Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E
- Li Lin†1,
- Chunyan Wei†2,
- Mingyue Chen†3,
- Hongcheng Wang3,
- Yuanyuan Li3,
- Yangrui Li1, 2,
- Litao Yang1, 2, 4Email author and
- Qianli An3Email author
© Lin et al.; licensee BioMed Central. 2015
Received: 14 January 2015
Accepted: 10 April 2015
Published: 8 May 2015
Klebsiella variicola strain DX120E (=CGMCC 1.14935) is an endophytic nitrogen-fixing bacterium isolated from sugarcane crops grown in Guangxi, China and promotes sugarcane growth. Here we summarize the features of the strain DX120E and describe its complete genome sequence. The genome contains one circular chromosome and two plasmids, and contains 5,718,434 nucleotides with 57.1% GC content, 5,172 protein-coding genes, 25 rRNA genes, 87 tRNA genes, 7 ncRNA genes, 25 pseudo genes, and 2 CRISPR repeats.
The species Klebsiella variicola was classified in 2004 and consisted of clinical and plant-associated isolates .The species K. singaporensis was classified in 2004 based on a single soil isolate  and was recently identified as a later junior heterotypic synonym of K. variicola . K. variicola is able to fix N2 . K. variicola strain At-22, one of the dominant bacteria in the fungus gardens of leaf-cutter ants, provides nitrogen source by N2 fixation  and carbon source by degrading leaf polymers to the ant-fungus symbiotic system . Former K. pneumoniae strain 342 (Kp342), which is phylogenomically close to strain At-22 [6,7] and has been identified as a strain of K. variicola , is able to colonize in plants and to provide small but critical amounts of fixed nitrogen to plant hosts .
K. variicola strain DX120E was isolated from roots of sugarcane grown in Guangxi, the major sugarcane production area in China . It is able to colonize in sugarcane roots and shoots, to fix N2 in association with sugarcane plants, and to promote sugarcane growth , and thus shows a potential as a biofertilizer. Here we present a summary of the features of the K. variicola strain DX120E (=CGMCC 1.14935) and its complete genome sequence, and thus provide a genetic background to understand its endophytic lifestyle, plant growth-promoting potentials, and similarities and differences to other plant-associated and clinical K. variicola isolates.
Classification and general features
Like typical members in the genera Klebsiella , K. variicola DX120E utilizes alanine, arabinose, D-arabitol, L-aspartate, D-cellobiose, citrate, D-fructose, L-fucose, D-galactose, gentiobiose, glucose, glycerol, myo-inositol, lactate, lactose, malate, maltose, D-mannitol, D-mannose, D-melibiose, L-proline, D-raffinose, L-rhamnose, L-serine, D-sorbitol, sucrose, and D-trehalose . DX120E does not utilize adonitol (also known as ribitol), which is a distinctive characteristic from K. pneumoniae .
Genome sequencing information
Genome project history
Classification and general features of Klebsiella variicola strain DX120E according to the MIGS recommendations 
Evidence code a
Species Klebsiella variicola
Type strain:F2R9T (ATCC BAA-830 = DSM 15968)
pH range; Optimum
Sucrose, citrate, fructose, galactose, glucose, lactose, malate, maltose, mannitol, mannose, rhamnose, & sorbitol
0 – 5% NaCl (w/v)
Daxin, Guangxi, China
0.1 – 0.2 m below the surface
Genome sequencing project information for Klebsiella variicola strain DX120E
PacBio 4 –10Kb library
Illumina 500 bp library
PacBio RS II
Illumina HiSeq 2000
PacBio 96 ×
Illumina 106 ×
HGAP in smrtanalysis-2.1.1SOAPdenovo 2.04
Gene calling method
CP009275 (plasmid pKV1)
CP009276 (plasmid pKV2)
Genbank Date of Release
January 1, 2015
Source Material Identifier
Agriculture, plant-microbe interactions
Growth conditions and DNA isolation
K. variicola DX120E was grown in liquid Luria-Bertani (LB) medium at 30°C to early stationary phase. The genome DNA was extracted from the cells by using a TIANamp bacterial DNA kit (Tiangen Biotech, Beijing, China). DNA quality and quantity were determined with a Nanodrop spectrometer (Thermo Scientific, Wilmington, USA).
Genome sequencing and assembly
The genome DNA of K. variicola DX120E was constructed into a 4 – 10 kb insert library and sequenced by the Pacific Biosciences’ (PacBio) Single Molecule, Real-Time (SMRT) sequencing technology  at the Duke University Genome Sequencing & Analysis Core Resource. Sequencing was run on single SMRT cell and resulted in 91,190 high-quality filtered reads with an average length of 6,196 bp. High-quality read bases were assembled by the Hierarchical Genome Assembly Process (HGAP) with smrtanalysis-2.1.1. The resulting draft genome consisted of 5,719,400 nucleotides and 5 contigs.
The genome DNA of K. variicola DX120E was also constructed into a 500-bp insert library and sequenced by an Illumina HiSeq 2000 sequencing system at BGI Tech, Shenzhen, China. The Illumina HiSeq 2000 sequencing resulted in 6,699,933 high-quality filtered reads with an average length of 90 bp. The sequencing data were assembled by the Short Oligonucleotide Analysis Package (SOAPdenovo 2.04) . The resulting draft genome consisted of 5,695,362 nucleotides and 27 scaffolds.
The two draft genomes were aligned by Mauve . The Illumina scaffold 1 bridged the PacBio contig 1 and contig 2; the Illumina scaffold 3 bridged the PacBio contig 1, contig 2, and contig 3; the Illumina scaffold 11 bridged the circular PacBio contig 4; the Illumina scaffold 16 bridged the circular PacBio contig 5. The genome sequencing was completed by PCR and Sanger sequencing to close the contig gaps of the PacBio-sequenced genome.
Automated genome annotation was completed by the NCBI Prokaryotic Genome Annotation Pipeline. Product description annotations were obtained by searching against the KEGG, InterPro, and COG databases. Genes with signal peptides were predicted by SignalP . Genes with transmembrane helices were predicted by TMHMM . Genes for tRNA were found by tRNAScanSE . Ribosomal RNAs were found by BLASTN vs. ribosomal RNA databases; 5S rRNA hits were further refined by Cmsearch . Thirteen disrupted genes were replaced by the complete gene sequences obtained from the Illumina HiSeq 2000 sequencing.
Summary of genome: one chromosome and two plasmids
% of total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosomepartitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights from the genome sequence
The genome of K. variicola DX120E contains genes contributing to multiple plant-beneficial functions. In accordance with previously detected N2 fixation, indole-3-acetic acid production, siderophore production, and phosphate solubilization , the genome of K. variicola DX120E contains nif cluster, indole-3-pyruvate decarboxylase, siderophore enterobactin synthesis genes (entABCDEF) and enterobactin exporter gene (entS), and pyrroloquinoline quinone synthesis genes (pqqBCDEF) contributing to these functions. Moreover, the genome of K. variicola DX120E contains the budABC operon for the synthesis of acetoin and 2,3-butanediol , and thus may induce plant systemic resistance to pathogens .
DX120E contains plasmids similar to those in Klebsiella relatives. The plasmid pKV1 is most similar to the plasmid pKp5-1 of the K. pneumoniae strain 5–1 (Kp5-1)  with a 97% identity of 56% coverage (Additional file 1: Figure S1); the similar regions mainly encode transposase/recombinases and proteins functioning in plasmid replication, partitioning, and conjugal transfer. The plasmid pKV2 is most similar to the plasmid pKOXM1C of the K. oxytoca strain M1 with a 96% identity of 89% coverage (Additional file 2: Figure S2); the similar regions mainly encode proteins for plasmid partitioning and phage functions.
The genome of K. variicola DX120E has high average nucleotide identities (ANI)  about 99% to the available genomes of K. variicola strains DSM 15968T, At-22, Bz19, and Kp342. Bz19 was isolated from faeces of a hospitalized patient . The plant-beneficial strain Kp342 is able to infect mouse organs, although it is less virulent than typical clinical K. pneumoniae isolates . Kp5-1, which has the plasmid pKp5-1 close to pKV1, is a cotton pathogen causing boll-rot disease . The genome of strain Kp5-1 has ANI values about 99% to the genomes of the known K. variicola strains and thus belongs to K. variicola . These drive concerns about potential pathogenicity of DX120E to animals and plants. Therefore, DX120E’s pathogenic potentials to animals and plants should be determined before using DX120E as a biofertilizer in the field.
The complete genome sequence of K. variicola DX120E provides the genetic background for understanding the bacterial mechanisms to adapt endophytic life and to promote plant growth. The high degree of whole-genome and plasmid similarities between DX120E and phytopathogenic and clinical Klebsiella isolates suggests the risk of using DX120E as a biofertilizer. The available genome sequences of the K. variicola strains allow an in-depth comparative analysis to understand the subtle pathogenicity mechanisms of the pathogens and to predict pathogenic risks for the plant-beneficial strain DX120E.
This work was supported by the National Natural Science Foundation of China (31171504 and 31240056), Guangxi Provincial Natural Science Foundation of China (2011GXNSFF018002 and 2013GXNSFBA019061), Science and Technology Development Foundation of Guangxi Academy of Agricultural Sciences (2013JZ11), Guangxi Special Funds for Bagui Scholars and Distinguished Experts (2013).
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