2026 Volume 41 Issue 1 Article ID: ME25079
Nitrous oxide (N2O) is a potent greenhouse gas, and the enzyme Nos catalyzes its reduction to dinitrogen (N2). Bradyrhizobium ottawaense exhibits strong N2O-reducing activity with high nosZ expression. To investigate whether promoter sequences affect nosZ expression, we constructed reciprocal promoter-swapped mutants between B. ottawaense and B. diazoefficiens. The swapping of promoters did not significantly affect expression levels. B. ottawaense mutants maintained approximately 200-fold higher expression levels than B. diazoefficiens, and the introduction of the B. ottawaense promoter into B. diazoefficiens did not increase expression levels. Therefore, the present results indicate that promoter sequence differences are not the primary factor affecting nosZ expression, suggesting regulation by other factors.
Nitrous oxide (N2O) is a long-lived atmospheric gas that acts both as a potent greenhouse gas, with a global warming potential that is approximately 300-fold that of CO2, and as an ozone-depleting substance (Ravishankara et al., 2009; IPCC, 2023). Its atmospheric concentration has risen by >20% since the pre-industrial era, mainly due to agriculture, which accounts for more than half of anthropogenic emissions (Tian et al., 2020). Therefore, reducing N2O emissions from croplands is a critical challenge in the mitigation of climate change.
N2O reduction occurs in some bacteria and archaea that possess nitrous oxide reductase (NosZ) (Hallin et al., 2018). Since the 1980s, nosZ genes and their enzymatic activities have been characterized (Bhandari and Nicholas, 1984). More recently, the discovery of novel nosZ clades, Clades II and III (Sanford et al., 2012; He et al., 2025), has expanded our understanding of microbial N2O reduction and opened up possibilities for agricultural mitigation technologies using N2O-reducing bacteria (Itakura et al., 2013; Hiis et al., 2024; Nishida et al., 2025).
Several rhizobia, including Bradyrhizobium diazoefficiens, harbor the classical Clade I nosZ and are capable of growing with N2O as the sole electron acceptor (Sameshima-Saito et al., 2006; Wasai-Hara et al., 2023). Field applications using a nosZ-overexpressing strain of B. diazoefficiens have demonstrated the potential for “microbial N2O mitigation” (Itakura et al., 2013). However, wild-type strains exhibit insufficient N2O-reducing activity, making naturally occurring high-activity strains desirable for practical applications.
The expression of the nos operon is regulated by FixLJK2 and RegSR in response to low oxygen levels (Torres et al., 2017; Sciotti et al., 2003), and by NasST in response to the presence of nitrate (Sánchez et al., 2014, 2017). A recent study suggested that when nitrate (NO3–) and N2O are both present, N2O is preferentially reduced, highlighting the role of electron allocation and the possible involvement of novel transcriptional regulatory mechanisms (Gao et al., 2021).
We previously isolated the rhizobial strain, Bradyrhizobium ottawaense, and found that its N2O-reducing activity was seven- to eight-fold higher than that of B. diazoefficiens (Wasai-Hara et al., 2020, 2023). This high activity is considered to be attributable to the up-regulated expression of nosZ (Wasai-Hara et al., 2023). Furthermore, sequence analyses and mutant-based experiments suggested that known regulatory systems (FixLJK2, RegSR, and NasST) are not responsible for this up-regulated expression. Low sequence conservation (48% homology) has been observed in the promoter sequence upstream of nosZ. In addition, two transcription start sites were detected in B. ottawaense under N2O-respiring conditions, whereas B. diazoefficiens possessed only a single start site, indicating that sequence variations in this region affect the transcriptional levels of the nos operon (Wasai-Hara et al., 2023).
We herein examined the contribution of promoter sequences to nosZ expression by constructing reciprocal promoter-swapped mutants between B. ottawaense SG09 (high activity) and B. diazoefficiens USDA110T (low activity). We compared nosZ expression levels in these mutants to investigate whether promoter differences explain the up-regulated expression observed in B. ottawaense.
The construction of promoter-swapped mutants is described below. The promoter including the region and untranslated region (UTR), as defined in the present study, are shown in Fig. 1A and B. Transcription start sites were previously identified (Pd1 and Pd2), corresponding to the two promoter-including regions (Promoter #1/#2) in B. ottawaense SG09. Mutants, in which only the promoter sequence or both the promoter and UTR sequences were replaced, were constructed by first deleting the 286 bp (80+133+73 bp) upstream of the transcription start site of SG09 using the in-frame markerless deletion method (Fig. 1C) (Wasai-Hara et al., 2023). The up- and downstream regions of the promoter and UTR region were amplified with the primers listed in Table S1 and S2 and with Prime STAR® Max DNA Polymerase (Takara Bio). The fragments were combined by overlap extension PCR and cloned into the SmaI site of the suicide vector pK18mobsacB-Ω using an In-fusion HD Cloning Kit (Takara Bio). The sequences of the introduced fragments were confirmed by sequencing. Transmission of the plasmid to B. ottawaense SG09 and homologous recombination of the promoter region were achieved by triparental mating. Transconjugants were selected based on resistance to streptomycin (100 μg mL–1), spectinomycin (100 μg mL–1), and polymyxin (100 μg mL–1) and sensitivity to 10% sucrose. Single crossover strains were then cultured in HM medium without antibiotics, and deletion mutants—lacking the promoter and/or UTR and exhibiting sensitivity to Sp/Sm and resistance to sucrose—were obtained. The alternative promoter (80 bp) and UTR (83 bp) from B. diazoefficiens USDA110T were then inserted into the genome at the original locus (ΔPnos::110, ΔPnos::110+UTR) using the same in-frame markerless method. The validity of mutant constructs was confirmed by DNA sequencing. Additional mutants lacking either of the two promoters were also generated (ΔP1 and ΔP2). To assess the effects of upstream sequences, a partial ferritin deletion mutant (Δftn [Δ763+IGR488]) was constructed, retaining the promoter, but lacking the upstream ferritin and intergenic regions (Fig. 1C).
Promoter and upstream regions of the nos operon in Bradyrhizobium ottawaense SG09 and B. diazoefficiens USDA110T.
(A, B) Upstream sequences and gene organization of the nos operon are shown. Pd1, Pd2, and P indicate the transcription start sites in SG09 and USDA110T. The –35 and –10 consensus sequences preceding each transcription start site are underlined. A putative FixK box is shown in the box. The translational start codon (ATG) of nosR is indicated in bold type. (A) In SG09, two promoters—the upstream region with promoter #1 (80 bp) and the downstream region with promoter #2 (133 bp)—followed by a 73-bp untranslated region (UTR), are arranged upstream of the nos operon in the 5′ direction. Upstream of these elements, a ferritin gene is located on the reverse strand. (B) In USDA110T, a single promoter and an 83-bp UTR are located upstream of the nos operon in the 5′ direction. Similarly, a ferritin gene is located on the reverse strand. (C) The design of the mutants constructed in the present study. The deleted region and inserted elements are shown.
In B. diazoefficiens USDA110T, mutants with swapped promoter regions alone, swapped promoter and UTR regions together, and promoter deletion mutants were constructed. Furthermore, a deletion mutant encompassing part of the ferritin gene and the upstream intergenic region (Δftn[Δ877+IGR99]) was generated (Fig. 1C).
nosZ gene expression levels were measured by RT-qPCR as previously described (Wasai-Hara et al., 2023). B. ottawaense SG09, B. diazoefficiens USDA110T, and their mutant lines were cultured in 10 mL of HM medium (Cole and Elkan, 1973) in 75-mL glass tubes at 28°C with shaking at 180 rpm. The headspace was replaced with N2, and 3.25 mL of 100% N2O was added as the sole electron acceptor, resulting in a final concentration of 5% (Wasai-Hara et al., 2023). Pre-cultured cells were inoculated into the HM medium at an initial OD660=0.05, and total RNA was extracted after overnight cultivation (final OD reached approximately 2.0±0.5). Fold changes in gene expression were calculated using the ΔΔCt method. Raw Ct values were initially normalized to the internal reference gene (sigA), and the fold change from the control condition was then calculated.
The nosZ transcript level in B. ottawaense SG09 wild-type was 220±37-fold higher than that in B. diazoefficiens USDA110T (Fig. 2A and S2A), consistent with previous findings (Wasai-Hara et al., 2023). The replacement of the nosZ promoter in SG09 with that from USDA110T (SG09ΔPnos::110) did not significantly affect expression levels, which remained 279±60-fold higher. Therefore, promoter replacement was considered to not affect nosZ expression levels. In contrast, swapping of both the promoter and UTR sequences (SG09ΔPnos::110+UTR) resulted in a significant reduction in nosZ expression (Fig. 2A). This may attributed to the mRNA of the UTR region in USDA110T forming a hairpin secondary structure under nitrate-free conditions, which may sterically hinder the access of transcription machinery and thereby inhibit transcription (Sánchez et al., 2017). Therefore, the introduction of the USDA110T-derived UTR sequence in this study was considered to have inhibited transcription to a similar extent. The negative effects of the UTR were attenuated under nitrate-respiring conditions (Fig. S1 and S2B)
NosZ expression and growth rate in promoter-swapped strains under N2O-reducing conditions.
(A) Relative nosZ transcript levels in Bradyrhizobium ottawaense SG09 background strains—wild-type (WT), promoter-swapped strain (ΔPnos::110), promoter and UTR-swapped strain (ΔPnos:110+UTR:), single promoter-deletion mutants (ΔP1 and ΔP2), and an upstream deletion mutant without promoter deletion (Δftn[Δ763+IGR488])—as well as in B. diazoefficiens USDA110T background strains—WT, promoter-swapped strain (ΔPnos::SG09), promoter and UTR-swapped strain (ΔPnos::SG09+UTR), and an upstream deletion mutant (Δftn[Δ877+IGR99]). Transcript levels were quantified by RT-qPCR and values are shown relative to the USDA110T WT strain (set to 1). Bars represent means, and error bars indicate SD (n=4–6). Different letters above the bars represent significant differences between the inoculation treatments analyzed using Tukey’s test after an analysis of variance (ANOVA; P<0.05). (B, C) Growth of SG09 and USDA110T background strains under N2O-respiring conditions. Error bars indicate SD (n=3).
The deletion of either promoter individually (SG09ΔP1 and ΔP2) resulted in a significant decrease in nosZ expression (Fig. 2A). Therefore, both promoters in SG09 were functionally active under N2O-respiring conditions. In contrast, the deletion of an upstream region (SG09Δftn[Δ763+IGR488]) that left both promoter sequences intact did not cause a significant reduction in nosZ transcript levels (Fig. 2A). Based on these results, the upstream region defined in the present study was not considered to affect nosZ expression.
In B. diazoefficiens USDA110T, the replacement of the promoter region with that of SG09 (USDA110TΔPnos::SG09) or with both the promoter and UTR (USDA110TΔPnos::SG09+UTR) resulted in no significant change in expression (Fig. 2A). Similarly, the deletion of the upstream gene region (USDA110TΔftn[Δ877+IGR99]) did not affect nosZ expression. These results, similar to those observed for SG09, indicate that in the USDA110T background, the promoter sequence itself did not affect nosZ expression.
Furthermore, the complete deletion of the promoter region abolished growth under N2O-respiring conditions in both species, although the mutants grew normally under nitrate-respiring or aerobic conditions, confirming that the presence of the promoter is indispensable for nosZ expression and, thus, N2O respiration (Fig. 2B, C, and S3).
Collectively, the present results demonstrate that the promoter region is essential for nosZ expression in both B. ottawaense SG09 and B. diazoefficiens USDA110T, whereas the promoter sequence itself does not affect expression levels. Since promoter activity is generally considered to be a key factor affecting gene expression (Hawley and McClure, 1983; Wang et al., 2023), these results are unexpected and noteworthy. Heterologous promoters may be substituted with similar efficiency, indicating that differences in nosZ expression between the two strains are primarily governed by trans-acting factors rather than by promoter cis-elements. Such regulation may involve transcriptional activators recognizing conserved motifs within the nos promoter between B. ottawaense and B. diazoefficiens, or, alternatively, species-specific differences in transcriptional or translational resource allocation or in mRNA stability (Zhu and Dai, 2025). Possible approaches to identify factors that regulate expression levels in B. ottawaense include screening for proteins that bind to the nos promoter site, narrowing down candidate factors that are co-expressed with the nos gene through a transcriptome analysis, and the genome-wide screening of regulatory elements using random transposon mutagenesis. On the other hand, it remains unclear whether high nosZ expression in B. ottawaense reflects species-specific activation or repression in B. diazoefficiens. To elucidate the mechanisms underlying differential nosZ expression in Bradyrhizobium species, future studies need to incorporate comparative analyses across lineages and examine the interactions between nosZ and other denitrification genes. A more detailed understanding of these regulatory mechanisms will provide a basis for controlling nosZ expression and engineering efficient N2O-reducing bacteria.
Wasai-Hara, S., Shimoda, Y., Mitsui, H., Sato, S., Imaizumi-Anraku, H., and Minamisawa, K. (2026) Promoter Sequences Do Not Solely Govern nosZ Expression Differences between Bradyrhizobium ottawaense and B. diazoefficiens. Microbes Environ 41: ME25079.
https://doi.org/10.1264/jsme2.ME25079
This work was supported by a Grant-in-Aid for JSPS Fellows (Grant Numbers: 22J01397) and the project JPNP18016, commissioned by the New Energy and Industrial Technology Development Organization (NEDO). We would like to thank Yukiko Fujisawa and Rie Morohashi for their technical support.
Conflicts of interestThe authors declare that there are no conflicts of interest.
