- Short genome report
- Open Access
High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Cupriavidus sp. strain UYPR2.512
Standards in Genomic Sciences volume 10, Article number: 13 (2015)
Cupriavidus sp. strain UYPR2.512 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida grown in soils from a native forest of Uruguay. Here we describe the features of Cupriavidus sp. strain UYPR2.512, together with sequence and annotation. The 7,858,949 bp high-quality permanent draft genome is arranged in 365 scaffolds of 369 contigs, contains 7,411 protein-coding genes and 76 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.
Legumes establish symbiotic associations with a group of soil bacteria, rhizobia, able to fix atmospheric nitrogen (N2). Rhizobia elicit the formation of a symbiotic organ called a nodule comprising differentiated plant and bacterial cells. Differentiated rhizobia within nodules are termed bacteroids, and acquire the ability to fix nitrogen. Rhizobia are phylogenetically diverse including genera from the Alphaproteobacteria (Allorhizobium, Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, Rhizobium, etc.) as well as from the Betaproteobacteria (Burkholderia, Cupriavidus) [1, 2].
The biological nitrogen fixation process significantly contributes to the development of sustainable agriculture reducing the use of supplies dependent on fuel and alleviating environmental impacts produced by the addition of chemical fertilizer . Moreover, forestation with leguminous trees associated with rhizobia, “nitrogen-fixing trees”, has been successfully used for recovering degraded soils .
Parapiptadenia rigida (Benth.) Brenan, is a “nitrogen-fixing tree” belonging to the Piptadenia group from the Mimosoideae subfamily . It is a multipurpose tree, very appreciated because of its timber and therefore used in high quality furniture and construction. It is also used for gums, tannins and essential oil extraction, has medicinal properties and is included in agroforestry and reforestation programs [4, 6, 7]. Taulé et al. demonstrated that this species could be nodulated either by Alpha-rhizobia (Rhizobium) or by Beta-rhizobia (Burkholderia and Cupriavidus) with Burkholderia being the preferred natural symbiont of this legume. In the case of Cupriavidus sp. UYPR2.512, this strain was isolated from a nodule of a P. rigida plant grown in soils collected from Mandiyú native forest in Artigas, Uruguay. Isolated bacterial colonies of Cupriavidus sp. UYPR2.512 were able to nodulate and to promote the growth of P. rigida, as well as Mimosa pudica plants .
To our knowledge, the only published sequenced genome of a Beta-rhizobia belonging to the Cupriavidus genus so far is that of C. taiwanensis LMG 19424T. Interestingly, the closest relative of Cupriavidus sp. UYPR2.512 is C. necator ATCC 43291T. Here, we present the description of the Cupriavidus sp. UYPR2.512 high-quality permanent draft genome sequence and its annotation.
Classification and features
Cupriavidus sp. strain UYPR2.512 is a motile, Gram-negative, non-spore-forming rod (Figure 1 Left, Center) in the order Burkholderiales of the class Betaproteobacteria. The rod-shaped form varies in size with dimensions of 0.5-0.7 μm in width and 0.9-1.2 μm in length (Figure 1 Left). It is fast growing, forming 0.5-0.8 mm diameter colonies after 24 h when grown on TY  at 28°C. Colonies on TY are white-opaque, slightly domed, moderately mucoid with smooth margins (Figure 1 Right).
Figure 2 shows the phylogenetic relationship of Cupriavidus sp. strain UYPR2.512 in a 16S rRNA gene sequence based tree. This strain is the most similar to Cupriavidus necator ATCC 43291T, Cupriavidus oxalaticus DSM 1105T and Cupriavidus taiwanensis LMG 19424T based on the 16S rRNA gene alignment with sequence identities of 99.32%, 98.49% and 98.42%, respectively, as determined using the EzTaxon-e server . Cupriavidus necator ATCC 43291T has been isolated from soil and is a non-obligate predator causing lysis of various Gram-positive and Gram-negative bacteria in the soil . Cupriavidus taiwanensis LMG 19424T is a plant symbiont and was isolated from root nodules of Mimosa pudica collected from three fields at Ping-Tung Country in the southern part of Taiwan . Minimum Information about the Genome Sequence (MIGS) is provided in Table 1 and Additional file 1: Table S1.
Cupriavidus sp. strain UYPR2.512 was isolated from Parapiptadenia rigida, a Mimosoideae legume native to Uruguay . This tree is native to South America, including south Brazil, Argentina, Paraguay, and Uruguay, and used by locals for timber and as a source of gums, tannins and essential oils . Cupriavidus sp. strain UYPR2.512 is able to renodulate its original host and is highly efficient in fixing nitrogen with this host . A selection of other host plants, including Trifolium repens, Medicago sativa, Peltophorum dubium and Mimosa pudica were tested for their ability to nodulate with UYPR2.512. Of these plants, strain UYPR2.512 was only able to nodulate and fix nitrogen effectively with M. pudica.
Genome sequencing information
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 Genomic Encyclopedia of Bacteria and Archaea, The Root Nodulating Bacteria chapter (GEBA-RNB) project at the U.S. Department of Energy, Joint Genome Institute . The genome project is deposited in the Genomes OnLine Database  and the high-quality permanent draft genome sequence in IMG . Sequencing, finishing and annotation were performed by the JGI using state of the art sequencing technology . A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
Cupriavidus sp. strain UYPR2.512 was grown to mid logarithmic phase in TY rich media  on a gyratory shaker at 28°C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method .
Genome sequencing and assembly
The draft genome of Cupriavidus sp. UYPR2.512 was generated at the DOE Joint Genome Institute . An Illumina Std shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 29,312,424 reads totaling 4,396.9 Mbp . All general aspects of library construction and sequencing performed at the JGI can be found at the JGI web site . 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, Han J. unpublished). Artifact filtered sequence data was then screened and trimmed according to the k–mers present in the dataset. High–depth k–mers, presumably derived from MDA amplification bias, cause problems in the assembly, especially if the k–mer depth varies in orders of magnitude for different regions of the genome. Reads with high k–mer coverage (>30x average k–mer depth) were normalized to an average depth of 30x. Reads with an average kmer depth of less than 2x were removed. Following steps were then performed for assembly: (1) normalized Illumina reads were assembled using Velvet version 1.1.04  (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim  (3) normalized Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r41043). Parameters for assembly steps were: 1) Velvet (velveth: 63 –shortPaired and velvetg: -very clean yes –exportFiltered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) 2) wgsim (-e 0 –1 100 –2 100 –r 0 –R 0 –X 0) 3) Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std_shredpairs TARGETS = standard VAPI_WARN_ONLY = True OVERWRITE = True). The final draft assembly contained 369 contigs in 365 scaffolds. The total size of the genome is 7.9 Mbp and the final assembly is based on 839.6 Mbp of Illumina data, which provides an average of 106.8x coverage.
Genes were identified using Prodigal , as part of the DOE-JGI genome annotation pipeline [36, 37] 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 National Center for Biotechnology Information (NCBI) non-redundant 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-Expert Review (IMG-ER) system  developed by the Joint Genome Institute, Walnut Creek, CA, USA.
The genome is 7,858,949 nucleotides with 65.25% GC content (Table 3) and comprised of 365 scaffolds and 369 contigs (Figure 3). From a total of 7,487 genes, 7,411 were protein encoding and 76 RNA only encoding genes. The majority of genes (75.64%) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COG functional categories is presented in Table 4.
Cupriavidus sp. UYPR2.512 belongs to a group of Beta-rhizobia isolated from Parapiptadenia rigida, a native tree from Uruguay belonging to the Mimosoideae legume group . This tree is also native to the south of Brazil, Argentina and Paraguay . Greenhouse experiments from previous studies have shown that Cupriavidus sp. UYPR2.512 is also able to nodulate and fix nitrogen with Mimosa pudica, an invasive species in many regions around the world . Phylogenetic analysis revealed that UYPR2.512 is the most closely related to Cupriavidus necator ATCC 43291T, Cupriavidus oxalaticus DSM 1105T and Cupriavidus taiwanensis LMG 19424T . In contrast to the other two strains, Cupriavidus taiwanensis LMG 19424T is a microsymbiont that is able to nodulate and fix nitrogen in association with Mimosa species . In total five Cupriavidus strains (AMP6, LMG 19424T, STM6018, STM6070 and UYPR2.512), which can form a symbiotic association have now been sequenced. A comparison of these strains reveals that UYPR2.512 has the largest genome (7.9 Mbp), with the highest KOG count (1398), the lowest G + C (65.25%) and signal peptide (9.3%) percentages in this group. All of these genomes share the nitrogenase-RXN MetaCyc pathway catalyzed by a multiprotein nitrogenase complex. Out of five Cupriavidus strains (AMP6, LMG 19424T, STM6018, STM6070 and UYPR2.512), which contain the N-fixation pathway, only Cupriavidus sp. UYPR2.512 has been shown to nodulate and fix effectively with Parapiptadenia rigida. The genome attributes of Cupriavidus sp. UYPR2.512 will therefore be important for ongoing molecular analysis of the plant microbe interactions required for the establishment of leguminous tree symbioses with this host.
Chen WM, Laevens S, Lee TM, Coenye T, De Vos P, Mergeay M, et al.: Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 2001, 51:1729–1735. 10.1099/00207713-51-5-1729
Moulin L, Munive A, Dreyfus B, Boivin-Masson C: Nodulation of legumes by members of the beta-subclass of Proteobacteria. Nature 2001, 411:948–950. 10.1038/35082070
Crews TE, Peoples MB: Legume versus fertilizer sources of nitrogen: ecological tradeoffs and human needs. Agric Ecosyst Environ 2004, 102:279–297. 10.1016/j.agee.2003.09.018
Farias JA, Hoppe JM, Vivian JAC: Comportamento de mudas de Parapiptadenia rigida (Bentham) Brenan, submetidas a diferentes índices de luminosidade e em função de diferentes dimensões de recipientes. Caderno de Pesquisa Série Biologia 2005, 17:69–80.
Jobson RW, Luckow M: Phylogenetic study of the genus Piptadenia (Mimosoideae : Leguminosae) using plastid trnL-F and trnK/matK sequence data. Syst Bot 2007, 32:569–575. 10.1600/036364407782250544
Schmidt CA, Murillo R, Bruhn T, Bringmann G, Goettert M, Heinzmann B, et al.: Catechin derivatives from Parapiptadenia rigida with in vitro wound-healing properties. J Nat Prod 2010, 73:2035–2041. 10.1021/np100523s
de Souza GC, Haas AP, von Poser GL, Schapoval EE, Elisabetsky E: Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J Ethnopharmacol 2004, 90:135–143. 10.1016/j.jep.2003.09.039
Taule C, Zabaleta M, Mareque C, Platero R, Sanjurjo L, Sicardi M, et al.: New betaproteobacterial rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan. Appl Environ Microbiol 2012, 78:1692–1700. 10.1128/AEM.06215-11
Amadou C, Pascal G, Mangenot S, Glew M, Bontemps C, Capela D, et al.: Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Res 2008, 18:1472–1483. 10.1101/gr.076448.108
Beringer JE: R factor transfer in Rhizobium leguminosarum . J Gen Microbiol 1974, 84:188–198. 10.1099/00221287-84-1-188
Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, et al.: Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 2012, 62:716–721. 10.1099/ijs.0.038075-0
Makkar NS, Casida LE: Cupriavidus necator gen. nov., sp. nov. - a nonobligate bacterial predator of bacteria in soil. Int J Syst Evol Microbiol 1987, 37:323–326.
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, 28:2731–2739. 10.1093/molbev/msr121
Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, et al.: The Genomes OnLine Database (GOLD) v. 4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2012, 40:D571–579. 10.1093/nar/gkr1100
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M, Angiuoli SV, Ashburner M, Axelrod N, Baldauf S, Ballard S, Boore JL, Cochrane G, Cole J, Dawyndt P, de Vos P, de Pamphilis C, Edwards R, Faruque N, Feldman R, Gilbert J, Gilna P, Glöckner FO, Goldstein P, Guralnick R, Haft D, Hancock D, et al.: Towards a richer description of our complete collection of genomes and metagenomes "Minimum Information about a Genome Sequence" (MIGS) specification. Nat Biotechnol 2008, 26:541–547. 10.1038/nbt1360
Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, et al.: The Genomic Standards Consortium. PLoS Biol 2011, 9:e1001088. 10.1371/journal.pbio.1001088
Woese CR, Kandler O, Wheelis ML: Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Nat Acad Sci USA 1990, 87:4576–4579. 10.1073/pnas.87.12.4576
Chen WX, Wang ET, Kuykendall LD: The Proteobacteria. New York: Springer - Verlag; 2005.
Validation of publication of new names and new combinations previously effectively published outside the IJSEM Int J Syst Evol Microbiol 2005, 55:2235–2238.
Garrity GM, Bell JA, Lilburn TE: Class II. Betaproteobacteria. In Bergey's Manual of Systematic Bacteriology. Volume 2. 2nd edition. Edited by: Garrity GM, Brenner DJ, Krieg NR, Staley JT. New York: Springer - Verlag; 2005.
Garrity GM, Bell JA, Lilburn TE: Order 1. Burkholderiales . In Bergey's Manual of Systematic Bacteriology. Volume 2. 2nd edition. Edited by: Garrity GM, Brenner DJ, Krieg NR, Staley JT. New York: Springer - Verlag; 2005.
Garrity GM, Bell JA, Lilburn TE: Family I. Burkholderiaceae . In Bergey's Manual of Systematic Bacteriology. Volume 2. 2nd edition. Edited by: Garrity GM, Brenner DJ, Krieg NR, Staley JT. New York: Springer - Verlag; 2005.
Balkwill DL: Genus I. Cupriavidus . In Bergey's Manual of Systematic Bacteriology. Volume 2. 2nd edition. Edited by: Garrity GM, Brenner DJ, Krieg NR, Staley JT. New York: Springer - Verlag; 2005.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25:25–29. 10.1038/75556
Reeve W, Ardley J, Tian R, Eshragi L, Yoon J, Ngamwisetkun P, et al.: A genomic encyclopedia of the root nodule bacteria: Assessing genetic diversity through a systematic biogeographic survey. Stand Genomic Sci 2015, 10:14. 10.1186/1944-3277-10-14
Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Pillay M, et al.: IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 2014, 42:D560-D567. 10.1093/nar/gkt963
Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, et al.: The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS ONE 2012, 7:e48837. 10.1371/journal.pone.0048837
GOLD ID Cupriavidus sp. UYPR2.512. [https://gold.jgi-psf.org/projects?id=9663]
CTAB DNA extraction protocol. [http://jgi.doe.gov/collaborate-with-jgi/pmo-overview/protocols-sample-preparation-information/]
Bennett S: Solexa Ltd. Pharmacogenomics 2004, 5:433–438. 10.1517/146224126.96.36.1993
JGI Website. [http://www.jgi.doe.gov]
Zerbino D, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008, 18:821–829. 10.1101/gr.074492.107
Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, et al.: High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Nat Acad Sci 2011, 108:1513–1518. 10.1073/pnas.1017351108
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010, 11:119. 10.1186/1471-2105-11-119
Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC: The DOE-JGI Standard Operating Procedure for the annotations of microbial genomes. Standards Genomic Sci 2009, 1:63–67. 10.4056/sigs.632
Chen IM, Markowitz VM, Chu K, Anderson I, Mavromatis K, Kyrpides NC, et al.: Improving microbial genome annotations in an integrated database context. PLoS ONE 2013, 8:e54859. 10.1371/journal.pone.0054859
Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, et al.: GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010, 7:455–457. 10.1038/nmeth.1457
Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25:955–964. 10.1093/nar/25.5.0955
Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al.: SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007, 35:7188–7196. 10.1093/nar/gkm864
INFERNAL. Inference of RNA alignments. [http://infernal.janelia.org]
Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC: IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009, 25:2271–2278. 10.1093/bioinformatics/btp393
Chen WM, Moulin L, Bontemps C, Vandamme P, Bena G, Boivin-Masson C: Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature. J Bacteriol 2003, 185:7266–7272. 10.1128/JB.185.24.7266-7272.2003
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.
The authors declare that they have no competing interests.
EF supplied the strain and background information for this project, PVB supplied DNA to JGI, TR performed all imaging, SDM and WR drafted the paper, JH provided financial support and all other authors were involved in sequencing the genome and editing the final manuscript. All authors read and approved the final manuscript.