High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Cupriavidus sp. strain UYPR2.512
© De Meyer et al.; licensee BioMed Central. 2015
Received: 26 November 2014
Accepted: 5 December 2014
Published: 11 April 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.
KeywordsRoot-nodule bacteria Nitrogen fixation Rhizobia Betaproteobacteria GEBA-RNB
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
Species Cupriavidus sp.
pH range; Optimum
Soil, root nodule on host
Nodule collection date
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
Genome sequencing project information for Cupriavidus sp. strain UYPR2.512
Illumina Std PE
Illumina HiSeq 2000
106.8 X Illumina
Velvet 1.1.04, ALLPATHS-LG V.r41043
Gene calling methods
Genbank Date of Release
September 16, 2013
Source Material Identifier
Symbiotic N2fixation, agriculture
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.
Genome statistics for Cupriavidus sp. strain UYPR2.512
% of total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of protein coding genes of Cupriavidus sp. strain UYPR2.512 associated with the general COG functional categories
% of total (5,426)
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking, secretion, and vesicular transport
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 metabolite biosynthesis, transport and catabolism
General function prediction only
Not in COGS
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.
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.
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