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Draft genome of Paraburkholderia caballeronis TNe-841T, a free-living, nitrogen-fixing, tomato plant-associated bacterium
Standards in Genomic Sciencesvolume 12, Article number: 80 (2017)
Paraburkholderia caballeronis is a plant-associated bacterium. Strain TNe-841T was isolated from the rhizosphere of tomato (Solanum lycopersicum L. var. lycopersicum) growing in Nepantla Mexico State. Initially this bacterium was found to effectively nodulate Phaseolus vulgaris L. However, from an analysis of the genome of strain TNe-841T and from repeat inoculation experiments, we found that this strain did not nodulate bean and also lacked nodulation genes, suggesting that the genes were lost. The genome consists of 7,115,141 bp with a G + C content of 67.01%. The sequence includes 6251 protein-coding genes and 87 RNA genes.
Paraburkholderia caballeronis was isolated in the State of Mexico, Mexico from the tomato rhizosphere as a free-living, nitrogen-fixing bacterial species . It was described as B. caballeronis and found to nodulate Phaseolus vulgaris L. . Most nodulating bacteria are isolated from root nodules but this was not the case for B. caballeronis , which was isolated from rhizospheric soil. Given the ability of this bacterium to fix nitrogen under both free-living and symbiotic conditions, this type strain was selected for genome sequencing to study its nitrogen-fixing and other plant-growth promoting activities. However, after analyzing the genome, we found that the genes for fixing nitrogen were present but nodulation genes were not. We carried out several unsuccessful tests to check the ability of this strain to nodulate P. vulgaris, strongly suggesting that the strain had lost the nod genes. The genome sequence of P. caballeronis TNe-841T was obtained in cooperation with JGI-DOE. The type species is TNe-841T (= LMG 26416 T = CIP 110324 T).
Classification and features
Burkholderia caballeronis TNe-841T has been proposed to belong to the newly described genus Paraburkholderia. The last years, Burkholderia sensu lato has been subjected to some taxonomical changes, where the genus has been split to Burkholderia, Paraburkholderia, Caballeronia and Robbsia andropogonis [3,4,5]. However, this division has caused some skepticism, which has been expressed by The International Committee on Systematics of Prokaryotes, through the Subcommittee for the Taxonomy of Rhizobium and Agrobacterium discussed during the 12th Nitrogen Fixation Conference held in Budapest, Hungary on 25 August 2016 . The Subcommittee stated: “Research efforts directed towards robust characterization and taxonomy of Burkholderia sensu lato species can help in realizing this agricultural potential. Clearly, large-scale phylogenomic study is required for resolving these taxa”. In order to analyze this issue and to provide generic limits in Burkholderia sensu lato, a large phylogenomic analysis was carried out using the amino acid and nucleotide sequence of 106 conserved proteins from 92 species . The analysis performed with maximum likelihood unambiguously supported five different lineages: Burkholderia sensu stricto, Paraburkholderia, Caballeronia, Robbsia andropogonis and B. rhizoxinica. To check the position of P. caballeronis within Paraburkholderia , the 16S rRNA gene sequence (ca. 1500 bp) was amplified and sequenced at Macrogen  with the universal primers fD1/rD1 . The nucleotide sequence (accession number EF139186) was compared to other Paraburkholderia species using Muscle 3.57 for alignment . A phylogenetic analysis was performed with ML using the PhyML program . Among-site rate variation was modeled by a gamma distribution with four rate categories  with each category being represented by its mean under the GTR + G model. Tree searches were initiated from a BioNJ seed tree retaining the best tree among those found with NNI (Nearest Neighbor Interchange). The robustness of the ML topologies was evaluated using a Shimodaira-Hasegawa (SH)-like test . The ML tree was obtained with the program MEGA version 5 . The position of P. caballeronis in the ML tree shows that it is close to P. kururiensis (Fig. 1). The colony morphology on BSE medium was uniform, 1 mm diameter, with entire margins that were convex, whitish, and translucent transparent. The cells are strictly aerobic Gram-negative, non-spore forming rod (0.49–0.69 μm × 1.2–2.7 μm) and have flagella (Fig. 2). Other phenotypic traits for this strain have been published before . The strain has the following enzymes: arginine dihydrolase, urease catalase, and nitrogenase and associated proteins. It is also able to assimilate D-glucose, DL-arabinose, D-mannose, D-mannitol, N-acetyl glucosamine, gluconate, capric acid, malate acetate, D-ribose, D-xylose, D-adonitol, D-galactose, D-fructose, L-rhamnose, inositol, D-sorbitol, D-cellobiose, D-turanose, D-xylose, D-fucose, D-arabitol, potassium 2-ketogluconate, and potassium 5-ketogluconate (Table 1). Oxidase activity was weak. The strain grew on MacConkey agar plates at 29 °C and 37 °C, but weakly at 42 °C. P. caballeronis TNe-841T grew on LB and BSE agar plates at 15, 29, 37, and 42 °C and on LB plates at 29 °C with up to 5.0% NaCl.
The following fatty acids were detected in strain TNe-841T : C14:0 (4.46%), C16:0 (21.77%), C16:0 2OH (2.3%), C16:0 3OH (6.2%), C16:1 2OH (3.81%), C17:0 cyclo (12.43%), C18:1 2OH (1.5%), C18:1 ω 7c (16.62%), C19:0 cyclo ω 8c (14.89%), summed feature 2 (5.9%), and summed feature 3 (8.3%). Summed feature two corresponds to C14:0 3OH and/or 16:1 ISO I, an unidentified fatty acid with equivalent chain length value of 10.928 12:0 ALDE or any combination of these fatty acids. Summed feature three corresponds to C16:1 w7c and/or C15:0 ISO 2OH.
Genome sequencing information
Genome project history
P. caballeronis TNe-841T was sequenced at the JGI-DOE as a part of the project “Root nodule microbial communities of legume samples collected from USA, Mexico and Botswana” directed by Dr. Ann M. Hirsch. The goal of this project was to identify the microbial community housed within nodules of native legumes living in three arid or semi-arid, nutrient-poor environments in Mexico, Botswana, and the United States. Both Paraburkholderia and Rhizobium bacteria had been previously isolated from Mexico. P. caballeronis TNe-841T was chosen as the reference strain for a study of bacteria associated with native legume soils and nodules.
Growth conditions and genomic DNA preparation
P. caballeronis TNe-841T cells were grown in 5 ml of LB minus NaCl at 30 °C for 18 h at 120 rpm. The DNA extraction was done using Invitrogen’s Purelink™ Genomic DNA Mini Kit. The purified DNA was monitored for integrity by gel electrophoresis, and then sent to the JGI for sequencing.
Two surface-sterilized and rinsed seeds of Phaseolus vulgaris L. c.v. Negro Chapingo were planted per pot in surface-sterilized black pots (29.5 cm tall; 17 cm diameter) filled with autoclaved vermiculite:perlite (2:1) and watered with autoclaved 1/4 strength Hoagland’s –N medium. Two separate experiments were performed. The pots were either left uninoculated (sterilized water or Hoagland’s –N medium was added), inoculated with 10 ml of P. caballeronis TNe-841T diluted to OD600 = 0.2 or with B. tuberum DUS833, which was a positive control. Some pots were also watered with 1/4 strength Hoagland’s + N medium as an additional positive control. The appropriate medium was added twice weekly and the plants grown in a Conviron growth chamber under 16 h days/8 h nights at 24 °C.
Genome sequencing and assembly
The draft genome of P. caballeronis was generated using the PacBio sequencing technology . A Pacbio SMRTbell™ library was constructed and sequenced on the PacBio RS platform, which generated 194,884 filtered sub-reads totaling 879.3 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at . The raw reads were assembled using HGAP (version: 2.3.0 p5 protocol version = 2.3.0 method = RS HGAP Assembly.3 smrtpipe.py v1.87.139483) . The final draft assembly contained 3 contigs in 3 scaffolds totaling 7.115 Mbp in size. The input read coverage was 62.2X.
Genes were identified using Prodigal  followed by a round of manual curation using GenePRIMP  for finished genomes and draft genomes in fewer than 10 scaffolds. The predicted CDSs were translated and used to search the NCBI nonredundant database, UniProt, TIGRFam, Pfam, 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 platform  developed by the JGI Walnut Creek CA USA .
The genome was also manually annotated at IPN and UCLA using the IMG platform .
The final draft assembly of P. caballeronis TNe-841T contained 3 contigs in 3 scaffolds accumulating 7,115,141 bp in size (Table 3). The G + C content of the genome was 67.01%, which is very close to the one determined during the description of the species (66.0%) . The genome was predicted to encode 6338 genes including 6251 protein-coding genes and 87 RNA genes (15 rRNAs 60 tRNAs and 12 ncRNA). The number of genes associated with general COG functional categories is shown in Table 4, in addition to other functions such as extracellular structures and mobilome.
Insights from the genome sequence
P. caballeronis was originally described as a free-living, nitrogen-fixing bacteria with the ability to form nodules on Phaseolus vulgaris L. roots . Although nitrogen fixation genes are present, nodulation genes were not found in the sequenced genome. Moreover, after the initial experiments, P. vulgaris nodulation was no longer detected in greenhouse bioassays in two different laboratories. This nodulation instability seems to be more frequent than originally assumed because a similar loss of nodulation ability has been reported with other Burkholderia strains isolated from nodules. The strains CCGE1002 and CCGE1003 (Marco Antonio Rogel CCG-UNAM, pers. comm.) also lost the ability to nodulate, but strain CCGE1002, which retains the ability to nodulate, was recovered from a stored sample. Its symbiotic plasmid was subsequently sequenced (NCBI BioSample PRJNA37719). In contrast, nodulation genes were no longer detected in the genome of strain CCGE1003 (NCBI BioSample PRJNA37721). A similar loss of nodulation genes was reported for two Burkholderia strains isolated from Kennedia coccinea  and Gastrolobium capitatum  in Australia.
Strain TNe-841T also contains genes for degrading a large number of xenobiotics including aminobenzoate, atrazine, benzoate, bisphenol, caprolactam, chloroalkane, chloroalkene, chlorohexane, chlorobenzene, dioxin, ethylbenzene, fluorobenzoate, naphthalene, nitrotoluene, polycyclic aromatic hydrocarbons, styrene, toluene, and xylene.
ANI calculation was used to compare the genome of P. caballeronis TNe-841T and other Paraburkholderia species (Table 5). The ANI results showed that strains TNe-851T correspond to a different species since the highest ANI value was 83.32. The accepted ANI cut-off for species is 95-96%, which corresponds to a DNA-DNA hybridization of 70% [24, 25].
P. caballeronis TNe-81T, is a plant-associated bacteria species with the ability to fix nitrogen, although the ability to nodulate legumes as shown in the original description was apparently lost. This nodulation instability seems to be rather common among nodulating bacteria, particularly Burkholderia / Paraburkholderia . Our interest in studying the genome of P. caballeronis TNe-841T started when we found that this bacterium, isolated from the tomato rhizosphere, was able to nodulate bean. This led us to find out the identity of the original host for this species. Our work team has recently isolated a P. caballeronis strain from bean nodules used as a trap with soil from an area where Mimosoideae plants are present (unpublished results). We are characterizing additional isolates from Mimosoideae plant nodules to try to establish if this plant might be the host of P. caballeronis TNe-841T.
Average nucleotide identity
Department of energy
Integrated microbial genomes
Joint Genome Institute
National center for biotechnology information
Caballero-Mellado J, Onofre-Lemus J, Estrada-De Los Santos P, Martínez-Aguilar L. The tomato rhizosphere, an environment rich in nitrogen-fixing Burkholderia species with capabilities of interest for agriculture and bioremediation. Appl Environ Microbiol. 2007;73(16):5308–19.
Martínez-Aguilar L, Salazar-Salazar C, Méndez RD, Caballero-Mellado J, Hirsch AM, Vásquez-Murrieta MS. Estrada-de Los Santos P. Burkholderia caballeronis sp. nov., a nitrogen fixing species isolated from tomato (Lycopersicon esculentum) with the ability to effectively nodulate Phaseolus vulgaris. Anton Leeuw Int J G. 2013;104(6):1063–71.
Sawana A, Adeolu M, Gupta RS. Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division fo this genus into the emended genus Burkholderia containing pathogenic organisms and new genus Paraburkholderia gen. Nov. harboring environmental species. Front Genet. 2014;5:429.
Dobritsa AP, Samadpour M. Transfer of eleven Burkholderia species to the genus Paraburkholderia and proposal of Caballeronia gen. Nov., a new genus to accommodate twelve species of Burkholderia and Paraburkholderia. Int J Syst Evol Microbiol. 2016;66(8):2836–46.
Lopes-Santos L, Castro DBA, Ferreira-Tonin M, Corrêa DBA, Weir BS, Park D, Ottoboni LMM, Neto JR, Destéfano SAL. Reassessment of the taxonomic position of Burkholderia andropogonis and description of Robbsia andropogonis gen. Nov., comb. nov. Anton Leeuw Int J G. 2017;110(6):727–36.
de Lajudie PM, Young JPW. International committee on systematics of prokaryotes subcommittee for the taxonomy of rhizobium and agrobacterium minutes of the meeting, budapest, 25 august 2016. Int J Syst Evol Microbiol. 2017;67:2485–94.
Beukes C, Palmer M, Manyaka P, Chan WY, Avontuur J, van Zyl E, Huntemann M, Clum A, Pillay M, Palaniappan K. Genome data provides high support for generic boundaries in Burkholderia sensu lato. Front Microbiol. 2017;8:1154.
MACROGEN INC. [http://foreign.macrogen.com/eng/].
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697–703.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Res. 2004;32(5):1792–7.
Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52(5):696–704.
Yang Z. Among-site rate variation and its impact on phylogenetic analyses. Trends Ecol Evol. 1996;11(9):367–72.
Anisimova M, Gascuel O. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst Biol. 2006;55(4):539–52.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731–9.
Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B. Real-time DNA sequencing from single polymerase molecules. Science. 2009;323(5910):133–8.
JGI. Joint Genome Institute [http://www.jgi.doe.gov/].
Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. 2013;10(6):563–9.
Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11(1):119.
Pati AI, Mikhailova N, Ovchinikova N, Hooper G, Lykidis S, Kyrpides A, GenePRIMP N. A gene prediction improvement pipeline for microbial genomes. Nat Methods. 2010;7(6):455–7.
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acid Res. 1997;25(5):955–64.
Markowitz VM, Chen IMA, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Huntemann M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides N. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acid Res. 2013;42:D560–D567.
Walker R, Watkin E, Tian R, Bräu L, O’Hara G, Goodwin L, Han J, Lobos E, Huntemann M, Pati A, et al. Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park, Australia. Stand Genomic Sci. 2014;9(3):551–61.
Walker R, Watkin E, Tian R, Bräu L, O’Hara G, Goodwin L, Han J, Reddy T, Huntemann M, Pati A. Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2232 from Karijini National Park, Australia. Stand Genomic Sci. 2014;9(3):1168.
Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 2009;106(45):19126–31.
Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57(1):81–91.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541–7.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990;87(12):4576–9.
Garrity GM, Bell JA, Lilburn TE. Class II. Betaproteobacteria. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005.
Garrity GM, Bell JA, Lilburn TE. Class II. Betaproteobacteria. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 29.
Garrity GM, Bell JA, Lilburn TE. Order 1. Burkholderiales. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 30.
Garrity GM, Bell JA, Lilburn TE, Family I. Burkholderiaceae. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005.
Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, Hashimoto Y, Ezaki T, Arakawa M. Proposal of Burkholderia gen. Nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol. 1992;36(12):1251–75.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1):25–9.
EYTG and FURR are recipients of a fellowship from CONACyT. PES is recipient of SNI, EDI and COFAA fellowships. We thank Dr. E.O. Lopez-Villegas (Escuela Nacional de Ciencias Biológicas, IPN) for transmission electron microscopy analysis.
The genome sequence was conducted by the U.S. Department of Energy, Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Sciences of the U.S. Department of Energy under the proposal 1572 and contract No. DE-AC02-05CH11231.
The authors declare that they have no competing interests.
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