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  Standards in Genomic Sciences (2013) 7: 469-482   DOI:10.4056/sigs.3517166 The Genomic Standards Consortium  Complete genome sequence of   Nitrosomonas sp.  Is79, an ammonia oxidizing bacterium adapted to low ammonium concentrations Annette Bollmann 1* , Christopher J. Sedlacek 1 , Jeanette Norton 2 , Hendrikus J. Laanbroek 3 , Yuichi Suwa 4 , Lisa Y. Stein 5 , Martin G. Klotz 6 , Daniel Arp 7 , Luis Sayavedra-Soto 7 , Megan Lu 8 , David Bruce 8 , Chris Detter 8 , Roxanne Tapia 8 , James Han 9 , Tanja Woyke 9 , Susan M. Lu-cas 9 , Sam Pitluck 9 , Len Pennacchio 9 , Matt Nolan 9 , Miriam L. Land 10 , Marcel Huntemann 9 , Shweta Deshpande 9 , Cliff Han 8 , Amy Chen 9 , Nikos Kyrpides 9 , Konstantinos Mavromatis 9 , Victor Markowitz 9 , Ernest Szeto 9 , Natalia Ivanova 9 , Natalia Mikhailova 9 , Ioanna Pagani 9 , Amrita Pati 9 , Lin Peters 9 , Galina Ovchinnikova 9 , and Lynne A. Goodwin 8   1  Miami University, Oxford, Ohio, USA 2  Utah State University, Logan, Utah, USA 3  Netherlands Institute for Ecology, Wageningen, The Netherlands 4  Chuo University, Tokyo, Japan 5  University of Alberta, Edmonton, Alberta, Canada 6  University of North Carolina, Charlotte, North Carolina, USA 7  Oregon State University, Corvallis, Oregon, USA 8  Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 9  DOE Joint Genome Institute, Walnut Creek, California, USA 10  Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA * Corresponding author : Annette Bollmann (bollmaa@miamioh.edu) Keywords :  Nitrosomonas , Ammonia-oxidizing bacteria, Ammonia oxidation, nitrification, ni-trogen cycle, freshwater, oligotrophic Nitrosomonas sp.  Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family  Nitrosomonadaceae  within the phylum  Proteobacteria . Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately re-sulting in the production of nitrate.  Nitrosomonas sp.  Is79 is an ammonia oxidizer of high in-terest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006. Introduction Nitrosomonas sp.  Is79 is a betaproteobacterial am-monia-oxidizer. The genus name  Nitrosomonas  de-rived from nitrosus (Latin: nitrous) and monad (Greek: a unit) meaning nitrite producing unit. Nitrosomonas sp.  Is79 was enriched and isolated from freshwater sediment [1]. Closely related strains can be found in freshwater environments around the world [2-6]. Other  Nitrosomonas  spe-cies have been isolated from freshwater and ma-rine systems, wastewater treatments plants and soils [7,8]. The genome sequence of   Nitrosomonas sp.  Is79 is the fifth genome of the betaproteobacterial ammonia oxidizers that has been completed by DOE-Joint Genome Institute (CP002876.1) [9-12]. Here we present summary classification and a set of features for  Nitrosomonas sp.  Is79, together with the description of the com-plete genome sequence and annotation. Classification and features Fourteen species with valid published names are currently assigned to the  Nitrosomonadaceae  [13-19]. Besides these described species, many un-described isolates are available [7,20-22]. These strains were isolated from freshwater, marine sys-tems, wastewater and soils, share the traits of aer-obic chemolithoautotrophic metabolism using am-monia as an electron donor, and carbon dioxide as carbon source (Table 1). Strain Is79 was isolated into pure culture by A. Bollmann in 2001 and maintained in liquid stock  Nitrosomonas sp . Is79 470 Standards in Genomic Sciences cultures since then, being transferred to fresh me-dium approximately once per month. The strain has not been deposited in a culture collection, but can be obtained from A.B. upon request. Based on 16S rRNA gene sequences, the strains most closely related to  Nitrosomonas sp.  Is79 are  Nitrosomonas oligotropha  Nm10 with 97.8% sequence identity and Nitrosomonas ureae  Nm45 with 97% se-quence identity (Figure 1). The sequence of the single 16S rRNA gene copy in the genome of Nitrosomonas sp.  Is79 differs by two nucleotides from the previously published 16S rRNA gene se-quence (AJ621026), both of which are insertions into the whole genome sequence. Growth studies show that   Nitrosomonas sp.  Is79 has a chemolithoautotrophic metabolism using ammonia as energy source producing nitrite. The strain is strictly aerobic and fixing carbon autotrophically from carbon dioxide via the Calvin cycle [37].  Nitrosomonas sp.  Is79 is adapted to low ammonium concentrations and has been isolated after enrichment in continuous culture under ammonium-limited conditions [1]. Further exper-iments showed that   Nitrosomonas sp.  Is79 was able to grow and outcompete Nitrosomonas europaea  under ammonium-limited conditions [38] and has K s  and K m  values for ammonium low-er than other ammonia-oxidizing bacteria [Bollmann unpublished]. Figure 1 . Phylogenetic tree showing the position of   Nitrosomonas sp.  Is79 relative to the other described strains within the family.  Nitrosomonas sp.  AL212 is not a formally described strain, but was included be-cause the whole genome of this strain became recently available [12]. The tree was constructed from 1,272 aligned characters of the 16S rRNA gene sequence under the maximum likelihood criterion and rooted in ac-cordance with a current taxonomy using the software package MEGA [35]. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates (left) and from 1,000 maximum parsimony bootstrap replicates (right) if larger than 60% [35]. Strains with whole genome sequencing projects registered in GOLD [36] are shown in red and the published in red-bold:  Nitrosomonas europaea  (AL954747), Nitrosomonas eutropha  (CP000450), Nitrosospira multiformis  (CP000103), Nitrosomonas sp.  AL212 (CP002552) and Nitrosomonas sp.  Is79 (CP02876).    Bollman et al.  http://standardsingenomics.org 471 Table 1 . Classification and general features of   Nitrosomonas sp.  Is79 according to the MIGS recommendations [23] MIGS ID Property Term Evidence code Domain  Bacteria  TAS [24] Phylum  Proteobacteria  TAS [25] Class  Betaproteobacteria  TAS [26,27] Current classification Order  Nitrosomonadales  TAS [27,28] Family  Nitrosomonadaceae  TAS [27,29] Genus  Nitrosomonas  TAS [13,30-32] Species  Nitrosomonas  sp IDA Type strain Is79 IDA Gram stain negative NAS Cell shape rod-shaped, short NAS Motility not reported Sporulation none NAS Temperature range mesophile NAS Optimum temperature not reported Salinity < 50mM NaCl, very sensitive to salt TAS [1,33] MIGS-22 Oxygen requirement aerobic TAS [1] Carbon source carbon dioxide TAS [1] Energy source ammonia TAS [1] Energy metabolism chemolithoautotroph TAS [1] MIGS-23 Isolation and growth conditions Isolation after enrichment in chemostat under low substrate concentrations, adapted to low ammonium concentrations in the medium TAS [1] MIGS-6 Habitat freshwater TAS [1] MIGS-15 Biotic relationship free-living NAS MIGS-14 Pathogenicity None NAS Biosafety level 1 TAS [34] Isolation freshwater sediment TAS [1] MIGS-4 Geographic location Lake Drontermeer (Netherlands) TAS [1] MIGS-4.1 Latitude 52°58’N NAS MIGS-4.2 Longitude 5°50’E NAS MIGS-4.3 Depth 0.5 m (root zone in the littoral zone of the lake) TAS [1] MIGS-4.4 Altitude around sea level TAS [1] MIGS-5 Sample collection time Fall 1997 TAS [1] Evidence codes – IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e. a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e. not directly observed for living, iso-lated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from a living isolate by one of the authors or an expert mentioned in the acknowledgements. Genome sequencing and annotation Genome project history The organism was selected for sequencing as part of DOE-JGI program CSP 2006 because it is adapted to growth at low ammonium concentra-tions. The genome sequence is deposited in the Genome OnLine Database [36] and the complete genome is deposited in GenBank. Sequencing, fin-ishing and annotation were performed by DOE-Joint Genome Institute (JGI). A summary of the project information is shown in Table 2. Growth conditions and DNA isolation The strain  Nitrosomonas sp.  Is79 was grown in mineral salts medium with 5mM ammonium at 27°C until all ammonium was consumed [39]. DNA was isolated using the protocol recommended by JGI (Bacterial genomic DNA isolation using CTAB). Size and quality of the bulk DNA was determined according to DOE-JGI guidelines. The size of the gDNA was larger than 23 kbp as determined by agarose gel electrophoresis.  Nitrosomonas sp . Is79 472 Standards in Genomic Sciences Table 2.  Genome sequencing project information MIGS ID Property Term MIGS-31 Finishing quality Finished MIGS-28 Libraries used Three 454 pyrosequence libraries, standard and two paired end (9 and 7 kb average insert size) and one Illumina library MIGS-29 Sequencing platforms 454 Titanium, Illumina MIGS-31.2 Sequencing coverage 454 Titanium: 36.6 × and Illumina: 910.8 x MIGS-30 Assemblers Newbler version 2.3; VELVET version 1.0.13 MIGS-32 Gene calling method Prodigal 1.4, GenePRIMP INSDC ID CP002876 GenBank Date of Release July 05, 2010 GOLD ID Gc01870 NCBI project ID 52837 Database: IMG 2505679045 MIGS-13 Source material identifier Nitrosomonas sp.  Is79 Project relevance Environmental strain, nitrogen cycle Genome sequencing and assembly The draft genome of   Nitrosomonas sp.  Is79 was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [40] and 454 technologies [41]. For the genome, we constructed and sequenced an Illumina GAii shotgun library which generated 46,913,976 reads totaling 3,565.5 Mb, a 454 Titanium standard library which generated 252,425 reads and 2 paired end 454 libraries with an average insert size of 7 kb, and 9 kb which generated 401,484 reads totaling 173.6 Mb of 454 data. All general aspects of li-brary construction and sequencing performed at the JGI can be found at the JGI website [42]. The initial draft assembly contained 250 contigs in 3 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3-Prerelease-6/30/2009. The Newbler consensus sequences were computa-tionally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data were assem-bled with VELVET, version 1.0.13 [43] and the consensus sequence were computationally shred-ded into 1.5kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using par-allel phrap, version SPS – 4.24 (High Performance Software, LLC). The software Consed [44-46] was used in the following finishing process. Illumina data were used to correct potential base errors and increase the consensus quality using the software Polisher developed at JGI [Lapidus, un-published]. Possible mis-assemblies were correct-ed using gapResolution [Han, unpublished], Dupfinisher [47] or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR [Cheng, unpublished] primer walks. A total of 667 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total size of the ge-nome is 3,783,444 bp and the final assembly is based on 138.9 Mb of 454 draft data, which pro-vide an average 36.6 coverage of the genome and 3,461Mb of Illumina draft data, which provide av-erage 910.8× coverage of the genome. Genome annotation Genes were identified using Prodigal [48] as part of the Oak Ridge National Laboratory genome an-notation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [49]. The predicted CDSs were translated and used to search the National Center for Biotechnology In-formation (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases These data sources were com-bined to assert a product description for each predicted protein. Non-coding genes and miscel-laneous features were predicted using tRNAscan-SE [50], RNAmmer [51], Rfam [52], TMHMM [53], and signal P [54].
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