Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism with implications for function in soil environments.


Introduction
The first step in the aerobic nitrification process is the oxidation of ammonia to nitrite, mediated mainly by AOB or AOA in soil environments. The most numerous AOB isolated or detected by non-cultural methods in aerobic agricultural surface soils are consistently members of the Nitrosospira genus [1]. Nitrosospira briensis C-128 [2] is a chemolithoautotrophic ammoniaoxidizing betaproteobacterium (order Nitrosomonadales, family Nitrosomonadaceae, genus Nitrosospira [3][4][5][6][7][8][9]) isolated from a fertilized soil under cultivation for blueberry in Falmouth, Massachusetts, USA in 1971. The genome of Nitrosospira briensis C-128 is the third genome sequence from the genus Nitrosospira [8][9][10] to be published [11][12][13] and thus provides an important comparison among Nitrosospira. This report includes a summary of the genome sequence and selected features for Nitrosospira briensis C-128 and results are publically available in GenBank accession CP012371.

Organism information
Classification and features Nitrosospira briensis was described by Winogradsky and & Winogradsky in 1933 [8] as an ammonia-oxidizing bacterium isolated from soil. The genus name, Nitrosospira, is derived from two Latin roots: nitrosus, meaning nitrous, and spira, indicating spiral. The species name briensis, refers to the original isolation location near Brie, France. The culture described by Winogradsky & Winogradsky [8] was not maintained and reisolation of a replacement strain was reported by Watson in 1971 [14]. At approximately the same time, N. briensis strain C-128 was isolated by enrichment culturing [15] from a surface soil sample (pH 6.2) collected from a fertilized blueberry patch in East Falmouth, Massachusetts in 1971 (Frederica Valois). In 1993, the genus Nitrosospira was emended to include the former genera of Nitrosovibrio and Nitrosolobus [9] based on the high identities of the 16S rRNA gene sequences. Nitrosospira briensis was designated the type species for the genus with strain C-76 as the type strain (also known as strain Nsp10 [16] 1 ). The full-length 16S rRNA gene sequence of N. briensis C-128 is 99 % identical to the N. briensis strain C-76/ Nsp10 sequence (Fig. 1). The culture of N. briensis strain C-128 was received in the Norton laboratory from F. Valois (Woods Hole Oceanographic Institution) in 1995. Nitrosospira briensis C-128 is presently maintained in a culture collection at WHOI and may be obtained upon request from J.M. Norton. Classification and general features of Nitrosospira briensis C-128 are provided as Minimum Information about the Genome Sequence (MIGS) in Table 1. Electron micrographs of the pure culture organism are shown in Fig. 2 revealing the tight spirals visible with TEM negative staining and the convoluted surface of this Nitrosospira as revealed by SEM.

Genome sequencing information
Genome project history Nitrosospira briensis C-128 was chosen for sequencing through the Community Science Program (2010) of the DOE Joint Genome Institute as an important representative of the AOB to improve the scope and quality of intra-and inter-generic comparisons in the Nitrosomonadales. The chemolithotrophic metabolism of the AOB, the pathways for production of nitrous oxide and urea metabolism were additional motivating interests in sequencing this genome. Sequencing, finishing, and annotation were accomplished by JGI. The genome sequence has been deposited in the Genome OnLine Database [17] and is part of the NCBI Reference Sequence Collection [18]. A summary of the project information is found in Table 2.

Growth conditions and genomic DNA preparation
Nitrosospira briensis C-128 was grown in a 25 mM ammonium medium pH 7 containing mineral salts and phenol red at 28°C in 100 ml of media in 500 ml flasks as described previously [19]. The pH was adjusted to neutral using 0.5 M KHCO 3 as needed during growth. Early stationary phase cultures were checked at harvest  Fig. 1 The phylogenetic tree highlighting the position of Nitrosospira briensis C-128 relative to other Nitrosomonadaceae [48] and Spirillum volutans (outgroup). The tree was inferred from 1417 aligned characters of the 16S rRNA gene sequence by the neighbour-joining method [49] using the sotware MEGA [50]. Support values (%) at branch points are from 1000 NJ bootstrap replicates and shown only for values exceeding 60 %. GenBank references are for genomes (full-length 16S rRNA gene extracted) or are the near full-length 16S rRNA sequences [51]. Bold denotes a genome sequence available (NCBI or GOLD), whereas bold blue denotes the published genomes: Nitrosomonas europaea [52], "Nitrosomonas eutropha" [53], "Nitrosomonas communis" [54], Nitrosomonas sp. AL212 [55], "Nitrosomonas ureae" [56], Nitrosomonas sp. Is79A3 [57], Nitrosospira briensis C-128 (this study), Nitrosospira lacus APG3 [11] and Nitrosospira multiformis [12] for heterotrophic contamination by plating 0.1 mL on ¼ strength nutrient agar plates and incubating for two weeks. Cells were harvested from four 100 mL cultures by centrifugation (13,000 RCF for 30 min). Bacterial genomic DNA (gDNA) was isolated using the CTAB protocol recommended by JGI [20]. Size and quality of the gDNA was assessed via gel electrophoresis and amplification of the V4 region of the 16S rRNA gene using universal primers [21] followed by sequencing at the Center for Integrative Biosystems, USU on the ABI PRISM™ 3730 DNA Analyzer using BigDye terminator chemistry. The gDNA was of the expected size (greater than 23 kbp) and no contaminating organisms were detected by partial 16S rRNA gene sequencing of 10 replicate reactions or by plating. Approximately 20 μg of DNA was submitted to JGI for sequencing.

Genome sequencing and assembly
The genomic DNA of Nitrosospira briensis C-128 was sequenced at the DOE JGI using the Pacific Biosciences (PacBio) sequencing technology [22]. All general aspects of sample handling, library construction and sequencing Phylum Proteobacteria TAS [45] Class Betaproteobacteria TAS [7,46] Order Nitrosomonadales TAS [5,46] Family Nitrosomonadaceae TAS [4,46] Genus Nitrosospira TAS [6,8] Species Nitrosospira briensis TAS [6,8] Strain C-128 IDA Gram stain negative TAS [14] Cell shape Spiral/vibrioid IDA Motility motile TAS [14] Sporulation Non-sporulating TAS [14] Temperature range 15-30°C TAS [14] Optimum temperature 25-28°C TAS [14] pH range; Optimum 6.0-8.2;7.0 TAS [14] Carbon source carbon dioxide; carbonate TAS [14] Energy source ammonia oxidation TAS [14] Energy metabolism chemolithotroph TAS [14] MIGS 1000 nm a b Fig. 2 Electron micrographs of N. briensis. A) TEM prepared by negative staining as previously described [14,15]. Scale is 1000 nm. B) SEM of Nitrosospira briensis C-128. Glass coverslips were placed in a growing culture for approximately one month, removed and then fixed with 2 % glutaraldehyde in 0.1 % HEPES buffer overnight. The samples were subjected to alcohol series dehydration (50-100 % ethanol) and then chemically dried using hexamethyldisilazane. The image shows presumptive invaginations of the membranes of the cell. Scale is 500 nm followed JGI isolate sequencing protocols. A PacBio SMRTbell™ library was constructed and sequenced on the PacBio RS platform, which generated 148,206 reads totaling 519.8Mbp. Raw reads were assembled using HGAP v. 2.2.0.p1 [23]. The final draft assembly contained one contig in one scaffold, totaling 3.2 Mbp in size. The input read coverage was 176.1×. An earlier version of the genome was sequenced using the Illumina Hi-Seq 2000 platform. However, this earlier sequence assembly JHVX00000000.1 remained in 31 scaffolds (sequences JHVX01000001.1-JHVX01000031.1) with the nearly identical repeats of several key catabolic gene clusters remaining unresolved. Previously, genome closure for Nitrosospira [12] was achieved only after extensive directed finishing to correctly assemble long nearly identical repeats of gene clusters encoding key catabolic modules including ammonia monooxygenase (amo) for the activation of substrate and hydroxylamine dehydrogense (haoA) and hemecytochrome c proteins (cycAB) for the extraction of electrons and their delivery to the quinone pool in the membrane [24]. The long read capability of the PacBio platform and our depth of coverage enabled sufficient discrimination of repeats to assemble across multiple nearly identical regions into a single contig representing the chromosome of the bacterium. For predicted genes outside of gaps and repeat regions the PacBio and the Illumina predicted genes were 100 % identical. Therefore, we did not combine the Illumina Hi-Seq data with the PacBio data for the complete genome sequence CP012371 reported here.

Genome annotation
Genes were identified using Prodigal [25], as part of the JGI's Microbial annotation pipeline followed by a round of manual curation using GenePRIMP [26]. The predicted CDSs were translated and used to search the NCBI nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. Transfer RNA genes were identified using the tRNAScanSE tool [27]. Ribosomal RNA genes were found by searches against models of the ribososmal RNA genes built from SILVA [28]. Other non-coding RNAs were found using INFER-NAL [29]. Further gene prediction and manual curation was performed within the Integrated Microbial Genomes (IMG) platform [30] developed at JGI.

Genome properties
The genome of Nitrosospira briensis C-128 contains 3,210,113-bp in one chromosome with a GC content of 53.25 % and no plasmids (Fig. 3). The genome contains one complete ribosomal RNA operon similar to other AOB [3]. Coding bases (2,758,471) comprised 85.93 % of the total. We identified 3018 protein encoding genes, 55 RNA genes and 130 pseudogenes. For the identified genes, 74.23 % had a function prediction associated with them. The two-way average nucleotide identity [31] between the chromosomes of Nitrosospira multiformis ATCC 25196 [9,32,33] and Nitrosospira briensis C-128 was found to be 77.2 % confirming species delineation [34]. The genome statistics are summarized in Table 3 and genes associated with COG functional categories are summarized in Table 4.
CRISPR/Cas System Nitrosospira briensis C-128 contains a CRISPR/Cas system located at F822_1846-1851 suggestive of phage interactions [39]. The CRISPRassociated (CAS) proteins belong to the subtype 1-F (Yersinia pestis type) [40]. The CRISPR contains 11 spacers each with 32 bp. No matches between these spacers and protospacers in viral genomes were detected in the NCBI non-redundant database. The direct repeat sequence in the CRISPR is 28 bp: TTTCTGAGCTGCCTATGCGG-CAGTGAAC. As soil viral metagenomes become better characterized, associations between viral protospacers and the spacers found in N. briensis' CRISPR may help to identify possible phage types of N. briensis.

Conclusions
Nitrosospira briensis C-128 has a suite of genes enabling it to survive in soil environments as a chemolithoautotroph. The completion of several genomes in the Nitrosospira genus will facilitate a comprehensive analysis of the genetic toolkit that enables these AOB to co-inhabit the terrestrial niche. Further experiments elucidating gene function, especially those involved in the metabolism of nitrogen oxides and related to nitrosative stress [41], will increase the relevance of the completed genome of Nitrosospira briensis C-128. The evolutionary relationships in the genera of the Nitrosomonadaceae are currently under reconsideration. Endnotes 1 Editor's note -Readers are advised that the published record regarding the type strain and a proposed neotype strain of Nitrosospira briensis is problematic. Although