormant auxillary shoot buds.

Recently, the root nodule bacteria (RNB) microsymbionts capable of fixing nitrogen in symbiotic associations with Tephrosia have been characterized [5]. Both Bradyrhizobium and Ensifer were present within nodules, but a particularly high incidence of Ensifer was noted [5]. Ensifer was found to occupy the nodules of all four species of Tephrosia examined [5]. Here we present a preliminary description of the general features of the T. wallichii (Biyani) microsymbiont Ensifer sp. TW10 together with its genome sequence and annotation.

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 1 shows the phylogenetic neighborhood of Ensifer sp. strain TW10 in a 16S rRNA sequence based tree. This strain has 99% sequence identity at the 16S rRNA sequence level to E. kostiense LMG 19227 and 100% 16S rRNA sequence identity to other Indian Thar Desert Ensifer species (JNVU IC18 from a nodule of Indigofera and JNVU TF7, JNVU TP6 and TW8 from nodules of Tephrosia).
Figure 1.

Phylogenetic tree showing the relationship of Ensifer sp. TW10 (shown in bold print) to other Ensifer spp. 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, version 5 [19]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [20]. Bootstrap analysis [21] 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 [22]. Published genomes are indicated with an asterisk.

Table 1.

Classification and general features of Ensifer sp. TW10 according to the MIGS recommendations [6]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [7]

 

Phylum Proteobacteria

TAS [8]

 

Class Alphaproteobacteria

TAS [9,10]

 

Order Rhizobiales

TAS [10,11]

 

Family Rhizobiaceae

TAS [12,13]

 

Genus Ensifer

TAS [1416]

 

Species Ensifer sp.

IDA

 

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 [5]

 

Carbon source

Varied

NAS

 

Energy source

Chemoorganotroph

NAS

MIGS-6

Habitat

Soil, root nodule, on host

TAS [5]

MIGS-15

Biotic relationship

Free living, symbiotic

TAS [5]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

 

Biosafety level

1

TAS [17]

 

Isolation

Root nodule of Tephrosia wallichii

TAS [5]

MIGS-4

Geographic location

Jodhpur, Indian Thar Desert

TAS [5]

MIGS-5

Soil collection date

Oct, 2009

IDA

MIGS-4.1

Longitude

73.021177

IDA

MIGS-4.2

Latitude

26.27061

IDA

MIGS-4.3

Depth

15cm

 

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 [18].

Classification and general features

Ensifer sp. strain TW10 is a Gram-negative rod (Figure 2, and Figure 3) in the order Rhizobiales of the class Alphaproteobacteria. It is fast growing, forming white-opaque, slightly domed and moderately mucoid colonies with smooth margins within 3–4 days at 28°C when grown on YMA [23].
Figure 2.

Image of Ensifer sp. TW10 using scanning electron microscopy.

Figure 3.

Image of Ensifer sp. TW10 using transmission electron microscopy.

Symbiotaxonomy

Ensifer sp. TW10 has the ability to nodulate (Nod+) and fix nitrogen (Fix+) effectively with a wide range of perennial native (wild) legumes of Thar Desert origin and with species of crop legumes (Table 2). Ensifer sp. TW10 is symbiotically competent with these species when grown in alkaline soils. TW10 can nodulate the wild tree legume Prosopis cineraria of the Mimosoideae subfamily. However, it does not form nodules on the Mimosoid hosts Mimosa hamata and M. himalayana even though these hosts are known to be nodulated by Ensifer species [5,24]. TW10 was not compatible with the host Phaseolus vulgaris, a legume of the Phaseolae tribe.
Table 2.

Compatibility of Ensifer sp. TW10 with different wild and cultivated legume species

Species Name

Family

Wild/Cultivar

Common Name

Habit/Growth Type

Nod

Fix

Tephrosia falciformis Ramaswami

Papilionoideae

Wild

Rati biyani

Under-shrub Perennial

+

+

Tephrosia purpurea(L.) Pers. sub sp.leptostachya DC.

Papilionoideae

Wild

-

Herb Annual/Perennial

+

+

Tephrosia purpurea (L.) Pers. sub sp.purpurea (L.) Pers

Papilionoideae

Wild

Biyani, Sarphanko

Herb Annual/Perennial

+

+

Tephrosia villosa (Linn.) Pres.

Papilionoideae

Wild

Ruvali-biyani

Herb Annual/Perennial

+

+

Prosopis cineraria(Linn.) Druce.

Mimosoideae

Wild/Cultivar

Khejari

Tree Perennial

+

+

Mimosa hamata Willd.

Mimosoideae

Wild

Jinjani, Jinjanio

Shrub Perennial

M. himalayana Gamble

Mimosoideae

Wild

Hajeru

Shrub Perennial

Vigna radiata (L.) Wilczek

Papilionoideae

Cultivar

Moong bean

Annual

+

+

Vigna aconitifolia(Jacq.) Marechal

Papilionoideae

Cultivar

Moth bean

Annual

+

+

Vigna unguiculata(L.) Walp.

Papilionoideae

Cultivar

Cowpea

Annual

+

+

Macroptilium atropurpureum(DC.) Urb.

Papilionoideae

Cultivar

Siratro

Annual

+

+

Phaseolus vulgarisL.

Papilionoideae

Cultivar

Common bean

Annual

Nod: “+” means nodulation observed, “−” means no nodulation

Fix: “+” means fixation observed, “−” means no fixation

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 [22] and standard 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.

Genome sequencing project information for Ensifer sp. strain TW10.

MIGS ID

Property

Term

MIGS-31

Finishing quality

Standard draft

MIGS-28

Libraries used

1× Illumina library

MIGS-29

Sequencing platforms

Illumina HiSeq2000

MIGS-31.2

Sequencing coverage

330× Illumina

MIGS-30

Assemblers

Allpaths, LG version r42328, Velvet 1.1.04

MIGS-32

Gene calling methods

Prodigal 1.4,

 

GenBank

pending

 

Genbank Date of Release

pending

 

GOLD ID

Gi08835

 

NCBI project ID

210334

 

Database: IMG

2509276019

 

Project relevance

Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

Ensifer sp. TW10 was cultured to mid logarithmic phase in 60 ml of TY rich medium [25] 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 [26].

Genome sequencing and assembly

The genome of Ensifer sp. TW10 was generated at the Joint Genome Institute (JGI) using Illumina [27] technology. An Illumina std shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 14,938,244 reads totaling 2,241 Mbp.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [26]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, Copeland, A, and Han, J, unpublished).

The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [28] (version 1.1.04), (2) 1–3 kb simulated paired end reads were created from Velvet contigs using wgsim (https://github.com/lh3/wgsim), and (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG (version r42328) [29]. Parameters for assembly steps were: 1) Velvet (velveth: 63 -shortPaired and velvetg: -veryclean yes -exportFiltered yes -mincontiglgth 500 -scaffolding no-covcutoff 10) 2) wgsim (-e 0 -1 100 -2 100 -r 0 -R 0 -X 0) 3) Allpaths-LG (PrepareAllpathsInputs:PHRED64=1 PLOIDY=1 FRAGCOVERAGE=125 JUMPCOVERAGE=25 LONGJUMPCOV=50, RunAllpath-sLG: THREADS=8 RUN=stdshredpairs TARGETS=standard VAPIWARNONLY=True OVERWRITE=True). The final draft assembly contained 57 contigs in 57 scaffolds. The total size of the genome is 6.8 Mbp and the final assembly is based on 2241Mbp of Illumina data, which provides an average 330× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [30] as part of the DOE-JGI annotation pipeline [31]. 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. The tRNAScanSE tool [7] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [32]. 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 [33]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform) [34,35].

Genome properties

The genome is 6,802,256 nucleotides with 61.56% GC content (Table 4) and comprised of 57 scaffolds (Figure 4) of 57 contigs. From a total of 6,546 genes, 6,473 were protein encoding and 73 RNA only encoding genes. The majority of genes (77.44%) 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 5.
Figure 4.

Graphical map of five of the largest scaffolds from the genome of Ensifer sp. TW10. 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 4.

Genome Statistics for Ensifer sp. TW10

Attribute

Value

% of Total

Genome size (bp)

6,802,256

100.00

DNA coding region (bp)

5,800,968

85.28

DNA G+C content (bp)

4,187,461

61.56

Number of scaffolds

57

 

Number of contigs

57

 

Total gene

6,546

100.00

RNA genes

73

1.12

rRNA operons

1

 

Protein-coding genes

6,473

98.88

Genes with function prediction

5,069

77.44

Genes assigned to COGs

5,069

77.44

Genes assigned Pfam domains

5,282

80.69

Genes with signal peptides

539

8.23

Genes with transmembrane helices

1,419

21.68

Table 5.

Number of protein coding genes of Ensifer sp. TW10 associated with the general COG functional categories.

Code

Value

%age

Description

J

198

3.55

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

481

8.61

Transcription

L

237

4.24

Replication, recombination and repair

B

3

0.05

Chromatin structure and dynamics

D

37

0.66

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

66

1.18

Defense mechanisms

T

262

4.69

Signal transduction mechanisms

M

298

5.34

Cell wall/membrane biogenesis

N

77

1.38

Cell motility

Z

0

0.00

Cytoskeleton

W

1

0.02

Extracellular structures

U

132

2.36

Intracellular trafficking and secretion

O

192

3.44

Posttranslational modification, protein turnover, chaperones

C

322

5.77

Energy production conversion

G

538

9.63

Carbohydrate transport and metabolism

E

606

10.85

Amino acid transport metabolism

F

96

1.72

Nucleotide transport and metabolism

H

194

3.47

Coenzyme transport and metabolism

I

199

3.56

Lipid transport and metabolism

P

251

4.49

Inorganic ion transport and metabolism

Q

139

2.49

Secondary metabolite biosynthesis, transport and catabolism

R

678

12.14

General function prediction only

S

578

10.35

Function unknown

-

1,477

22.56

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 funding received from the Murdoch University Strategic Research Fund through the Crop and Plant Research Institute (CaPRI), the GRDC National Rhizobium Program (UMU00032), the Council of Scientific and Industrial Research (CSIR) for a fellowship for Nisha Tak, the Department of Biotechnology (India) for a research grant (BT/PR11461/AGR/21/270/2008) and the Commonwealth of Australia for an Australia India Senior Visiting Fellowship for Ravi Tiwari.

Authors’ Affiliations

(1)
BNF and Stress Biology Lab, Department of Botany, JNV University
(2)
Centre for Rhizobium Studies, Murdoch University
(3)
School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University
(4)
Bioscience Division, Los Alamos National Laboratory
(5)
DOE Joint Genome Institute
(6)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory

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Copyright

© The Author(s) 2013