- Short genome report
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High quality draft genome sequence of the type strain of Pseudomonas lutea OK2T, a phosphate-solubilizing rhizospheric bacterium
Standards in Genomic Sciencesvolume 11, Article number: 51 (2016)
Pseudomonas lutea OK2T (=LMG 21974T, CECT 5822T) is the type strain of the species and was isolated from the rhizosphere of grass growing in Spain in 2003 based on its phosphate-solubilizing capacity. In order to identify the functional significance of phosphate solubilization in Pseudomonas Plant growth promoting rhizobacteria, we describe here the phenotypic characteristics of strain OK2T along with its high-quality draft genome sequence, its annotation, and analysis. The genome is comprised of 5,647,497 bp with 60.15 % G + C content. The sequence includes 4,846 protein-coding genes and 95 RNA genes.
Phosphorus, one of the major essential macronutrients for plant growth and development, is usually found in insufficient quantities in soil because of its low solubility and fixation [1, 2]. Since phosphorus deficiency in agricultural soil is limits plant growth, the release bound phosphorus from soils by microbes is an important aspect that can be used to improve soil fertility for increasing crop yields .
Phosphate-solubilizing microorganisms, a group of soil microorganisms capable of converting insoluble phosphate to soluble forms, have received attention as efficient bio-fertilizers for enhancing the phosphate availability for plants . As one of the representative phosphate-solubilizing bacteria , rhizosphere-colonizing pseudomonads are of interest owing to the benefits they offer to plants. Besides increasing the phosphate accessibility, they promote plant development by facilitating direct and indirect plant growth promotion through the production of phytohormones and enzymes or through the suppression of soil-borne diseases by inducing systemic resistance in the plants [5–7].
Pseudomonas lutea OK2T (=LMG 21974 T, CECT 5822 T) with insoluble phosphate-solubilizing activity was isolated from the rhizosphere of grass growing in northern Spain . Characteristics of the whole genome sequence and a brief summary of the phenotype for this type strain are presented in this study.
Classification and features
A 16S rRNA gene sequence of P. lutea OK2T was compared to those of other type strains of the genus Pseudomonas using BLAST on NCBI . The 16S rRNA gene sequence showed highest similarity (99 % identity) to that of P. graminis DSM 11363T , followed by similarity to the 16S rRNA gene sequence of P. rhizosphaerae IH5 T (98 % identity) , P. protegens CHA0 T (98 % identity) [12, 13], P. rhodesiae CIP 104664 T (97 % identity) , and P. argentinensis CH01 T (97 % identity) . Species showing full-length 16S rRNA gene sequences in BLAST analysis were considered for further phylogenetic analyses. A phylogenetic tree was constructed using the neighbor-joining method , and the bootstrap value was set as 1,000 times random replicate sampling. The consensus phylogenetic neighborhood of P. lutea OK2T within the genus Pseudomonas is shown in Fig. 1.
P. lutea OK2T is a motile, strictly aerobic, non-spore forming, gram-negative bacterium that belongs to the family Pseudomonadaceae of the class Gammaproteobacteria . The cells are rod-shaped with a diameter of approximately 0.75 μm and a length of 1.2–1.6 μm (Fig. 2). The strain produces yellow, translucent, circular convex colonies of 1–2 mm diameter on plates containing YED-P medium (per liter: 7.0 g of glucose, 3.0 g of yeast extract, 3.0 g of bicalcium phosphate, and 17.0 g of agar) within 2 days at 25 °C . P. lutea OK2T is capable of oxidizing glucose in media containing ammonium nitrate as a nitrogen source and hydrolyzes aesculin . The strain OK2T is positive for catalase, but negative for oxidase, gelatinase, caseinase, urease, β-galactosidase, arginine dehydrolase, tryptophan deaminase, and indole/H2S . Further, it can utilize galactose, ribose, mannose, glycerol, D-fructose, D-xylose, D-/L-arabinose, D-/L-arabitol, D-/L-fucose, L-lyxose, melibiose, inositol, mannitol, adonitol, xylitol, caprate, malate, gluconate, 2-ketogluconate, and citrate as sole carbon sources, but cannot utilize maltose, lactose, sucrose, trehalose, cellobiose, starch, glycogen, inulin, sorbitol, D-tagatose, D-raffinose, L-xylose, L-sorbose, L-rhamnose, N-acetylglucosamine, salicin, and erythritol . Unlike other pseuodomonads, the strain OK2T does not produce fluorescent pigments .
The important non-polar fatty acids present in P. lutea OK2T include hexadecenoic acid (16:1, 39.0 %), hexadecanoic acid (16:0, 29.0 %), and octadecenoic acid (18:1, 18.6 %). In addition, the strain OK2T has hydroxy fatty acids such as 3-hydroxydodecanoic acid (3-OH 12:0, 3.3 %), 2-hydroxydodecanoic acid (2-OH 12:0, 2.7 %), and 3-hydroxydecanoic acid (3-OH 10:0, 2.4 %) . The whole-cell fatty acid profile of this strain is similar to that observed in other representative strains of the genus Pseudomonas , such as P. graminis  and P. rhizosphaerae . The general characteristics of the strain are summarized in Table 1.
Genome sequencing information
Genome project history
P. lutea OK2T was selected as a novel-phosphate solubilizing strain for the genome-sequencing project of agriculturally useful microbes undertaken at Kyungpook National University. Genome sequencing was performed in September 2014, and the results of the Whole Genome Shotgun project have been deposited at DDBJ/EMBL/GenBank under the accession number JRMB00000000. The version described in this study is the first version, indicated as JRMB00000000.1. The information obtained from the genome sequencing project is registered on the Genome Online Database  with the GOLD Project ID Gp0107463. A summary of this information and its association with the Minimum Information about a Genome Sequence (MIGS) version 2.0 compliance  are presented in Table 2.
Growth conditions and genomic DNA preparation
The strain was cultured in tryptic soy broth (Difco Laboratories Inc., Detroit, MI) at 30 °C on a rotary shaker at 200 rpm. Genomic DNA was isolated using a QIAamp® DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's standard protocol. The quantity and purity of the extracted genomic DNA were assessed using a Picodrop Microliter UV/Vis Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) and Qubit® 2.0 Fluorometer (Fisher Scientific Inc., Pittsburgh, PA), respectively.
Genome sequencing and assembly
The isolated genomic DNA of P. lutea OK2T was sequenced using the SMRT DNA sequencing platform and the Pacific Biosciences RS II sequencer with P4 polymerase-C2 sequencing chemistry (Pacific Biosciences, Menlo Park, CA) . After shearing the genomic DNA, a 10-kb insert SMRT-bell library was prepared and loaded on two SMRT cells. During the 90 min of movie time, 654,270,150 read bases were generated from 300,584 reads. All the obtained bases were filtered to remove any reads shorter than 100 bp or those having accuracy values less than 0.8. Subsequently, 461,880,761 nucleotides were obtained from 116,562 reads, with a read quality of 0.843. These bases were assembled de novo using the RS HGAP assembly protocol version 3.3 on the SMRT analysis platform version 2.2.0 . The HGAP analysis yielded five contigs corresponding to five scaffolds, with a 67.58-fold coverage. The maximum contig length and N50 contig length were identical: 2,839,280 bp. The total length of the P. lutea OK2T genome was found to be 5,647,497 bp.
The protein coding sequences were determined using the NCBI PGAP version 2.8 (rev. 447021) . Additional gene prediction and functional annotation analyses were performed on the RAST server  and IMG-ER pipeline, respectively, by the Department of Energy-Joint Genome Institute .
The average G + C content of the genome was 60.15 %. The genome was predicted to encode 4,941 genes including 4,846 protein-coding genes and 95 RNA genes (24 rRNAs, 70 tRNAs, and 1 ncRNA). Putative functions were assigned to 4,102 of the protein-coding genes, and 3,507 genes (approximately 70.98 %) were assigned to the COG functional categories. The most abundant COG category was "Amino acid transport and metabolism" (10.36 %), followed by "General function prediction only" (8.71 %), “Transcription" (8.34 %), and “Signal transduction mechanisms” (6.52 %). The category for “Mobilome: prophages, transposons” (0.92 %) was also classified with functional genes for transposase (LT42_00515, LT42_05870, LT42_07855, LT42_10965, LT42_14240, LT42_14330, LT42_18595, LT42_19270, LT42_21870, LT42_21925), integrase (LT42_17205), terminase (LT42_06460, LT42_17145, LT42_17150), and plasmid stabilization protein (LT42_19025, LT42_24175). The genome statistics of strain OK2T are presented in Table 3 and Fig. 3. The gene distribution within the COG functional categories is presented in Table 4.
Insights from the genome sequence
Microorganisms that show phosphate-solubilizing activity are generally known to be involved in either of the following two biochemical mechanisms: production of organic acids for the acidification of external surroundings for plants and production of enzymes for direct solubilization [24, 25]. Genes encoding functional enzymes with these biochemical properties were predicted using the KO database via IMG-ER pipeline [26, 27]. The genome of P. lutea OK2T was annotated with several genes involved in phosphate solubilization. For example, ldhA (D-lactate dehydrogenase, KO:K03778) and icd (isocitrate dehydrogenase, KO:K00031) were found to be involved in the production of organic acids, and phoD (alkaline phosphatase D, KO:K01113) was involved in direct phosphate solubilization. Direct oxidation of glucose to gluconic acid by a periplasmic membrane-bound glucose dehydrogenase is also known to be one of the major metabolic steps for phosphate solubilization in pseudomonads . In relation to this process, the gcd gene coding for a cofactor pyrroloquinoline quinone-dependent glucose dehydrogenase (=quinoprotein glucose dehydrogenase, KO:K00117) was revealed (Table 5). Phosphate solubilization is normally a complex phenomenon depending on conditions such as bacterial, nutritional, physiological, and growth variations . Given that phosphate solubilization can occur through various microbial processes/mechanisms , the predicted genes on the genome being described could compositely contribute to this activity.
P. lutea OK2T is also expected to possess functional traits related to plant growth promotion [29–32]. As shown in Table 5, genes coding for functional enzymes with various PGPR effects such as “antibiotic resistance”, “metal ion resistance”, “toxin production”, “siderophore production”, “attachment and colonization in the plant rhizosphere”, and “plant hormone auxin production” were revealed. Although nif gene clusters involved in nitrogen-fixing activity were not found in the strain OK2T, a gene encoding for the nitrogen-fixation protein NifU (KO:K04488) was identified .
Within the genus Pseudomonas sensu stricto, P. lutea OK2T is presented as a group phylogenetically closest to P. graminis DSM 11363 T  and P. rhizosphaerae IH5T  (shown in Fig. 1). The majority of the genes in P. lutea OK2T were predicted based on the genome of P. rhizosphaerae IH5T (=DSM 16299 T, IMG Genome ID 2593339263) . However, genes such as ldhA (D-lactate dehydrogenase, KO:K03778), penP (beta-lactamase class A, KO:K17836), marC (multiple antibiotic resistance protein, KO:K05595), rcnA (nickel/cobalt exporter, KO:K08970), arsH (arsenical resistance protein ArsH, KO:K11811), fic (cell filamentation protein, KO:K04095), and chpA (chemosensory pili system protein ChpA, KO:K06596) and the gene clusters coding for enzymes with type IV secretion systems were only annotated in OK2T. Furthermore, pertinent gene clusters for type VI secretion systems, known as a complex multicomponent secretion machine, with bacterial competitions [35–37] were only predicted in the strain OK2T. The type VI secretion system may be related to possible features of bacterial motility/adaptation/competition in the strain. Although the strain P. graminis DSM 11363 T had similar general features and biochemical properties as strain OK2T, its genome sequence is not yet available.
Average Nucleotide Identity calculations  were used to compare the genomes of P. lutea OK2T and other sequenced Pseudomonas species (Table 6). The strain was found to be most closely related to Pseudomonas syringae ATCC 19310 T (77.31 % identity), followed by Pseudomonas kilonensis 520-20T (76.96 % identity). These values are under the acceptable range of species cutoff values of 95–96 % , indicating that P. lutea OK2T is different from other sequenced Pseudomonas species.
We presented here the first genome sequence of P. lutea OK2T, a phosphate-solubilizing bacterium isolated from the rhizosphere of grass in northern Spain . This study showed that P. lutea OK2T has potential traits including phosphate-solubilizing capability, making it as an effective pseudomonad-PGPR.
Considering a variety of complex conditions that occur in rhizospheres , the environmental adaptability of PGPR in in situ rhizosphere became an important factor for improved plant growth-promoting capacity. In addition, initial studies focusing on the functional properties of PGPR [31, 32] have led to interest in the comparative analyses of pan-/core-genomes of these bacteria, which are of ecological importance for elucidating the fundamental genotypic features of PGPR under diverse rhizosphere conditions [41, 42]. The genetic information obtained for P. lutea OK2T will improve our understanding of the genetic basis of phosphate-solubilizing pseudomonad-PGPR activities and further provide insights into the practical applications of the strain as a biocontrol agent in the field of agriculture.
Hierarchical genome assembly process
Integrated microbial genomes-expert review
Kyoto encyclopedia of genes and genomes Orthology
Prokaryotic genome annotation pipeline
Plant growth-promoting rhizobacteria
Rapid annotation using subsystems technology
Single molecule real-time
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This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2015R1D1A1A01057187).
YK performed the genomic sequencing, genomic analyses, phenotypic characterization of the bacterium, and drafted the manuscript. GP performed the genomic analyses and drafted the manuscript. JHS conceived the study, participated in its design and coordination, and drafted the manuscript. All the authors have read and approved the final manuscript.
The authors declare that they have no competing interests.