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
© The Author(s) 2013
Published: 20 December 2013
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.
Keywordsroot-nodule bacteria nitrogen fixation rhizobia Alphaproteobacteria
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 . 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 . Despite these harsh conditions, the Thar Desert has very rich plant diversity in comparison to other desert landscapes . 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 .
The Indian Thar desert harbors several native and exotic plants of the Leguminoseae family  including native legume members of the sub-families Caesalpinioideae, Mimosoideae and Papilionoideae that have adapted to the harsh Thar desert environment . 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 . Both Bradyrhizobium and Ensifer were present within nodules, but a particularly high incidence of Ensifer was noted . Ensifer was found to occupy the nodules of all four species of Tephrosia examined . 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.
Classification and general features of Ensifer sp. TW10 according to the MIGS recommendations 
Species Ensifer sp.
Soil, root nodule, on host
Free living, symbiotic
Root nodule of Tephrosia wallichii
Jodhpur, Indian Thar Desert
Soil collection date
Classification and general features
Compatibility of Ensifer sp. TW10 with different wild and cultivated legume species
Tephrosia falciformis Ramaswami
Tephrosia purpurea(L.) Pers. sub sp.leptostachya DC.
Tephrosia purpurea (L.) Pers. sub sp.purpurea (L.) Pers
Tephrosia villosa (Linn.) Pres.
Prosopis cineraria(Linn.) Druce.
Mimosa hamata Willd.
M. himalayana Gamble
Vigna radiata (L.) Wilczek
Vigna aconitifolia(Jacq.) Marechal
Vigna unguiculata(L.) Walp.
Macroptilium atropurpureum(DC.) Urb.
Genome sequencing and annotation
Genome project history
Genome sequencing project information for Ensifer sp. strain TW10.
1× Illumina library
Allpaths, LG version r42328, Velvet 1.1.04
Gene calling methods
Genbank Date of Release
NCBI project ID
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  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 .
Genome sequencing and assembly
The genome of Ensifer sp. TW10 was generated at the Joint Genome Institute (JGI) using Illumina  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 . 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  (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) . 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.
Genes were identified using Prodigal  as part of the DOE-JGI annotation pipeline . 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  was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA . 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 . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform) [34,35].
Genome Statistics for Ensifer sp. TW10
% of Total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of scaffolds
Number of contigs
Genes with function prediction
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of protein coding genes of Ensifer sp. TW10 associated with the general COG functional categories.
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production conversion
Carbohydrate transport and metabolism
Amino acid transport metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolite biosynthesis, transport and catabolism
General function prediction only
Not in COGS
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.
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