High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Burkholderia sp. strain UYPR1.413

Burkholderia sp. strain UYPR1.413 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida collected at the Angico plantation, Mandiyu, Uruguay, in December 2006. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated predominantly by Burkholderia microsymbionts. Moreover, Burkholderia sp. strain UYPR1.413 is a highly efficient nitrogen fixing symbiont with this host. Currently, the only other sequenced isolate to fix with this host is Cupriavidus sp. UYPR2.512. Therefore, Burkholderia sp. strain UYPR1.413 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 GEBA-RNB project. Here we describe the features of Burkholderia sp. strain UYPR1.413, together with sequence and annotation. The 10,373,764 bp high-quality permanent draft genome is arranged in 336 scaffolds of 342 contigs, contains 9759 protein-coding genes and 77 RNA-only encoding genes.


Introduction
Rhizobia are soil bacteria that have acquired the ability to establish symbiotic associations with plants, mainly from the Fabaceae family, and carry out the Biological Nitrogen Fixation (BNF) process. BNF is catalyzed by the rhizobial nitrogenase complex, whereby N 2 is reduced to ammonium.
Burkholderia sp. UYPR1.413 strain has been isolated from a root nodule of Parapiptadenia rigida (Benth.) Brenan found in an angico plantation in Artigas, Uruguay [18]. P. rigida belongs to the Mimosoideae subfamily and is a woody species, which can reach 30 m in height and a diameter of 60 to 80 cm [19]. The wood is of excellent quality, heavy, elastic, very hard and quite durable, rich in tannins and has medicinal properties [20]. There are six different species of Parapiptadenia in the Americas of which only P. rigida is present in Uruguay. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated by rhizobia belonging to the genera Burkholderia, Cupriavidus and Rhizobium, of which the Burkholderia microsymbionts predominated [18]. Burkholderia sp. UYPR1.413 strain belongs to a group of microsymbionts that were able to  Fig. 2 Phylogenetic tree highlighting the position of Burkholderia sp. strain UYPR1.413 (shown in blue print) relative to other type and non-type strains in the Burkholderia genus using 1046 bp internal region of the 16S rRNA gene. Several Alpha-rhizobia sequences were used as outgroup. All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5.05 [47]. The tree was built using the maximum likelihood method with the General Time Reversible model. Bootstrap analysis with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Strains with a genome sequencing project registered in GOLD [30] have the GOLD ID provided after the strain number. Finished genomes are designated with an asterisk nodulate and fix nitrogen with P. rigida [18]. In this work we present the description of the Burkholderia sp. UYPR1.413 high-quality permanent draft genome sequence and its annotation.

Organism information Classification and features
Burkholderia sp. strain UYPR1.413 is a motile, Gramnegative, non-spore-forming rod ( Fig. 1 Left, Center) in the order Burkholderiales of the class Betaproteobacteria. The rod-shaped form varies in size with dimensions of 0.3-0.5 μm in width and 1.0-2.0 μm in length ( Fig. 1 Left). It is fast growing, forming 0.5-1 mm diameter colonies after 24 h when grown on TY [21] at 28°C. Colonies on TY are white-opaque, slightly domed, moderately mucoid with smooth margins (Fig. 1 Right). Figure 2 shows the phylogenetic relationship of Burkholderia sp. strain UYPR1.413 in a 16S rRNA gene sequence based tree. This strain is phylogenetically most related to Burkholderia sabiae Br3407 T , Burkholderia caribensis MWAP64 T and Burkholderia phymatum STM815 T with sequence identities to UYPR1.413 16S rRNA gene sequence of 98.96, 98.64 and 98.56 %, respectively, as determined using the EzTaxon-e server [22]. Burkholderia sabiae Br3407 T was first isolated from root nodules of Mimosa caesalpiniifolia, a native tree to Brazil [6]. Burkholderia caribensis MWAP64 T was first isolated from vertisol in Martinique [5] and related strains have been identified as a plant growth promoting bacteria for grain Amaranth and Mango trees [23,24] and nitrogen fixing root nodule bacteria for several Mimosa species [25,26]. Burkholderia phymatum STM815 T is also known to nodulate effectively with several Mimosa species [27]. Minimum Information , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [56] about the Genome Sequence (MIGS) [28] is provided in Table 1.

Symbiotaxonomy
Burkholderia sp. strain UYPR1.413 was isolated from Parapiptadenia rigida, a Mimosoideae legume native to Uruguay [18]. 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 [18]. Burkholderia sp. strain UYPR1.413 is able to renodulate its original host and is highly efficient in fixing nitrogen with this host [18]. A selection of host plants, including Trifolium repens, Medicago sativa, Peltophorum dubium and Mimosa pudica were investigated previously for their ability to nodulate with UYPR1.413 and only M. pudica plants were nodulated by UYPR1.413, albeit ineffectively [18].

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 (JGI) for projects of relevance to agency missions [29]. The genome project is deposited in the Genomes OnLine Database [30] and the high-quality permanent draft genome sequence in IMG [31]. Sequencing, finishing and annotation were performed by the JGI using state of the art sequencing technology [32]. A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation
Burkholderia sp. strain UYPR1.413 was grown to mid logarithmic phase in TY rich media [21] 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 [33].

Genome sequencing and assembly
The draft genome of Burkholderia sp. UYPR1.413 was generated at the DOE Joint genome Institute (JGI) using state of the art technology [32]. An Illumina Std shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 23,255,298 reads totaling 3488.3 Mbp. All general aspects of library  construction and sequencing performed at the JGI can be found at the JGI web site [34]. 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). The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet version 1.1.04 [35] (2) 1-3 Kbp simulated paired end reads were created from Velvet contigs using wgsim [36] (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG (version r41043) [37]. Parameters for assembly steps were: 1) Velvet (velveth: 63-shortPaired and velvetg: -very clean yes -exportFiltered yes -min contig lgth 500 -scaf-

Genome annotation
Genes were identified using Prodigal [38], as part of the DOE-JGI genome annotation pipeline [39,40] followed by a round of manual curation using GenePRIMP [41] 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 [42] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [43]. 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 INFER-NAL [44]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes-Expert Review (IMG-ER) system [45] developed by the Joint Genome Institute, Walnut Creek, CA, USA.

Genome properties
The genome is 10,373,764 nucleotides with 62.28 % GC content ( Table 3) and comprised of 336 scaffolds and 342 contigs (Fig. 3). From a total of 9836 genes, 9759 were protein encoding and 77 RNA only encoding genes. The majority of genes (75.92 %) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4.

Conclusion
Burkholderia sp. UYPR1.413 belongs to a group of Beta-rhizobia isolated from Parapiptadenia rigida, a native tree from Uruguay belonging to the Mimosoideae The total is based on the total number of protein coding genes in the genome legume group [18]. This tree is also native to the south of Brazil, Argentina and Paraguay [18]. Phylogenetic analysis revealed that UYPR1.413 is most closely related to Burkholderia sabiae Br3407 T , Burkholderia caribensis MWAP64 T and Burkholderia phymatum STM815 T . Interestingly, Br3407 T was isolated from nitrogenfixing nodules on the roots of Mimosa caesalpiniifolia, a legume tree native to Brazil [6]. MWAP64 T has not been reported to nodulate legume plants, however B. caribensis TJ182 is able to nodulate and fix nitrogen with Mimosa pigra [7]. STM815 T was originally isolated from Macroptilium atropurpureum but could not be authenticated on this host [1]. Additional studies showed that STM815 T is instead able to nodulate a wide range of Mimosa species [27]. Glasshouse experiments from previous studies have shown that Burkholderia sp. UYPR1.413 is also able to nodulate Mimosa pudica seedlings, albeit ineffectively [18]. However, it is different from the other microsymbiont in that it can form an effective association with Parapiptadenia rigida. The only other sequenced isolate to fix with this host is Cupriavidus sp. UYPR2.512 [46]. There are in total 13 Burkholderia strains that are known legume symbionts; four (WSM3556 T , WSM4176, WSM5005 T , STM678 T ) nodulate South African papilionoid species, in contrast to the other nine (BR3459, CCGE1002, DSM 21604, JPY251, JPY366, LMG 23256 T , STM815, STM3621 and UYPR1.413) that are able to nodulate mimosoid species. A comparison of the mimosoid nodulating strains reveals that UYPR1.413 has the largest genome (10.4 Mbp), with the highest KOG count (1670) and the lowest GC (65.28 %) percentage in this group. All 13 of these genomes share the nitrogenase-RXN MetaCyc pathway catalyzed by a multiprotein nitrogenase complex. However, only Burkholderia sp. UYPR1.413 has been shown to fix effectively with Parapiptadenia rigida. The genome attributes of Burkholderia sp. UYPR1.413 will therefore be important for ongoing molecular analysis of the plant microbe interactions required for the establishment of leguminous tree symbioses with this host.