ABSTRACT
The complete genome sequences of two chemoautotrophic nitrite-oxidizing bacteria of the genus Nitrospina are reported. Nitrospina gracilis strain Nb-211 was isolated from the Atlantic Ocean, and Nitrospina sp. strain Nb-3 was isolated from the Pacific Ocean. We report two highly similar ~3.07-Mbp genome sequences that differ by the presence of ferric iron chelator (siderophore) biosynthesis genes.
ANNOUNCEMENT
Nitrospina species are aerobic, chemoautotrophic, nitrite-oxidizing bacteria that have so far only been found in marine habitats (1), where they play an important role in the nitrogen cycle (2). Nitrospina gracilis Nb-211 (ATCC 25379), first described in 1971 (3), was isolated from surface waters (13-m depth) of the Atlantic Ocean approximately 200 miles from the mouth of the Amazon River. Nitrospina sp. strain Nb-3 was isolated from the Pacific Ocean off the coast of Peru and has not been validly described; however, its 16S rRNA gene sequence was published in 1994 (4). Both strains belong to the Nitrospinaceae family within the Nitrospinae/Nitrospinota (Joint Genome Institute/Genome Taxonomy Database [JGI/GTDB]) phylum.
For genomic sequencing, cultures were grown in 2-L glass bottles in artificial seawater medium containing 2 mM nitrite and incubated in the dark without agitation as described previously (5). Cells were collected via centrifugation (1 h, 15,000 × g, 10°C), and DNA was extracted from the cell pellets using a cetyltrimethylammonium bromide (CTAB)-phenol-chloroform protocol (6). Draft genomes were generated at the DOE JGI using the Pacific Biosciences (PacBio) sequencing technology (7). Genomic DNA was sheared to 10 kb using g-TUBE columns (Covaris) and subjected to library preparation using the SMRTbell Express template prep 2.0 kit. The PacBio SMRTbell library was purified and size-selected using AMPure PB beads and sequenced on the PacBio Sequel platform, which generated 123,391 subreads (5,002.7 ± 3,337.2 bp) totaling 617,291,847 bp for strain Nb-211 and 89,053 subreads (6,935.6 ± 5,348.8 bp) totaling 617,633,853 bp for strain Nb-3. Reads of >5 kb were assembled with HGAP (v. smrtlink/8.0.0.80529, HGAP 4 [1.0]) using default settings (8). The input read coverage was 188.4× for strain Nb-211 and 189.5× for strain Nb-3. The final draft genome sequences consisted of one scaffold each, with a total size of 3,069,626 bp for strain Nb-211 and 3,075,869 bp for strain Nb-3 (Table 1). We confirmed complete circularization with the Circlator pipeline v.1.5.5 (9), which uses nucmer v.3.1 (10) to check for alignment between assembled contigs at opposite ends of the assembly, identifying a 50,007-bp alignment with 100% identity for strain Nb-211 and 50,012 bp with 99.99% identity for strain Nb-3.
TABLE 1.
Genome features of Nitrospina gracilis Nb-211 and Nitrospina sp. Nb-3
| Strain | GenBank accession no. | JGI taxon ID | Genome size (bp) | G+C content (%) | No. of DNA scaffolds | No. of total genes | No. of protein coding genes | No. of rRNA operons | No. of trRNA operons | CRISPR count |
|---|---|---|---|---|---|---|---|---|---|---|
| N. gracilis Nb-211 | JAKJKD010000001 | 2917506613 | 3,069,626 | 57.43 | 1 | 2,939 | 2,879 | 1 | 50 | 2 |
| Nitrospina sp. Nb-3 | JAKJKC010000001 | 2929071401 | 3,075,869 | 56.21 | 1 | 2,939 | 2,846 | 1 | 49 | 0 |
Both genomes were annotated using the Integrated Microbial Genomes (IMG) Annotation Pipeline (IMGAP) v.5.0.22/3. The genome of Nitrospina gracilis Nb-211 contains 2,939 coding DNA sequences (CDS), and that of Nitrospina sp. Nb-3 contains 2,905 CDS. Both genome sequences share an average nucleotide identity (ANI) of 85.5%, well below the intraspecies threshold of 96.5% (11). Strain Nb-3 shares 99.98% ANI with the previously published draft genome sequence of Nitrospina gracilis strain 3/211 (12), indicating that the latter likely derives from the culture originally designated strain Nb-3 (4). Strain Nb-3 encodes a putative iron chelator (siderophore) biosynthesis gene cluster, which is absent in strain Nb-211, potentially reflecting adaptations to differences in iron availability in the respective ocean basins the strains were isolated from. Strain Nb-211 was isolated near the Amazon River, which is a source of iron to the Atlantic Ocean (13), while strain Nb-3 was isolated from the relatively iron-deplete North Pacific (14).
Data availability.
The whole-genome shotgun sequencing project of Nitrospina gracilis Nb-211 has been deposited at DDBJ/ENA/GenBank under BioProject number PRJNA708439 and accession number JAKJKD000000000. The whole-genome shotgun sequencing project of Nitrospina sp. Nb-3 has been deposited at DDBJ/ENA/GenBank under BioProject number PRJNA783628 and accession number JAKJKC000000000. The NCBI Sequence Read Archive (SRA) accession numbers for the raw reads are SRR17430281 for strain Nb-211 and SRR17430190 for strain Nb-3.
ACKNOWLEDGMENTS
This work was supported by a Simons Foundation Early Career Investigator Award (3435889) and a U.S. National Science Foundation award OCE-1924512 to A.E.S. B.B. was supported by the Austrian Science Fund (FWF) project number J4426-B. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under contract number DE-AC02-05CH11231. These data were generated for JGI proposal number 506203 to B.B. and A.E.S.
We thank Christa Pennacchio, who managed the project at JGI.
Contributor Information
Barbara Bayer, Email: bbayer@ucsb.edu.
Alyson E. Santoro, Email: asantoro@ucsb.edu.
Frank J. Stewart, Montana State University
REFERENCES
- 1.Daims H, Lücker S, Wagner M. 2016. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends Microbiol 24:699–712. doi: 10.1016/j.tim.2016.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Kuypers MMM, Marchant HK, Kartal B. 2018. The microbial nitrogen-cycling network. Nat Rev Microbiol 16:263–276. doi: 10.1038/nrmicro.2018.9. [DOI] [PubMed] [Google Scholar]
- 3.Watson SW, Waterbury JB. 1971. Characteristics of two marine nitrite oxidizing bacteria, Nitrospina gracilis nov. gen. nov. sp. and Nitrococcus mobilis nov. gen. nov. sp. Archives of Microbiology 77:203–230. [Google Scholar]
- 4.Teske A, Alm E, Regan JM, Toze S, Rittmann BE, Stahl DA. 1994. Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. J Bacteriol 176:6623–6630. doi: 10.1128/jb.176.21.6623-6630.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bayer B, Saito MA, McIlvin MR, Lücker S, Moran DM, Lankiewicz TS, Dupont CL, Santoro AE. 2021. Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions. ISME J 15:1025–1039. doi: 10.1038/s41396-020-00828-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.JGI. 2012. Bacterial genomic DNA isolation using CTAB. https://jgi.doe.gov/wp-content/uploads/2014/02/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf.
- 7.Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, et al. 2009. Real-time DNA sequencing from single polymerase molecules. Science 323:133–138. doi: 10.1126/science.1162986. [DOI] [PubMed] [Google Scholar]
- 8.Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. doi: 10.1038/nmeth.2474. [DOI] [PubMed] [Google Scholar]
- 9.Hunt M, De Silva N, Otto TD, Parkhill J, Keane JA, Harris SR. 2015. Circlator: automated circularization of genome assemblies using long sequencing reads. Genome Biol 16:294. doi: 10.1186/s13059-015-0849-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. 2004. Versatile and open software for comparing large genomes. Genome Biol 5:R12. doi: 10.1186/gb-2004-5-2-r12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, Mavrommatis K, Kyrpides NC, Pati A. 2015. Microbial species delineation using whole genome sequences. Nucleic Acids Res 43:6761–6771. doi: 10.1093/nar/gkv657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lücker S, Nowka B, Rattei T, Spieck E, Daims H. 2013. The genome of Nitrospina gracilis illuminates the metabolism and evolution of the major marine nitrite oxidizer. Front Microbiol 4:27. doi: 10.3389/fmicb.2013.00027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Subramaniam A, Yager PL, Carpenter EJ, Mahaffey C, Björkman K, Cooley S, Kustka AB, Montoya JP, Sañudo-Wilhelmy SA, Shipe R, Capone DG. 2008. Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean. Proc Natl Acad Sci USA 105:10460–10465. doi: 10.1073/pnas.0710279105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Moore JK, Braucher O. 2007. Observations of dissolved iron concentrations in the World Ocean: implications and constraints for ocean biogeochemical models. Biogeosciences 4:1241–1277. doi: 10.5194/bgd-4-1241-2007. [DOI] [Google Scholar]
Associated Data
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Data Availability Statement
The whole-genome shotgun sequencing project of Nitrospina gracilis Nb-211 has been deposited at DDBJ/ENA/GenBank under BioProject number PRJNA708439 and accession number JAKJKD000000000. The whole-genome shotgun sequencing project of Nitrospina sp. Nb-3 has been deposited at DDBJ/ENA/GenBank under BioProject number PRJNA783628 and accession number JAKJKC000000000. The NCBI Sequence Read Archive (SRA) accession numbers for the raw reads are SRR17430281 for strain Nb-211 and SRR17430190 for strain Nb-3.