Complete genome sequence of Afipia carboxidovorans strain SH125, a non-denitrifying nitrous oxide-reducing bacterium isolated from anammox biomass

Abstract

Here, we report a genome sequence of Afipia carboxidovorans strain SH125 isolated from an anammox reactor. This facultative anaerobic strain possesses the clade I-type nitrous oxide (N2O) reductase gene, devoid of nitrite- and nitric oxide reductase genes. Deciphering the genome will help explore N2O reducers instrumental in N2O mitigation.

Full text

| Environmental Microbiology | Announcement
Complete genome sequence of Apia carboxidovorans strain
SH125, a non-denitrifying nitrous oxide-reducing bacterium
isolated from anammoxbiomass
Kohei Oba,1 Shohei Yasuda,2 Akihiko Terada1,3
AUTHOR AFFILIATIONS See aliation list on p. 2.
ABSTRACT Here, we report a genome sequence of Apia carboxidovorans strain SH125
isolated from an anammox reactor. This facultative anaerobic strain possesses the clade
I-type nitrous oxide (N2O) reductase gene, devoid of nitrite- and nitric oxide reductase
genes. Deciphering the genome will help explore N2O reducers instrumental in N2O
mitigation.
KEYWORDS nitrous oxide, nosZ, anammox, denitrication, N2O-reducing bacteria
N2O-reducing bacteria are the primary consumers of N2O (1), a highly potent
greenhouse and ozone-depleting gas (2, 3). More descriptions regarding the
phylogeny, functions, and physiologies of N2O-reducing bacteria toward mitigating
N2O emissions are required (4, 5). Apia carboxidovorans strain SH125, detected as a
candidate for N2O sink in soils (6) and engineered systems (7, 8), was obtained from
anammox biomass enriched by exogenous N2O supply (9). The biomass was serially
diluted with 20× diluted phosphate-buered saline and spread onto 1.0 wt% gellan
gum plates containing the anammox medium (10). Cultures were anaerobically grown
using a jar lled with a deoxygenating reagent (Anaeropack, Mitsubishi Gas Chemical,
Tokyo, Japan) and N2O gas [5% (vol/vol)], followed by colony picking. The isolate showed
N2O consumption activity when N2O was added to Japan Collection of Microorganisms
(JCM) Medium No. 12 (nutrient broth medium with 0.5% NaCl, pH = 7.5) under an anoxic
condition (Fig. 1). This activity test, referring to reference (11), was initiated at 30°C by
adjusting the headspace gaseous N2O concentration of 1.7mg N/L.
After an aerobic incubation using JCM Medium No. 12, the genome was extracted
with a phenol-chloroform method (12, 13) and puried by a CTAB/NaCl solution. RNA as
a contaminant in the genomic DNA was decomposed by RNaseA (TaKaRa Bio, Inc., Shiga,
Japan). Barcoding and library preparation were conducted using Native Barcoding
Expansion 1–12 EXP-NBD104 [Oxford Nanopore Technologies (ONT), Oxford, UK] with
ONT Long Fragment Buer and ONT Ligation Sequencing Kit SQK-LSK109. Sequencing
was conducted on an R9.4.1 ow cell with the MinION Mk1B. Basecalling was performed
using Guppy v6.5.7 (https://community.nanoporetech.com/downloads) with a super-
accurate model (options –cong dna_r9.4.1_450bps_sup.cfg -x cuda:0). Subsequently,
demultiplex and barcode sequence removal was performed by applying Guppy’s
guppy_barcoder command. This resulted in 71,104 raw reads (1,373,410,240 bp), with an
N50 value of 41,150 bp. NanoFilt v2.8.0 (14) was used to lter low-quality reads (Q < 12)
and short reads (<10,000 bp). Error correction was performed using Canu v2.2 (15), and
genome assembly was generated by Flye v2.9.3 (16) (option –nano-corr). The assembly
was further polished using Medaka v1.11.2 (https://github.com/nanoporetech/medaka)
(option -m r941_min_sup_g507). Completeness (99.68%) and contamination (0.00%)
were evaluated with CheckM v1.2.2 lineage_wf (17). DDBJ Fast Annotation and
March 2024 Volume 13 Issue 3 10.1128/mra.01279-23 1
Editor J. Cameron Thrash, University of Southern
California, USA
Address correspondence to Akihiko Terada,
akte@cc.tuat.ac.jp.
The authors declare no conict of interest.
See the funding table on p. 3.
Received 11 January 2024
Accepted 13 February 2024
Published 22 February 2024
Copyright © 2024 Oba et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution 4.0 International license.

Submission Tool v1.6.0 (18, 19) was applied for annotation. Default parameters were used
for all software unless otherwise specied.
The genome consisted of a single circularized contig with a length of 3,743,720 bp
(284-fold coverage) and G + C content of 62.3%. The genome was predicted to encode
3,682 protein-coding sequences, 3 rRNA genes, and 51 tRNA genes. The genome
annotation did not identify any nitrite reductase and nitric-oxide reductase but a clade
I N2O reductase. The denitrifying genotype suggests the strain is a non-denitrifying
N2O-reducing bacterium. The genome sequence of A. carboxidovorans strain SH125 will
contribute to extending a comprehensive understanding of its role as an N2O sink.
ACKNOWLEDGMENTS
We thank Mr. Shohei Nagaoka, Dr. Megumi Kuroiwa, and the late Ms. Kanako Mori for
their experimental support.
This research was funded by the Grant-in-Aid for Scientic Research (Grant nos.
20H04362 and 23H03565), Fostering Joint International Research (20KK0243) from the
Japan Society for the Promotion of Science (JSPS), and the Kurita Water and Environment
Foundation (22T012).
AUTHOR AFFILIATIONS
1Department of Applied Physics and Chemical Engineering, Tokyo University of
Agriculture and Technology, Koganei, Tokyo, Japan
2Civil Engineering, School of Engineering, College of Science and Engineering, University
of Galway, Galway, Ireland
3Global Innovation Research Institute, Tokyo University of Agriculture and Technology,
Fuchu, Tokyo, Japan
FIG 1 Time course of N2O concentration by A. carboxidovorans strain SH125 during batch culture under anaerobic conditions. Solid circles represent N2O
concentrations. Each error bar represents a standard error of the mean. The experiment was conducted in triplicate.
Announcement Microbiology Resource Announcements
March 2024 Volume 13 Issue 3 10.1128/mra.01279-23 2

AUTHOR ORCIDs
Kohei Oba http://orcid.org/0009-0005-0862-5843
Shohei Yasuda http://orcid.org/0009-0007-2827-3675
Akihiko Terada http://orcid.org/0000-0002-9258-6912
FUNDING
Funder Grant(s) Author(s)
MEXT | Japan Society for the Promotion of
Science (JSPS)
20H04362, 23H03565,
20KK0243
Akihiko Terada
Kurita Water and Environment Foundation
(KWEF)
22T012 Akihiko Terada
AUTHOR CONTRIBUTIONS
Kohei Oba, Conceptualization, Data curation, Formal analysis, Investigation, Methodol
ogy, Resources, Software, Visualization, Writing – original draft | Shohei Yasuda, Data
curation, Investigation, Methodology, Supervision, Validation, Writing – review and
editing | Akihiko Terada, Conceptualization, Data curation, Funding acquisition, Project
administration, Resources, Supervision, Validation, Writing – review and editing
DATA AVAILABILITY
This genome sequence has been deposited on DDBJ under the accession number no.
AP029055. Sequencing data are available in the Sequence Read Archive under accession
number no. DRR517369.
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