Draft genome sequence of Nitrobacter vulgaris DSM 10236^T

Nitrite-oxidizing bacteria of the genus Nitrobacter play essential roles in nitrogen cycling in both terrestrial and aquatic environments. They are facultative lithoautotrophs that can grow in the presence or absence of oxygen (1 – 3). Nitrobacter vulgaris is a Gram-negative mesophile that has been isolated from many environments, including freshwater and soil (4). To date, genome sequencing has been performed on only one strain of N. vulgaris (Ab₁) (5). The type strain N. vulgaris DSM 10236^T (also known as N. vulgaris strain Z^T) was isolated from a Bauersberg waterworks sand filter in Hamburg, Germany (4). The genome sequence of N. vulgaris DSM 10236^T will support further study of its role in the nitrogen cycle.

N. vulgaris DSM 10236^T was grown in mixotrophic Nitrobacter medium DSMZ M.756a [https://mediadive.dsmz.de/medium/756a] at 28°C for 10 days. Genomic DNA was extracted using the MasterPure Gram-positive DNA Purification Kit (Lucigen) and sent to the Department of Energy, Joint Genome Institute for sequencing.

An Illumina short-insert DNA library was prepared with a PerkinElmer Sciclone robotic liquid handling system using a Roche KAPA Biosystems library preparation kit. DNA (200 ng) was sheared to 300 bp using a Covaris LE220, size-selected by double-SPRI, and then end-repaired, A-tailed, and ligated with Illumina-compatible sequencing adaptors containing a unique molecular index barcode. The library was quantified using KAPA Biosystems’ next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. The library was then multiplexed with other libraries, and the pool was sequenced on an Illumina NovaSeq 6000 using NovaSeq XP v1 reagent kits (Illumina), S4 flow cell, following a 2 × 150 indexed run recipe. In total, 17,899,282 sequence reads were generated. Raw sequences were quality filtered using BBTools v.38.86 per JGI standard operating practice (SOP) protocol 1061 (6), producing 1,499,468,893 bp of sequence. The filtered and normalized reads were assembled using SPAdes (version v3.13.0) with the assembly parameters ––phred–offset 33 ––cov–cutoff auto –t 16 –m 64 ––careful –k 25,55,95 (7). Contigs with lengths <1 kb were discarded (BBTools reformat.sh: minlength). The final draft assembly was then annotated using the IMG Annotation Pipeline v.5 (8) (Table 1).

Genome analyses were performed using IMG/M (9). The genome sequence of N. vulgaris DSM 10236^T has a pairwise average nucleotide identity of 98.8% and 86.5% with the sequences of N. vulgaris Ab₁ and N. hamburgensis X14, respectively (10). The genome contains all genes required for chemolithotrophic growth on nitrite (narK, nxrA, nxrB, nxrC, and nxrD), and its nitrite-oxidizing enzyme (NXR) operon is organized identically to the NXR operon in N. hamburgensis X14 and N. vulgaris Ab₁ (1). Interestingly, N. vulgaris DSM 10236^T appears to be the only Nitrobacter genome (of seven sequenced to date) with a predicted nitrous oxide reductase gene (nosZ, JGI gene ID 2829793416). It is located in an operon containing a nosR nitrous oxide reductase transcriptional regulator and a nitrous oxidase accessory protein. These genes are typically associated with denitrifying bacteria, and therefore further research is needed to explore possible connections between N. vulgaris DSM 10236^T and denitrification (11).

The work (proposal DOI: https://doi.org/10.46936/10.25585/60001087) was conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility that is supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231. This announcement was largely prepared by undergraduate students, and we gratefully acknowledge JGI for initiating and supporting it as an educational project (the “Adopt-a-genome” Project). We are also grateful to Meike Döppner, DSMZ, for DNA quality control.