N₂O Reduction by Gemmatimonas aurantiaca and Potential Involvement of Gemmatimonadetes Bacteria in N₂O Reduction in Agricultural Soils

Abstract

Agricultural soil is the primary N₂O sink limiting the emission of N₂O gas into the atmosphere. Although Gemmatimonadetes bacteria are abundant in agricultural soils, limited information is currently available on N₂O reduction by Gemmatimonadetes bacteria. Therefore, the effects of pH and temperature on N₂O reduction activities and affinity constants for N₂O reduction were examined by performing batch experiments using an isolate of Gemmatimonadetes bacteria, Gemmatimonas aurantiaca (NBRC100505^T). G. aurantiaca reduced N₂O at pH 5–9 and 4–50°C, with the highest activity being observed at pH 7 and 30°C. The affinity constant of G. aurantiaca cells for N₂O was 4.4‍ ‍μM. The abundance and diversity of the Gemmatimonadetes 16S rRNA gene and nosZ encoding nitrous oxide reductase in agricultural soil samples were also investigated by quantitative PCR (qPCR) and amplicon sequencing ana­lyses. Four N₂O-reducing agricultural soil samples were assessed, and the copy numbers of the Gemmatimonadetes 16S rRNA gene (clades G1 and G3), nosZ DNA, and nosZ mRNA were 8.62–9.65×10⁸, 5.35–7.15×10⁸, and 2.23–4.31×10⁹ copies (g dry soil)^–1, respectively. The abundance of the nosZ mRNA of Gemmatimonadetes bacteria and OTU91, OUT332, and OTU122 correlated with the N₂O reduction rates of the soil samples tested, suggesting N₂O reduction by Gemmatimonadetes bacteria. Gemmatimonadetes 16S rRNA gene reads affiliated with OTU4572 and OTU3759 were predominant among the soil samples examined, and these Gemmatimonadetes OTUs have been identified in various types of soil samples.

N₂O gas is a notorious greenhouse gas because of its strong global warming potential (265-fold greater than that of carbon dioxide) and persistence in the atmosphere (ca. 114 years) (Ravishankara et al., 2009; Montzka et al., 2011; IPCC, 2014). N₂O gas also contributes to the loss of stratospheric ozone and has been recognized as the dominant ozone-depleting substance (Ravishankara et al., 2009; Montzka et al., 2011). Terrestrial soils greatly contribute to N₂O emissions (i.e., 6–7 ton g year^–1, corresponding to ca. 60% of total N₂O emissions) (Butterbach-Bahl et al., 2013; Cui et al., 2013; Tian et al., 2020), and N₂O gas is discharged from soils as a net result of soil N₂O production and consumption (Holtan-Hartwig et al., 2000). The application of nitrogenous fertilizers to agricultural soils is a common practice, but markedly increases N₂O emissions from agricultural soils (Seitzinger et al., 2000; Liu and Greaver, 2009; Bouwman et al., 2013). More than 50% of the annual consumption of nitrogenous fertilizers is currently derived from urea-based fertilizer consumption (Glibert et al., 2014), and urea is hydrolyzed to ammonia and carbon dioxide by ureolytic microorganisms (Oshiki et al., 2018). The ammonia produced is subsequently oxidized to nitrite and/or nitrate by nitrification, followed by the reduction of nitrite and nitrate to nitrogen gas by denitrification and/or anammox processes (Isobe and Ohte, 2014; Oshiki et al., 2016). N₂O gas is produced biotically and abiotically by these nitrification and denitrification processes (Ishii et al., 2011). Regarding the consumption of N₂O, the biological reduction of N₂O is the only reaction that acts as a N₂O sink. This N₂O reduction reaction is catalyzed by the multicopper enzyme, nitrous oxide reductase (NosZ), which catalyzes the reduction of two electrons of N₂O to produce N₂ (Richardson et al., 2009; Simon and Klotz, 2013). The abundance and diversity of nosZ in soils have received a great deal of attention as a functional gene marker of N₂O reducers. nosZ has been found in various bacterial and archaeal genomes and classified into two phylogenetically distinct clades, nosZ clades I and II, based on sequence homology (Sanford et al., 2012; Jones et al., 2013). The abundance and diversity of nosZ clade I have been investigated in various types of soils, whereas those of nosZ clade II were largely overlooked until 2012 (Sanford et al., 2012). nosZ clade II was overlooked because the previously published oligonucleotide primer set (i.e., the nosZ1F and nosZ1R primers) (Henry et al., 2006) utilized for the PCR amplification of environmental nosZ sequences did not cover the sequence divergence of nosZ clade II. Recent molecular ana­lyses, including quantitative PCR (qPCR), an amplicon sequencing ana­lysis of nosZ clade II, and a metagenomic ana­lysis of soil DNA, revealed the abundant distribution of nosZ clade II in various types of soils, similar to nosZ clade I (Sanford et al., 2012; Jones et al., 2013; Jones et al., 2014; Orellana et al., 2014; Domeignoz-Horta et al., 2015; Samad et al., 2016; Juhanson et al., 2017). In addition to their phylogenetic differences, N₂O reducers carrying nosZ clade II showed an affinity for N₂O that was up to two orders of magnitude higher than those carrying nosZ clade I (Yoon et al., 2016; Suenaga et al., 2018), suggesting their significant contribution to N₂O mitigation from soils because the concentration of N₂O is generally low in soils (i.e., typically less than 1‍ ‍μM) (Schreiber et al., 2012). A linear regression ana­lysis (Domeignoz-Horta et al., 2015; 2018; Samad et al., 2016) and structural equation modeling and network ana­lysis (Jones et al., 2014) of the gene abundance and diversity of nosZ clade II in terrestrial soils indicated the greater contribution of N₂O reducers carrying nosZ clade II to the soil N₂O sink capacity than those carrying nosZ clade I.

The phylum Gemmatimonadetes is currently recognized as one of the nine dominant soil phyla (Janssen, 2006) because the Gemmatimonadetes 16S rRNA gene has been frequently and abundantly detected in various terrestrial environments (Janssen, 2006; DeBruyn et al., 2011). The phylum Gemmatimonadetes contains phylogenetically diverse bacterial members that have been classified into at least five sublineages (Hanada and Sekiguchi, 2014), and the following isolates have been described: Gemmatimonas aurantiaca (Zhang et al., 2003), G. phototrophica (Zeng et al., 2017), G. groenlandica (Zeng et al., 2021), Gemmatirosa kalamazoonensis (DeBruyn et al., 2013), Roseisolibacter agri (Pascual et al., 2018), and Longimicrobium terrae (Pascual et al., 2016). G. aurantiaca and Gt. kalamazoonesis carry nosZ clade II (accession numbers AP009153.1 and CP007128.1, respectively), and N₂O reduction by G. aurantiaca was recently demonstrated in a culture-dependent manner (Park et al., 2017; Chee-Sanford et al., 2019). Although G. aurantiaca has been characterized as an obligate aerobic heterotroph (Zhang et al., 2003), this bacterium reduced N₂O under not only aerobic, but also microaerobic and anoxic conditions when partially oxic conditions were present (Chee-Sanford et al., 2019). G. aurantiaca cells transcribed nosZ when they reduced N₂O (Park et al., 2017), suggesting that the abundance of Gemmatimonadetes nosZ mRNA correlates with that of a metabolically-active Gemmatimonadetes bacterial population. The metabolic capability of N₂O reduction by G. aurantiaca and the widespread distribution of Gemmatimonadetes bacteria in various types of soils including agricultural soils (Jones et al., 2014; Orellana et al., 2014; Samad et al., 2016) led us to expect the contribution of Gemmatimonadetes bacteria to N₂O mitigation from soils. However, the physiological characteristics of G. aurantiaca for N₂O reduction have not yet been examined in detail, and limited information is currently available on the involvement of Gemmatimonadetes bacteria in N₂O reduction in soils. Physiological pH and temperature ranges and affinity constants are key physiological information for obtaining a more detailed understanding of microbial activities in natural and man-made ecosystems (Oshiki et al., 2016).

Therefore, the present study investigated 1) the physiological characteristics of G. aurantiaca associated with N₂O reduction, and 2) the involvement of Gemmatimonadetes bacteria in N₂O reduction in agricultural soils. The effects of pH and temperature on N₂O reduction activities and affinity constants for N₂O reduction by G. aurantiaca were examined by performing batch incubations and assessing N₂O reduction activities. The involvement of Gemmatimonadetes bacteria in N₂O reduction in agricultural soils was then analyzed by a soil incubation experiment in which the relationship between the N₂O reduction rates of soils and the abundance of Gemmatimonadetes bacteria was investigated. Agricultural soil samples were incubated with the addition of ¹⁵NO₃^– to evaluate N₂O reduction rates, and the abundance and diversity of the Gemmatimonadetes 16S rRNA gene and nosZ in the soil samples tested were evaluated by qPCR and amplicon sequencing ana­lyses. The above DNA-based ana­lyses potentially detect metabolically-inactive Gemmatimonadetes bacteria; therefore, qPCR and amplicon sequencing ana­lyses of Gemmatimonadetes nosZ mRNA were also conducted, and the relationship between N₂O reduction rates and the abundance of nosZ mRNA was investigated. This is the first study to show a correlation between N₂O reduction rates and the abundance of Gemmatimonadetes nosZ mRNA in agricultural soils, and the potential involvement of Gemmatimonadetes bacteria in N₂O reduction in soil is discussed.

Materials and Methods

Bacterial culture and incubation conditions

G. aurantiaca (NBRC100505^T) cells were cultivated aerobically at 30°C with shaking at 90‍ ‍rpm. NBRC822 medium (L^–1: glucose, 0.5 g; peptone [BD Difco, Becton Dickinson and company], 0.5‍ ‍g; yeast extract [BD Difco], 0.5 g; sodium glutamate, 0.5 g; KH₂PO₄, 0.44 g; [NH₄]₂SO₄, 0.1 g; MgSO₄·7H₂O, 0.1 g; pH 7.0) was used for cultivation. Stationary-phase cells were harvested by centrifugation (13,420×g, 10‍ ‍min), washed, and resuspended in fresh NBRC822 medium. The cell suspension was subjected to the following activity tests.

Effects of pH and temperature on the N₂O reduction activity of G. aurantiaca

G. aurantiaca cells were incubated at pH 5 to 10 and at 4 to 80°C, and the consumption of N₂O was examined. Three milliliters of NBRC822 medium was dispensed into 7.7-mL serum glass vials (Nichiden-Rika Glass), which were then sealed with butyl rubber stoppers and aluminum caps. The pH of NBRC822 medium was adjusted in the range of pH 5 to 10 by adding the following pH buffer at a final concentration of 20‍ ‍mM: 2-morpholinoethanesulfonic acid, monohydrate (MES) for pH 5 to 6.5, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) for pH 7 to 7.5, and N-[Tris(hydroxymethyl)methyl]glycine (Tricine) for pH 8 to 10. After purging the liquid phase with argon gas for 3‍ ‍min, the headspace was replaced with pure He gas (>99.99995%). One hundred microliters of the G. aurantiaca cell suspension and N₂O gas (GL Science) were injected into the vials at a final concentration of 0.1–0.5‍ ‍mg protein mL^–1 and 180‍ ‍nmol (N vial)^–1, respectively, using a gas tight syringe. The vials were incubated at 4 to 80°C in the dark, and changes in N₂O concentrations in the headspace over time were examined.

Partial oxic conditions were essential for initiating N₂O reduction by G. aurantiaca, which occurred after the depletion of O₂ (Park et al., 2017). Although O₂ was not added externally to the above vials, certain amounts of oxygen were available in the vials due to 1) the incomplete removal of O₂ (e.g., 3‍ ‍min of an argon gas purge) and 2) the carryover of O₂ from the inoculum, which were adequate to initiate N₂O reduction by G. aurantiaca.

Affinity of G. aurantiaca cells for N₂O

The value of K_s for N₂O was calculated based on the N₂O consumption rate assessed using a N₂O-specific microsensor (N₂O-MR) and micro-respirometric system (Unisense) as previously described (Suenaga et al., 2018). Briefly, G. aurantiaca cells suspended in NBRC822 medium (pH 7) were dispensed into a 3.0-mL closed chamber (Unisense) and stirred with a dedicated stirrer bar at 300‍ ‍rpm. N₂O-saturated NBRC 822 medium (27–24‍ ‍mM N₂O at 20–25°C) was added using a Hamilton syringe to reach a final N₂O concentration of 30‍ ‍μM. The chamber was incubated at 30°C, and the N₂O concentration in the liquid phase was continuously monitored using SensorTrace Suite ver.2.8.0 (Unisense). N₂O concentration profiles were smoothed in Sigma Plot 13.0 to remove high frequency noise. The value of K_s was assessed by fitting N₂O concentrations and instantaneous consumption rates to the Michaelis-Menten equation using the solver function in Microsoft Excel. N₂O-MR was calibrated prior to incubations using aqueous N₂O solution as described in the manual provided by the supplier.

N₂O reduction by agricultural soils

Agricultural soils were incubated with the addition of ¹⁵NO₃^–, and the production of ^15-15N₂O and ^15-15N₂ gas was examined (Fukushi et al., 1984; Shan et al., 2016). Agricultural soils (designated Soils A, B, C, and D) were collected at four sites in Nagaoka city, Niigata, Japan (Table S1). Surface layers (0 to 5‍ ‍cm) were collected from five spots at each site (5×1 m) and sieved (pore diameter, 2‍ ‍mm) to remove concomitant gravel. The 10 g-wet of the sieved soils was dispensed into 50-mL serum glass vials, Na¹⁵NO₃^– was added at a final concentration of 71.4‍ ‍μmol (N vial)^–1, and the vials were sealed using a butyl rubber stopper and aluminum seal. The head space was replaced with pure He gas, and the vials were incubated in the dark at 30°C in quadruplicate. After an incubation for 69 h, incubated soils were collected from the vials using a sterile spatula and subjected to total DNA and RNA extraction.

Total DNA and RNA extractions

Total DNA and RNA extractions were performed using a Power Soil DNA Isolation and RNA PowerSoil Total RNA Isolation Kit (Qiagen Japan), respectively, according to the manufacturer’s protocols. Extracted RNA was transcribed to cDNA using a random 6mer primer and Prime Script RT Reagent Kit (TaKaRa Bio) (Kobayashi et al., 2017).

qPCR assay

The copy numbers of the 1) prokaryotic and 2) Gemmatimonadetes 16S rRNA gene (clades G1 and G3), 3) Gemmatimonadetes nosZ DNA, and 4) Gemmatimonadetes nosZ mRNA (i.e., synthesized cDNA) were assessed using the MiniOpticon Real-Time PCR System (Bio-Rad, Hercules). The reaction mixture (20‍ ‍μL) contained the KAPA SYBR FAST qPCR master mix (Nippon Genetics) (10‍ ‍μL), 0.8‍ ‍μL of each forward and reverse primer (10‍ ‍μM), and 1.6‍ ‍μL of extracted DNA or the synthesized cDNA template. The oligonucleotide primers used for PCR amplification were as follows: 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806r (5′-GGACTACHVGGGTWTCTAAT-3′) for the prokaryotic 16S rRNA gene (Caporaso et al., 2011), G1G3-673F (5′-GAATGCGTAGAGATCC-3′) and 907r (5′-CCGTCAATTCMTTTRAGTTT-3′) for the Gemmatimonadetes 16S rRNA gene affiliated to clades G1 and G3, which were previously described by DeBruyn et al., (2011), and nosZ-123-145-F (5′-AACAAGAACCSAAGGAYCG-3′) and nosZ-481-499-R (5′-ATRTCCCARTCCTGYTC-3′) for Gemmatimonadetes nosZ (the present study). Cycling conditions were as follows: 95°C for 30 s; 40 cycles at 95°C for 5‍ ‍s and 55°C for 10 s; and 65°C to 95°C in increments of 0.5°C for the melting curve ana­lysis. Negative controls (i.e., distilled water and an RNA template that was not reverse transcribed) were subjected to qPCR in parallel, and no amplicon was obtained from these negative controls. The genomic DNA of G. aurantiaca with a single copy of the 16S rRNA gene and nosZ was used as a standard for quantification. DNA concentrations were measured using the Qubit dsDNA BR assay kit and Qubit 3.0 fluorospectrometer (Thermo Fisher Scientific). Genomic DNA was serially diluted with distilled water to concentrations of 10⁵ to 10⁰ copies μL^–1.

The nosZ-123-145-F and nosZ-481-499-R primers were newly designed in the present study. We attempted to amplify the partial sequences of clade II nosZ with the nosZ-II-F and nosZ-II-R primers (Jones et al., 2013). However, no specific amplicon was obtained by PCR amplification using nosZ-II-F and nosZ-II-R primers from the DNA and cDNA samples prepared from the soil samples tested, even after the optimization of PCR conditions (i.e., Taq polymerase and the addition of DMSO and betaine) and cycling parameters (annealing temperature and extension time). A similar phenomenon was previously reported (Samad et al., 2016); therefore, we designed a new set of oligonucleotide primers for the specific detection of Gemmatimonadetes nosZ. The nosZ-123-145-F and nosZ-481-499-R primers were designed by performing a blastn search using the G. aurantiaca nosZ sequence as a query sequence against the nr database (NCBI, accessed on December 2016). The top 500 nosZ sequence hits were aligned using MUSCLE software under default conditions (18 iterations) (Edgar, 2004), and the conserved regions suitable for PCR primer design were manually examined. The coverage of the designed primers was examined by aligning Gemmatimonadetes nosZ sequences and the designed nosZ primer sequences, and by counting the numbers of primer-template mismatches.

Amplicon sequencing ana­lysis of the 16S rRNA gene and nosZ

The prokaryotic 16S rRNA gene and Gemmatimonadetes nosZ were amplified by PCR using the above oligonucleotide primers containing Illumina tag sequences at the 5′ end of the forward and reverse primers (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′, respectively). The PCR mixture had a volume of 20‍ ‍μL and contained 2‍ ‍μL of extracted DNA or synthesized cDNA, oligonucleotide primers (1‍ ‍μM each), dNTPs (200‍ ‍μM), 2% (v/v) DMSO, 1×PCR buffer, and ExTaq polymerase (0.025‍ ‍U‍ ‍μL^–1). Thermal cycling conditions were as follows: 35 cycles at 98°C for 10‍ ‍s, followed by 55°C for 30‍ ‍s, then 72°C for 30 s; and 72°C for 10‍ ‍min. PCR products were purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics). Purified PCR products were tagged with a sample-unique index and Illumina adapter sequences at their 5' ends (Nextera XT Index Kit v2; Illumina) by PCR. The PCR reaction mixture (20‍ ‍μL) contained 1×KAPA HiFi HS ReadyMix (Nippon Genetics), 1‍ ‍μL of each forward and reverse primer (10‍ ‍μM), and 2‍ ‍μL of the recovered PCR products. PCR was run under the following cycling conditions: 95°C for 3‍ ‍min, 10 cycles of 95°C for 20‍ ‍s, 65°C for 15‍ ‍s, and 72°C for 1‍ ‍min; and 72°C for 5‍ ‍min. After agarose gel electrophoresis, PCR products were excised from the gel and purified using the Agencourt AMPure XP Kit (Beckman Coulter). The tagged amplicons were pooled and sequenced on an Illumina MiSeq platform in a 250-bp paired-end sequencing reaction using the v2 reagent kit (Illumina).

Bioinformatics

The generated 16S rRNA gene and nosZ sequence reads were processed for the removal of adapter sequences using cutadapt and for quality trimming using Trimmomatic v0.33 (Bolger et al., 2014). Reads containing <50 bp or those associated with an average Phred-like quality score <30 were removed. Paired-end sequence reads were assembled in the paired-end assembler of the Illumina sequence software package (PANDAseq) (Masella et al., 2012). The nosZ reads obtained were subjected to a blastn search (threshold e-value; 10^–10) against the 22,647 nosZ sequences downloaded from the fungene database (http://fungene.cme.msu.edu/) to remove non-nosZ sequences. Regarding 16S rRNA, assembled sequence reads with ≥97% sequence identity were grouped into OTUs by UCLUST (Edgar, 2004). The phylogenetic affiliations of the OTUs were identified by a blastn search against reference sequences in Greengenes database version 13_5 (DeSantis et al., 2006) and in the nr database (NCBI). Regarding nosZ, sequence reads with ≥80% sequence identity were grouped into OTUs as previously reported (Palmer et al., 2009), and their phylogenetic affiliation was examined by a blastn search against the nr database. Putative chimeric sequences were removed using UCHIME (Edgar et al., 2011). Alpha diversity indices (observed species, Chao1, Good’s coverage, and Simpson’s index) were calculated in QIIME (Caporaso et al., 2010). Chao1 was computed at sampling depths of 6,500 and 2,700 reads for 16S rRNA and nosZ, respectively. The alignment of nucleic acid and protein sequences was performed using MUSCLE software (Edgar, 2004) with 18 iterations, and a phylogenetic tree was constructed in MEGA 7.0.26 (Kumar et al., 2016) using the maximum likelihood method (Jones-Taylor-Thornton model).

Chemical ana­lysis

^14-14N₂O, ^15-15N₂O, and ^15-15N₂ concentrations were measured by gas chromatography-mass spectrometry (GS/MS) as previously described (Isobe et al., 2011; Yoshinaga et al., 2011). Ten microliters of the headspace gas was collected using a 100-μL gas-tight glass syringe and immediately injected into the gas chromatograph GCMS-QP 2010 SE (Shimadzu) equipped with a fused silica capillary column (Agilent Technologies). Peaks at m/z=30, 44, and 46, corresponding to ^15-15N₂, ^14-14N₂O, and ^15-15N₂O, were monitored, and concentrations were calculated using standard curves prepared using the standard ^14-14N₂O gas for both ^14-14N₂O and ^15-15N₂O (Shimakyu) and the ^15-15N₂ gas (Cambridge Isotope Laboratories).

Biomass concentrations were measured by the Lowry method using the DC-protein assay kit (Bio-Rad) as previously reported (Oshiki et al., 2011). Bovine serum albumin was used to prepare calibration curves.

Correlation ana­lysis between N₂O reduction rates and physicochemical parameters or the abundance of the Gemmatimonadetes 16S rRNA gene or nosZ

A linear regression ana­lysis was performed using Microsoft Excel 16.57 to assess the coefficient of determination (R²) between N₂O reduction rates (16.6, 5.0, 24.3, and 9.5‍ ‍nmol-N [g dry soil]^–1 h^–1 in Soils A, B, C, and D, respectively, see below) and physicochemical parameters (Table S1) or the copy numbers of the prokaryotic 16S rRNA gene, Gemmatimonadetes 16S rRNA gene, Gemmatimonadetes nosZ DNA, and Gemmatimonadetes nosZ mRNA (copies [g dry soil]^–1) (Fig. 4). The copy numbers of Gemmatimonadetes nosZ mRNA affiliated to specific OTUs were calculated by multiplying the relative abundance of each OTU (%) by the copy numbers of Gemmatimonadetes nosZ mRNA.

Nucleotide sequence accession number

Raw sequence data obtained in the amplicon sequencing ana­lysis were deposited in the DDBJ nucleotide sequence database under accession number DRA006974. The sequence reads of each OTU are available under accession numbers LC390430 to LC401807 and IADF01000001 to IADF01000317 for the 16S rRNA gene and nosZ, respectively.

Results

pH and temperature ranges and affinity for N₂O reduction by G. aurantiaca

G. aurantiaca cells were incubated under different pH (pH 5 to 10) and temperature (4 to 80°C) conditions, and the activities of N₂O consumption were examined. As shown in Fig. 1a and b, G. aurantiaca cells consumed N₂O at pH 5–9 and 4–50°C, with the highest activity being observed at pH 7 and 30°C. The affinity of G. aurantiaca cells for N₂O was examined by continuously measuring N₂O concentrations using N₂O-MR. The relationship between N₂O reduction rates and N₂O concentrations is shown in Fig. 2, and the apparent affinity constant for N₂O (K_s) was 4.4‍ ‍μM.

Fig. 1.

Effects of pH and temperature on N₂O reduction by Gemmatimonas aurantiaca

a) G. aurantiaca cells (a 3-mL sample in a 7.7-mL glass vial) were incubated at 30°C and a pH range of 5–10 with ^14-14N₂O (180‍ ‍nmol [N vial]^–1). The highest activity, 0.0377‍ ‍nmol min^–1 [mg protein]^–1, was observed at pH 7. b) The incubation was repeated at pH 7 and 4–80°C. The highest activity, 0.0203‍ ‍nmol min^–1 (mg protein)^–1, was observed at 30°C. Error bars represent the range of standard deviations derived from three replicated vials.

Fig. 2.

Affinity constant of Gemmatimonas aurantiaca for N₂O reduction. G. aurantiaca cells were cultivated with the addition of 30‍ ‍μM N₂O, and N₂O consumption was monitored using a N₂O microsensor. Circle symbols correspond to the data set obtained by N₂O microsensor measurements, and the red line indicates a fitted Michaelis-Menten curve. The coefficient of determination (R²) was calculated at a range of 0.1 to 18‍ ‍μM N₂O.

N₂O reduction activities and abundance of Gemmatimonadetes in soil

Four agricultural soil samples collected in Nagaoka city, Niigata, Japan were incubated with the addition of ¹⁵NO₃^–. All soil samples reduced ¹⁵NO₃^– and produced ^15-15N₂O and ^15-15N₂ (Fig. 3). N₂O production was more prominent than N₂ production during the early phase of the incubation (up to 24 to 69 h), and N₂O concentrations then stabilized or decreased. In contrast, ^15-15N₂ concentrations continuously increased during the incubation, except for Soil C, which showed a decreased after an incubation for 93 h. ^15-15N₂O reduction rates were calculated as a slope of ^15-15N₂ concentrations during 0 to 93 h of the incubation, and were 16.6, 5.0, 24.3, and 9.5‍ ‍nmol N (g dry soil)^–1 h^–1 for Soil A, B, C, and D, respectively. These rates were similar or higher than those of Chinese paddy soils (2.37 to 8.31‍ ‍nmol N g^–1 h^–1) (Shan et al., 2016).

Fig. 3.

NO₃^– reduction to N₂O and N₂ during the batch incubation of agricultural soil samples. Agricultural soils (Soils A to D) were incubated in closed 50-mL glass vials with the addition of ¹⁵NO₃^– (71.4‍ ‍μmol [N vial]^–1), and the production of ^15-15N₂O (filled circles) and ^15-15N₂ (open circles) was examined. Error bars represent the range of standard deviations derived from four replicated incubations.

The abundance and diversity of the Gemmatimonadetes 16S rRNA gene and nosZ (DNA and mRNA) were examined using the above 4 soil samples collected after an incubation for 69 h. The copy numbers of the prokaryotic and Gemmatimonadetes (clades G1 and G3) 16S rRNA genes were 1.19–16.7×10¹⁰ and 8.62–9.65×10⁸ copies (g dry soil)^–1, respectively (Fig. 4). The new oligonucleotide primers nosZ-123-145-F and nosZ-481-499-R were designed to assess the copy number of Gemmatimonadetes nosZ, and the sequence coverage of the designed primers is shown in Fig. S1. The copy numbers of Gemmatimonadetes nosZ DNA and mRNA (i.e., cDNA) were 5.35–7.15×10⁸ and 2.23–4.31×10⁹ copies (g dry soil)^–1, respectively (Fig. 4). PCR amplicons of the prokaryotic 16S rRNA gene and Gemmatimonadetes nosZ mRNA were subjected to an amplicon sequencing ana­lysis. Overall, 6,572 to 12,252 sequence reads of the 16S rRNA gene were obtained from each soil sample and then clustered based on ≥97% sequence identity into 2,982 to 4,021 OTUs (Table S2a). Gemmatimonadetes 16S rRNA reads accounted for 4.7 to 8.9% of the total reads (Fig. S2), and the phylogeny of Gemmatimonadetes 16S rRNA reads is shown in Fig. 5. The Gemmatimonadetes 16S rRNA reads affiliated with OTU4572 and OTU3759 were abundant in the soils tested, whereas no OTU shared ≥97% sequence similarity with the G. aurantiaca 16S rRNA gene sequence.

Fig. 4.

Abundance of 16S rRNA and nosZ in agricultural soil samples assessed by quantitative PCR (qPCR). Error bars represent the range of standard deviations derived from quadruplicate qPCR assays. No amplicon was obtained from the negative controls (i.e., distilled water and the RNA template that was not reverse transcribed). A significant difference (Student’s t-test, 99% confidence interval) was observed between the copy numbers of Gemmatimonadetes nosZ DNA and mRNA, as shown with asterisks.

Fig. 5.

Phylogeny and abundance of Gemmatimonadetes 16S rRNA sequences detected in agricultural soil samples. 16S rRNA gene reads obtained by amplicon sequencing were clustered into species-level operational taxonomic units (OTUs) with ≥97% sequence identity, and a phylogenetic tree was constructed using the maximum likelihood method with the Jones-Taylor-Thornton model and the 16S rRNA of Escherichia coli (accession number BA000007.2) as an outgroup. Branching points that support a probability >80% in bootstrap ana­lyses (based on 500 replicates) are shown as filled circles. The scale bar represents 5% sequence divergence.

Regarding Gemmatimonadetes nosZ mRNA, 2,711 to 6,328 sequence reads were obtained (Table S2b). Gemmatimonadetes nosZ reads were screened from the total reads by a phylogenetic ana­lysis, and more than 72% of the total reads were affiliated with the Gemmatimonadetes nosZ clades; i.e., 74, 86, 72, and 76% of nosZ reads obtained from Soil A, B, C, and D, respectively, were affiliated with a putative Gemmatimonadetes nosZ clade. Gemmatimonadetes nosZ reads were clustered (≥80% sequence identity) (Palmer et al., 2009) into 90 Gemmatimonadetes nosZ OTUs. The phylogeny of the 44 major OTUs is shown in Fig. 6, and no OTU shared ≥80% sequence similarity with G. aurantiaca nosZ.

Fig. 6.

Phylogeny and abundance of 44 most abundant operational taxonomic units (OTUs) of Gemmatimonadetes nosZ mRNA. nosZ reads were clustered into species-level OTUs with ≥80% sequence identity (Palmer et al., 2009), and a phylogenetic tree was constructed using the maximum likelihood method with the Jones-Taylor-Thornton model and Robiginitalea biformata nosZ (accession number; CP001712) as an outgroup. Branching points that support a probability >80% in bootstrap ana­lyses (based on 500 replicates) are shown as filled circles. The phylogenetic positions of Gemmatimonadetes aurantiaca and Gemmatirosa kalamazoonesis nosZ in the Gemmatimonadetes nosZ clade tentatively proposed in the present study are shown in red. The scale bar represents 10% sequence divergence. The OTUs highlighted with a star symbol (i.e., OTU91, OTU332, and OTU122) are the major OTUs (>1% relative abundance in a soil sample) showing a strong correlation with soil N₂O reduction rates (Table 1).

The relationships between N₂O reduction rates and physicochemical parameters (Table S1) or the abundance of the Gemmatimonadetes 16S rRNA gene (clades G1 and G3) and nosZ were examined using a linear regression ana­lysis. As shown in Table 1, the abundance of the nosZ mRNA of Gemmatimonadetes bacteria, OTU91, OTU332, and OTU122 strongly correlated (R²; >0.84) with the N₂O reduction rates of the soil samples tested.

Table 1. Relationships between N₂O reduction rates and physicochemical parameters or the abundance of the Gemmatimonadetes 16S rRNA gene and nosZ in soil samples. The table shows R² values between the N₂O reduction rates of the soil samples examined and (upper) physicochemical parameters or (bottom) the abundance of the Gemmatimonadetes 16S rRNA gene and nosZ. Regarding Gemmatimonadetes nosZ mRNA OTUs, the major OTUs (>1% of relative abundance in a soil sample, Fig. 6) with R² values >0.8 are shown.

pH	Water content	TC	TN	P	NO3–	NO2–	NH4+
0.09	0.57	0.58	0.62	0.03	0.42	0.41	0.41

Prokaryotic 16S rRNA gene	Gemmatimonadetes
	16S rRNA gene	nosZ DNA	nosZ mRNA	OTU91 nosZ mRNA	OTU332 nosZ mRNA	OTU122 nosZ mRNA
0.11	0.02	0.36	0.91	0.97	0.84	0.92

Discussion

The relative abundance of the Gemmatimonadetes 16S rRNA gene in the soils examined in the present study ranged between 0.5 and 7.8% of the prokaryotic 16S rRNA copy number and between 4.7 and 8.9% of the total 16S rRNA gene amplicon reads, indicating that Gemmatimonadetes bacteria were the dominant soil bacteria. The abundance of Gemmatimonadetes 16S rRNA gene copy numbers to prokaryotic 16S rRNA gene copy numbers assessed by qPCR was generally lower than the relative abundance estimated by the amplicon sequencing ana­lysis. The G1G3-673F primer used for qPCR of the Gemmatimonadetes 16S rRNA gene targeted the 16S rRNA gene sequences of Gemmatimonadetes clades G1 and G3, but not G2 (DeBruyn et al., 2011), which may have resulted in an underestimation of the copy numbers of the Gemmatimonadetes 16S rRNA gene in the soils examined. On the other hand, although the nosZ-123-145-F and nosZ-481-499-R primers covered >92% of the Gemmatimonadetes nosZ sequences, the amplicon sequencing ana­lysis of Gemmatimonadetes nosZ mRNA revealed that between 14 and 28% of the total nosZ reads were not assigned to the putative Gemmatimonadetes nosZ clade. This result suggests that the copy numbers of Gemmatimonadetes nosZ mRNA (and potentially nosZ DNA) were overestimated by 28%. Therefore, caution is needed when comparing the copy numbers of the Gemmatimonadetes 16S rRNA gene and nosZ.

Previous DNA-based PCR and sequencing ana­lyses of nosZ clade II revealed metabolically inactive populations (i.e., dead and dormant cells) in soil. G. aurantiaca and other N₂O reducers transcribed nosZ mRNA when they reduced N₂O (Henderson et al., 2010; Mania et al., 2016; Park et al., 2017), and, thus, an mRNA-based ana­lysis of Gemmatimonadetes nosZ was herein performed to identify N₂O-reducing Gemmatimonadetes bacterial populations. Gemmatimonadetes nosZ mRNA was successfully detected in N₂O-reducing soils (Fig. 4 and 6), and correlations were observed between N₂O reduction rates and the abundance of Gemmatimonadetes nosZ mRNA (R²=0.91). On the other hand, a weak correlation was noted between N₂O reduction rates and the abundance of Gemmatimonadetes nosZ DNA (R²=0.36), which may have been due to the detection of a metabolically inactive Gemmatimonadetes population. Additionally, the abundance of the Gemmatimonadetes 16S rRNA gene (clades G1 and G3) did not show a correlation (R²=0.02). nosZ has been found in particular Gemmatimonadetes genomes and is not commonly conserved among this bacterial phylum, which may have contributed to the lack of a correlation between N₂O reduction rates and the abundance of the Gemmatimonadetes 16S rRNA gene. The correlations observed between the N₂O reduction rates of the soil samples tested and the abundance of the nosZ mRNA of Gemmatimonadetes bacteria, OTU91, OUT332, and OTU122 (Table 1) indicated the involvement of Gemmatimonadetes bacteria in N₂O reduction in these soil samples. This is the first study to show a correlation between N₂O reduction rates and the abundance of Gemmatimonadetes nosZ mRNA in soil. These correlations were examined using 4 agricultural soils in the present study, and, thus, more detailed studies are warranted using larger numbers and various types of soil samples. Furthermore, the soil samples examined in the present study contained phylogenetically diverse microorganisms other than Gemmatimonadetes bacteria (Fig. 2), and their contribution to N₂O reduction and other nitrogen transformation reactions in soils currently remains unclear. A metatranscriptomic ana­lysis other than target-specific qPCR is a powerful tool for investigating microbial nitrogen transformation reactions in soil ecosystems (Masuda et al., 2017).

To gain further insights into N₂O reduction by Gemmatimonadetes bacteria in soil, the effects of pH and temperature as well as affinity for N₂O reduction were examined using G. aurantiaca. The influence of pH and temperature on the N₂O reduction activities of Gemmatimonadetes bacteria have not yet been examined; nevertheless, this physiological information is key for understanding their involvement in N₂O reduction in soils. pH and temperature conditions markedly affected the N₂O reduction activities of G. aurantiaca in the present study; relative activity decreased by 50±28% at pH 6.5 (Fig. 1). G. aurantiaca reduced N₂O to N₂ at pH 5–9 and 4–50°C, and agricultural soils showed these pH and temperature ranges; i.e., pH 5.4–8.1 (Holtan-Hartwig et al., 2000; Domeignoz-Horta et al., 2015) and 3.6–25.8°C (Takata et al., 2011). The Ks value for N₂O (i.e., 4.4‍ ‍μM) was in the range of those previously reported in cultures of denitrifiers and bacteria catalyzing dissimilatory nitrite reduction to ammonium (0.324–100‍ ‍μM) (Betlach and Tiedje, 1981; Conrad, 1996; Yoon et al., 2016; Park et al., 2017; Suenaga et al., 2018) and using bulk agricultural soils (0.1–5.8‍ ‍μM) (Holtan-Hartwig et al., 2000). N₂O concentrations in agricultural soils were generally less than 1‍ ‍μM, but increased up to 400‍ ‍μM (Schreiber et al., 2012 and references therein) with nitrogenous fertilizer treatment, which was higher than the K_s value of G. aurantiaca. Collectively, these findings indicate that soil management practices, including pH and temperature control and nitrogenous fertilizer treatment, have a significant impact on N₂O reduction by Gemmatimonadetes bacteria (and likely N₂O emissions from soil). Although the aforementioned physiological characteristics of G. aurantiaca support our hypothesis that Gemmatimonadetes bacteria participated in N₂O reduction in the soils examined, it is important to note that the G. aurantiaca 16S rRNA gene and nosZ mRNA reads were not detected in the soil samples examined (Fig. 5 and 6); therefore, the physiological characteristics of soil-inhabiting Gemmatimonadetes bacteria need to be investigated in future studies. The 16S rRNA gene reads affiliated with OTU4572 and OTU3759 were commonly and abundantly found in the soils examined, and also from various types of European, American, and Asian soils as assessed by a blastn search against the nr database (Table S3a and b). Further studies are warranted to examine the physiological characteristics of the Gemmatimonadetes bacteria affiliated with OTU4572 and OTU3759.

Citation

Oshiki, M., Toyama, Y., Suenaga, T., Terada, A., Kasahara, Y., Yamaguchi, T., and Araki, N. (2022) N₂O Reduction by Gemmatimonas aurantiaca and Potential Involvement of Gemmatimonadetes Bacteria in N₂O Reduction in Agricultural Soils. Microbes Environ 37: ME21090.

https://doi.org/10.1264/jsme2.ME21090

Acknowledgements

This work was supported by JSPS KAKENHI Grant numbers 19K05805, 20H00641, and 20H02290 to M.O., T.Y., and N.A., respectively. The authors acknowledge Dr. Yuga Hirakata (National Institute of Advanced Industrial Science and Technology) for his technical assistance with library preparation and MiSeq sequencing. Additionally, the authors would like to thank Dr. Daisuke Hira (Sojo University), Dr. Yosuke Tashiro (Shizuoka University), and Dr. Hisashi Satoh (Hokkaido University) for their helpful discussions and valuable suggestions. Strain NBRC100505^T was provided by the National Institute of Technology and Evaluation. The authors declare no conflicts of interest associated with this manuscript.

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