2025 年 40 巻 4 号 論文ID: ME25032
Since nitrogenase is intrinsically sensitive to oxygen (O2), diverse aerobic diazotrophs need strategies to cope with nitrogenase damage by O2. In the present study, we investigated the mechanisms by which aerobic methane-oxidizing bacteria (methanotrophs) enable the concurrent activities of methane monooxygenase, which uses O2, and nitrogenase in the cytoplasm of the same cell. By using 15N labeling, we confirmed the capacity of alphaproteobacterial methanotroph Methylosinus sp. 3S-1 for nitrogen fixation and diazotrophic growth across a wide range of O2 concentrations <20%. When the initial O2 concentration was increased from 2 to 20% in a diazotrophic culture, similar decreases were observed in fixed nitrogen and NifH protein levels. In contrast, the mRNA levels of nitrogen fixation genes (nif genes) markedly increased and remained elevated for the duration of slow growth at high O2 concentrations. This pattern of nif expression in response to O2 may be attributed to the properties of the nif-specific transcriptional regulator NifA. The present results suggest that the increase in nif transcription is one of the strategies by which this methanotroph maintains nitrogen fixation on the background of aerobic methane oxidation.
Biological nitrogen fixation, facilitated by the complex metalloenzyme nitrogenase, is a process by which inert dinitrogen gas (N2) is metabolically converted into the tractable nitrogen form, ammonia, under physiological conditions. This enzyme consists of two components, the iron protein (NifH) and molybdenum–iron protein (NifDK): the former contains a simple iron-sulfur cluster [4Fe–4S] and functions to donate electrons to the latter component, while the latter contains two complex metalloclusters, the P cluster [8Fe–7S] and the Fe-Mo cofactor [7Fe–9S–Mo–C]-homocitrate, and provides a catalytic site for N2 reduction. Many of the genes associated with N2 fixation are designated as nif genes.
Previous studies suggested the evolutionary emergence of N2-fixing organisms, termed diazotrophs, from anaerobic archaea (Mus et al., 2019; Pi et al., 2022). The capacity for N2 fixation was so advantageous for habitat expansion that it spread over diverse phylogenetic groups of archaea and bacteria and eventually to obligate aerobes. However, all known nitrogenases are structurally related and are intrinsically sensitive to oxygen (O2) damage in their associated metalloclusters. Therefore, diazotrophs had to develop various strategies to cope with O2 damage under the selective pressure of increasing O2 concentrations in the biosphere. One of these strategies was to reduce intracellular O2 concentrations even under ambient conditions. This strategy was detected in Azotobacter vinelandii, an obligately aerobic and heterotrophic γ-proteobacterium used as a model in N2 fixation studies, and includes a process called respiratory protection (Jones et al., 1973; Martin del Campo et al., 2022). This bacterium possesses a complex respiratory electron transport chain consisting of several branches; following an increase in the concentration of O2, the branch coupling only partially with proton translocation is specifically up-regulated, thereby increasing O2 consumption before its penetration of the cell (Wu et al., 1997). Another strategy is the strict regulation of nitrogenase synthesis at the transcriptional level in response to surrounding O2 concentrations as well as the availability of fixed nitrogen and carbon sources. In many proteobacteria, the transcription of nif genes is driven by the transcriptional regulator NifA coupled with the RNA polymerase sigma factor σ54, and NifA activity is negatively modulated by excess O2. In γ- and β-proteobacteria, the NifL protein has been shown to form an inhibitory complex with NifA in response to O2 (Martinez-Argudo et al., 2004). In α-proteobacteria, the majority of which lack NifL, NifA activity is considered to be intrinsically sensitive to O2 (Dixon and Kahn, 2004).
Diverse aerobic diazotrophs appear to have developed their own strategies to specific physiological and metabolic conditions. Among them, aerobic methane (CH4)-oxidizing bacteria, or methanotrophs, are noteworthy. They are obligate aerobes that use CH4 as the sole carbon and energy source through the activity of methane monooxygenase (MMO). Two MMOs with different evolutionary origins are present in nature: soluble MMO (sMMO; the mmo gene product) in a subset of methanotrophs and intracytoplasmic membrane-bound particulate MMO (pMMO; the pmo gene product) in nearly all methanotrophs (Semrau et al., 2010). sMMO and pMMO catalyze the breaking of the strong C–H bond of CH4 by using O2 within the cell. Methanotrophs are classified into the classes Alphaproteobacteria (called type II), Gammaproteobacteria (called type I), and Methylacidiphilae (the phylum Verrucomicrobia) (called type III) (Knief, 2019). The capacity of diazotrophic growth and/or the presence of nif genes have been found in virtually all known type II methanotrophs and in the majority of known type I methanotrophs (Murrell and Dalton, 1983; Auman et al., 2001; Dedysh et al., 2004; Hara et al., 2022; Bao et al., 2025). Moreover, the co-occurrence of N2 fixation and CH4 oxidation was confirmed at the single-cell level for type II methanotrophs using a combination of fluorescence in situ hybridization and NanoSIMS (Hara et al., 2022). We inferred a constraint imposed on the protection of nitrogenase in aerobic methanotrophs because the strategy to reduce intracellular O2 concentrations may not be compatible with the efficiency of CH4 oxidation in the cytoplasm.
In the present study, we aimed to reveal a strategy employed by methanotrophs to cope with O2 damage to nitrogenase from the aspect of the regulatory mechanism of nif genes. We used Methylosinus sp. 3S-1, a type II methanotroph, which was previously shown to exhibit CH4 oxidation-dependent activity for N2 fixation under 10% (v/v) O2 conditions (Shinoda et al., 2019). The results obtained herein showed that the transcription of nif genes was markedly enhanced following an increase in the concentration of O2. This characteristic regulation may be involved in the strategy employed by Methylosinus sp. 3S-1 to maintain N2 fixation on the background of aerobic CH4 oxidation.
The genome DNA library of Methylosinus sp. 3S-1 was prepared with SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences of California) and with a cut-off at 20–25 kb using the BluePippin size selection system (Sage Science). The library was sequenced with the PacBio Sequel II system (Pacific Biosciences of California). Reads were assembled using SMRTlink v10.2. The circularity of contigs was confirmed by Circulator v1.5.5 (Hunt et al., 2015). Sequences were annotated using the DFAST web service (Tanizawa et al., 2018). Harr plots were created with GenomeMatcher (Ohtsubo et al., 2008). Average nucleotide identity (ANI) was calculated on the JSpeciesWS web server (Richter et al., 2016).
Bacterial growth conditionsMethylosinus sp. 3S-1 was grown in liquid nitrate mineral salt (NMS) medium (Whittenbury et al., 1970), in which the concentration of KNO3 was modified to 0.05% (w/v) (hereafter N-containing medium), in a bottle sealed with a butyl rubber stopper. CH4 was added into the bottle with a syringe to 20% (v/v) of the headspace, unless otherwise specified. The culture bottle was incubated at 30°C with shaking. When necessary, the atmosphere in the headspace was initially replaced with N2 at the appropriate tension using a vacuum line equipped with a pressure gauge (AVG-300; Okano Works) and other gases were then added with a syringe to the bottle at the necessary volume. The volume ratio of the medium to the headspace in the bottle was set to 1:10, and the initial concentrations of O2, CH4, and CO2 were set to 2–20% (v/v), 5% (v/v), and 0.5% (v/v), respectively, with N2 as the balance gas. To achieve diazotrophic growth, KNO3 was excluded from N-containing medium (hereafter N-free medium). To deprive the culture medium of nitrogen sources, bacterial cells grown to an optical density at 600 nm (OD600) of 0.3–0.5 were washed with saline (10 mM KCl and 4 mM MgSO4) by centrifugation, suspended in N-free medium to an OD600 of 0.05, and placed in a bottle to be sealed. To achieve different O2 concentrations during diazotrophic growth, bacterial cells grown in N-free medium to an OD600 of 0.15–0.22 were diluted with N-free medium to an OD600 of 0.05. To evaluate N2-fixing activity, the atmosphere within a 1,150-mL bottle containing 100 mL of the culture sample was initially replaced with argon gas and then with 15N-N2 (98 atom%; Shoko Science) at a tension corresponding to 74.5% (v/v) (in the case of the 20% O2 condition) or 92.5% (v/v) (in the case of the 2% O2 condition) of the headspace. O2, CH4, and CO2 were added with the syringe at the necessary volumes.
Treatment of culture samplesThe OD600 of culture samples was measured using a UV-1700 spectrophotometer (Shimadzu). Cell density was assessed by a microscopic count with a hemocytometer. In the RNA analysis, culture samples were quickly mixed with a phenol/ethanol solution to yield 1% (v/v) phenol, chilled on ice, and centrifuged. Pelleted cells were suspended in TRIzol Reagent (Invitrogen) and lysed by the FastPrep homogenizer with Lysing Matrix B (MP Biomedicals). Total RNA was prepared according to the instructions of TRIzol and purified further with DNase I (Qiagen) and RNA Clean & Concentrator-25 (Zymo Research) according to the manufacturers’ instructions (the protocol for the purification of >200 nt RNAs was used in the latter kit). RNA yield was measured with an Agilent 2200 TapeStation System (Agilent Technologies). In the protein analysis, culture samples were quickly mixed with trichloroacetic acid to 10% (w/v), chilled on ice, and centrifuged. Pelleted cells were washed with acetone, dried, suspended in a solution (25 mM TrisHCl, 0.1 mM EDTA, 40 mM NaCl, 0.5% Triton X-100, and 0.5% Tween-20, pH 7.5), and sonicated with a Bioruptor (Sonicbio). Total protein concentrations were assessed with a DC Protein Assay (Bio-Rad Laboratories). To measure CH4 and O2 consumption in the culture, the headspace gas was sampled at intervals. CH4 was evaluated with the GC-2025 gas chromatograph (Shimadzu) equipped with a SH-Alumina BOND/Na2SO4 column (Shimadzu) and flame ionization detector using N2 as the carrier gas and temperatures of 40, 100, and 130°C for the column, injector, and detector, respectively. O2 was measured using a GC-2014 gas chromatograph (Shimadzu) equipped with a Molecular Sieve 5A column and thermal conductivity detector using helium as the carrier gas and temperatures of 60, 100, and 100°C for the column, injector, and detector, respectively.
Measurement of N2 fixation using 15N2A culture (70 mL) grown in the presence of 15N2 was chilled on ice and centrifuged. Pelleted cells were washed with cold saline, suspended in water, transferred into a tin foil cup, and dried in a desiccator with P2O5. The concentration of 15N was measured by Shoko Science with the Flash2000-DELTAplus Advantage ConFloIII System (ThermoFisher Scientific).
Western blot analysisCell lysates containing 1 μg of total protein were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto an Immobilon-P PVDF membrane (Millipore). To detect NifH, anti-NifH chicken polyclonal IgY (Agrisera) and HRP-conjugated goat anti-chicken IgY (Arigo Biolaboratories) antibodies were used as the primary and secondary antibodies, respectively. To detect RpoH, anti-Sinorhizobium meliloti RpoH1 rabbit antiserum (Mitsui and Minamisawa, 2017) and a HRP-conjugated goat anti-rabbit IgG antibody (ProteinTech Group) were used. We detected chemiluminescence emitted from the target protein with Western Lightning ECL Pro (PerkinElmer) as a substrate using the ChemiDoc XRS+ System (Bio-Rad Laboratories).
Quantitative reverse-transcription PCR (qRT-PCR)Complementary DNA was synthesized from total RNA using the PrimeScript RT reagent kit with random hexamers (Takara Bio). Real-time PCR was performed using the ten-fold diluted cDNA solution as the template and the primer pairs listed in Table S1 on the CFX Connect Real-Time PCR Detection System (BioRad Laboratories) using KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Biosystems). To select a stably expressed gene during changes in growth conditions, the quantification cycle (Cq) values of candidate genes in the real-time PCR analysis were subjected to calculations with the geNorm2 (Vandesompele et al., 2002) and BestKeeper (Pfaffl et al., 2004) algorithms implemented in the R package ‘ctrlGene.’ Fold changes in gene expression were calculated by the ΔΔCq method after amplification efficiencies were confirmed to range between 95 and 99% for all the primers used.
Statistical and phylogenetic analysesStatistical analyses were performed using the base R functions aov() for an analysis of variance and TukeyHSD() for Tukey’s Honest Significant Difference post-hoc test, along with the dplyr package for data summarization (Wickham et al., 2023). Data visualization was performed using the ‘ggplot2’ package (Wickham, 2016) in the R computing environment (version 4.3.2; R Core Team, 2023). Phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis software (version 11; Tamura et al., 2021).
Accession numbers for the genome sequenceThe nucleotide sequences of the 3S-1 genome were deposited in DDBJ under accession numbers AP040855 to AP040857.
A draft genome sequence of Methylosinus sp. 3S-1 was previously reported (Bao et al., 2016). To conduct an extensive survey for N2 fixation-related genes, we finished the whole genome sequence using the PacBio RS II single-molecule real-time platform. The genome was 4,967,063 bp (65.9 mol% DNA G+C content) and consisted of a circular chromosome (4,502,247 bp; 66.0 mol% G+C) and two circular plasmids, p3S1-1 (284,541 bp; 66.0 mol% G+C) and p3S1-2 (180,275 bp; 63.6 mol% G+C). In comparisons with Methylosinus trichosporium OB3b, the genome of which consists of a chromosome (4,496,051 bp) and two plasmids (285,905 and 180,306 bp) (accession no. NZ_ADVE02000001.1 to NZ_ADVE02000003.1) (Stein et al., 2010), ANI values were 99.97, 99.98, and 99.99% for the chromosome, p3S1-1, and p3S1-2, respectively, to their counterpart replicons. A Harr plot analysis showed collinearity over the lengths of the respective pairs, except for a 52.8-kb inversion on the chromosome (Fig. S1). This result indicates that 3S-1 is a close relative of M. trichosporium OB3b.
Methylosinus sp. 3S-1 is highly tolerant of O2 in diazotrophic growthWe attempted to examine the effects of higher O2 concentrations on the growth of Methylosinus sp. 3S-1 in N-free medium. The culture was prepared with N-free medium in sealed bottles in which the headspace was set initially to 2, 6, 10, or 20% (v/v) O2, in combination with 5% (v/v) CH4 and 0.5% CO2 (v/v) with N2 as the balance gas, and OD600 and O2 and CH4 concentrations were monitored. OD600 increased in all cultures regardless of the initial O2 concentration; however, the initial rate of the increase was lower under higher O2 conditions (Fig. 1A). OD600 reached a plateau following O2 depletion in the cultures with initial O2 concentrations of 2 and 6% and following CH4 depletion in the culture with an initial O2 concentration of 10%. In the culture with an initial O2 concentration of 20%, the slowest, but most steady increase in OD600 was observed until 180 h with O2 remaining at >17% and CH4 at >1.8%. In this culture, the increase in OD600 accelerated earlier (~36 h) and then became steady even after the culture was continued by diluting and resetting the O2 concentration to 20% (Fig. 1A and B). When the same diluted culture was reset to 2% O2, rapid growth began immediately (Fig. 1B). We also tested 3S-1 growth in N-containing medium. In this case, the strain exhibited similar growth between the cultures with initial O2 concentrations of 2 and 20% (v/v) (Fig. S2), confirming that the negative effect of O2 on growth was only observed with N-free medium.
Effects of O2 on the growth of Methylosinus sp. 3S-1 in N-free medium. Growth was monitored over time using OD600 of the medium and O2 and CH4 concentrations in the headspace. CH4 was initially adjusted to 5% (v/v). (A) Cells grown in N-containing medium were rinsed and suspended in N-free medium to an OD600 of 0.05. To begin the culture, the O2 concentration was adjusted to 2, 6, 10, or 20% (v/v). (B) The 20% O2 culture with N-free medium was diluted to an OD600 of 0.05 for the growth test with 2 or 20% (v/v) O2. The same result was obtained from an independent experiment.
To comparatively characterize N2 fixation at low and high O2 concentrations, we conducted O2 shift experiments on 3S-1 from 2% to higher concentrations in N-free medium; the culture was additionally provided with CH4 and CO2 (initially 5 and 0.5% [v/v], respectively) with N2 as the balance gas. We used 15N-labeled N2 (98 atom%) as the balance gas when the culture grown at 2% O2 (OD600 of 0.21) was diluted to OD600 of 0.05 and the new O2 concentration was set to 2 or 20% (v/v). We measured 15N concentrations in cells grown under each O2 condition. In the culture with an initial O2 concentration of 2%, it increased to 72.2 atom% excess at 18 h from a natural abundance level, along with increases in OD600 and cell density (Table 1). In the culture with an initial O2 concentration of 20%, increases in the 15N concentration and cell density were negligible at 18 h whereas OD600 doubled during the same time. In an extension to 96 h, the 15N concentration reached 30.5 atom% excess (Table 1). Although it was not possible to specify a range of O2 concentrations that sustained diazotrophic growth, these results indicate that 3S-1 was capable of N2 fixation across a wide range of O2 concentrations. To examine the relationship between O2 concentrations and the rates of diazotrophic growth, a continuous culture, such as turbidostat, will be necessary in the future. The transiently accelerated increase in OD600 after the 20% O2 shift involved neither substantial N2 fixation nor a cell number increase, suggesting that cells were producing absorbance-contributing substances against the background of active CH4 oxidation before N2 fixation occurred.
Incorporation of 15N from 15N2 gas into Methylosinus sp. 3S-1 cells during growth in N-free mediuma
| Initial O2 conc |
OD600 and cell densityb | 15N conc in cells (atom% excess)c | |||||
|---|---|---|---|---|---|---|---|
| 0 h | 18 h | 96 h | 0 h | 18 h | 96 h | ||
| 2% | 0.05 (ND) | 0.17 (1.8×107) | ND (ND) | 0.0 c | 72.2 a | ND | |
| 20% | 0.10 (4.7×106) | 0.17 (1.1×107) | 1.4 c | 30.5 b | |||
a Samples for 15N measurements were taken from a preculture grown in N-free medium at 2% (v/v) O2 (0 h) and from a culture grown for 18 or 96 h after the dilution of the preculture to an OD600 of 0.05 and the adjustment of the O2 concentration to 2 or 20% (v/v) in the 15N2 balance; values are means from three separate cultures; ND, not determined.
b Cell density is shown in parentheses as a microscopic cell count mL–1.
c Based on 0.366 atom% of natural 15N abundance; a significant difference based on Tukey’s test (P<0.05) is presented by a different letter following a value.
We used the same 15N-fed culture to investigate whether nitrogenase protein and nif mRNA expression was consistent with the N2 fixation rate. A Western blot analysis using the anti-NifH antibody detected a single band close to the predicted molecular mass of NifH (31.7 kDa) from cells grown in N-free medium with an initial O2 concentration of 2% (in both the preculture and the control at 18 h), but no corresponding band from cells grown in N-containing medium (Fig. 2A). Eighteen hours after the shift to 20% O2, the NifH band showed an upshift in mobility and a decrease in intensity from that at 2% O2. By 96 h, the NifH band recovered partially in intensity with the upshifted position (Fig. 2A). In contrast, the band intensity of RpoH, the RNA polymerase sigma factor σ32 (33.4 kDa), remained constant among the same set of samples as that for NifH (Fig. 2B). These results indicate that the change in NifH levels correlated with that in N2 fixation rates. The band shift at high O2 concentrations was not unprecedented because NifH of M. trichosporium OB3b divided into two distinct bands, the upper of which became solely detected at a high O2 concentration in the mutant possessing constitutively expressed sMMO and increased O2 tolerance in N2 fixation (Kim and Graham, 2001). In the same study, the NifH band disappeared from the wild type at a high O2 concentration, which may correspond to 3S-1 where NifH levels initially decreased after the 20% O2 shift. Since 3S-1 and OB3b each harbor a single copy of nifH in their genomes, the differentiation of NifH by electrophoretic mobility was attributed to post-translational modifications.
Effects of O2 on NifH protein levels in Methylosinus sp. 3S-1 cells. A Western blot analysis was conducted using anti-NifH (panel A) and anti-RpoH (panel B) antibodies for cell lysates (0.5 μg total protein per lane for the NifH analysis and 1 μg total protein per lane for the RpoH analysis). Cells were grown in N-containing medium under an ambient condition supplemented with CH4 (5%, [v/v]) (lane 1) or in the same cultures as those in Table 1, where N-free medium was used with 5% (v/v) CH4 and the specified initial concentrations of O2 (lanes 2–5). Lane 2, cells from the preculture (2% O2) for samples in lanes 3–5; lane 3, cells grown for 18 h at 2% O2 (v/v); lane 4, cells grown for 18 h at 20% O2 (v/v); lane 5, cells grown for 96 h at 20% O2 (v/v). The positions of molecular weight markers are indicated on the left side of each panel. The same result was obtained from a set of separate cultures grown in parallel.
Regarding the mRNA levels of nif genes, we attempted to identify a suitable reference gene to normalize 3S-1 mRNA data from various growth conditions. We evaluated the stability in expression of eight housekeeping genes (clpX, dnaK, gyrB, recA, rho, rpmH, rpoB, and rpoD) in cells growing on CH4 in (i) N-containing medium at an ambient O2 concentration, (ii) N-free medium at 2% O2, and (iii) N-free medium at 20% O2 at three culture time points (for details, refer to the legend of Fig. S3). We selected recA as the reference because its expression was judged to be the most stable under the conditions tested (Fig. S3 and Table S2).
In the 3S-1 genome, many nitrogenase-related genes clustered into putative operons, each of which was preceded by a possible NifA-binding motif and σ54-recognized “–24/–12”-type promoter sequences (Fig. S4). This finding strongly suggests that the transcription of these genes is regulated by NifA, as is the case with many proteobacterial diazotrophs (Dixon and Kahn, 2004). We subjected nifH, nifB, the leading genes of the putative operons, and nifA to a qRT-PCR analysis with the O2 shift from 2 to 20% (v/v) in N-free medium. The results obtained showed that the mRNA levels of these nif genes markedly increased upon the O2 shift, in contrast to their protein levels and N2 fixation rates (Table 2). Therefore, O2 appeared to enhance, not repress, the transcription of the nif genes in 3S-1, which is opposite to its effects in many obligate aerobes and facultative anaerobes (Dixon and Kahn, 2004). High mRNA levels were maintained at 96 h, parallel with steady growth based on a low rate of N2 fixation (Table 2).
Changes in mRNA levels of nif genes in Methylosinus sp. 3S-1 cells grown at different O2 concentrationsa
| Gene | 2% O2b | 20% O2b | ||
|---|---|---|---|---|
| 18 h | 18 h | 96 h | ||
| nifH | 0.2±0.9 | 7.9±1.3 | 8.8±0.5 | |
| nifB | 0.9±0.3 | 9.0±1.2 | 9.6±0.4 | |
| nifA | 0.2±1.7 | 3.0±0.6 | 2.8±0.1 | |
a Cultures used for the quantitative reverse transcription PCR analysis are the same as those in Table 1.
b Values are the means±SD of the log2 fold change in mRNA levels relative to those in the preculture (2% O2) from three separate cultures.
To focus on changes during diazotrophic growth under different O2 conditions, we monitored 3S-1 growth in N-free medium over time to compare the expression of nif genes and pmoA. Following the O2 shift from 2 to 10% (v/v), the mRNA levels of nifH, nifB, and nifA were 18-, 96-, and 6-fold higher, respectively, after 15 h than those under 2% O2 culture conditions. The mRNA levels of these genes then returned to the 2% O2 culture levels or lower by 72 h while O2 and CH4 levels were decreasing (Fig. 3A). On the other hand, the NifH protein was detected at 15 h as a more intense and larger band upward of that from the 2% O2 culture. Only the upper portion of the band remained with decreased intensity at 36 h, and it disappeared below the detection limit by 72 h (Fig. 4A). Following the shift from 2 to 20% O2 (v/v), the mRNA levels of nif genes markedly increased at 15 h, as described above, and were 26- and 364-fold higher than 2% O2 culture levels for nifH and nifB, respectively (Fig. 3B). These high levels continued up to 144 h while O2 remained at >17% and CH4 at >2.5% (Fig. 3B). NifH protein levels decreased at 15 h and then gradually increased with an upshift in the band position (Fig. 4A). In contrast to the nif genes, the mRNA level of pmoA decreased at 15 h under 10 and 20% O2; it then recovered to the 2% O2 culture level in the 10% O2 culture, but only partially in the 20% O2 culture (Fig. 3A and B). As a control in the protein analysis, RpoH levels remained unchanged in both the 10% and 20% O2 experiments (Fig. 4B). Therefore, the changes observed in mRNA and protein levels were specific to nif genes and the NifH protein, respectively. These results indicate that O2 concentrations negatively correlated with nitrogenase protein levels and positively correlated with nif mRNA levels.
Time-course changes in mRNA levels of nif genes in Methylosinus sp. 3S-1 during growth in N-free medium following an increase in the O2 concentration. A 3S-1 preculture grown in N-free medium at 2% (v/v) O2 (Time 0 in the graph) was diluted to an OD600 of 0.05 and the culture was initiated at an O2 concentration 10% (v/v) (panel A) or 20% (v/v) (panel B). Culture fluid and headspace gas were taken at intervals to assess the OD600, mRNA levels of nifH, nifB, nifA, and pmoA, and O2 and CH4 concentrations. Values are the means±SD of four biologically independent measurements and error bars indicate SDs.
Time-course changes in NifH protein levels in Methylosinus sp. 3S-1 during growth in N-free medium following an increase in the O2 concentration. Cell lysate samples were taken from the same culture as that in Fig. 3 and subjected to a Western blot analysis using anti-NifH (panel A) and anti-RpoH (panel B) antibodies (a lysate containing 1 μg total protein was applied to each lane in both cases). Time (h) after an O2 shift to 10 or 20% (v/v) is indicated above each lane; lane P indicates the preculture, which was grown at 2% (v/v) O2. The positions of molecular weight markers are indicated on the left side of each panel.
The transcriptional pattern of nif genes was attributed to NifA properties. 3S-1 and other type II (α-proteobacterial) methanotrophs belonging to the genera Methylosinus, Methylocystis, and Methylocella form a distinct clade in the NifA phylogeny, the topology of which differs from that of the 16S rRNA phylogeny (Fig. 5). NifA generally has a domain structure consisting of an N-terminal GAF (cGMP-specific phosphodiesterases, Anabaena adenylate cyclases, and Escherichia coli FhlA) domain, a central σ54-interacting/AAA+ domain, and a C-terminal DNA-binding domain. α-Proteobacterial NifA is further characterized by conserved cysteine residues that are located near the end of the σ54-interacting domain and within a linker between the σ54-interacting domain and DNA-binding domain. These cysteine residues have been suggested to correlate with intrinsic O2 sensitivity (Dixon and Kahn, 2004). Although these cysteine residues are still conserved, Methylocystaceae NifAs vary from other NifAs mainly in the GAF domain and interdomain linker (Fig. S5), which may confer unique properties to methanotrophs. Moreover, the increase in nifA mRNA levels following the O2 upshift may have contributed to the enhanced transcription of other nif genes. In 3S-1, the autoactivation of nifA expression appeared to occur through a putative NifA/σ54-type promoter located across the upstream gene of nifA (Fig. S4).
Phylogenetic relationship between α-proteobacterial diazotrophs. Phylogenies based on the amino acid sequences of NifA (left) and nucleotide sequences of 16S rRNA (right) were analyzed using the maximum likelihood method. Species names in red and cyan indicate methanotrophs belonging to the families Methylocystaceae (genera Methylosinus and Methylocystis) and Beijerinckiaceae (genera Methylocella and Methylocapsa), respectively. Note that Methylocystis hirsuta and M. bryophila each possess two nifA homologs. Numerals above branches are the related bootstrap values (%; values ≥50 are shown). Scale bars indicate the substitution number per site.
The present study revealed a relationship between the surrounding O2 concentration and nif mRNA levels in Methylosinus sp. 3S-1 during diazotrophic growth. The transcription of nif genes in several obligate aerobes and facultative anaerobes is down-regulated as the concentration of O2 increases, which is consistent with the intrinsic sensitivity of nitrogenase to O2 (Dixon and Kahn, 2004; Martin del Campo et al., 2022). However, nif mRNA levels in 3S-1 in the present study were markedly higher at O2 concentrations of 10% and 20% (v/v) than 2% (v/v) (Table 2 and Fig. 3), whereas 2% O2 appeared to be more favorable for diazotrophic growth than the higher concentrations (Fig. 1). In consideration of the sequence motifs located upstream of the putative nif operons, the transcription of nif genes may be regulated by NifA and this regulator acts on nif promoters in an O2-insensitive manner through its intrinsic characteristics or the involvement of an additional protective factor. This notion is consistent with the distinctiveness of methanotrophs in terms of the NifA phylogeny of α-proteobacteria (Fig. 5). In this context, we considered two explanations for the cellular mechanisms underlying the up-regulated transcription of nif genes under increased O2 conditions. Specifically, O2 may promote NifA activity directly or through an unknown regulator that senses O2. Alternatively, nitrogenase damage from O2 may aggravate the starvation of fixed nitrogen and the imbalance in the cellular redox state, each of which could increase NifA activity (Fig. 6). The present results showed that N2 fixation decreased in 3S-1 as the concentration of O2 was increased from 2% (v/v) (Fig. 1 and Table 1). On the other hand, CH4 oxidation consistently functioned at both 2 and 20% (v/v) O2 (Fig. S2) to generate energy and reducing power to be utilized in cellular metabolism. In some α-proteobacteria, such as Rhodobacter capsulatus and Bradyrhizobium diazoefficiens, excess reducing equivalents contribute to the up-regulated expression of nif genes through the RegB/RegA (or homologous RegS/RegR) signal transduction pathway, which is plausible because N2 fixation functions as a major sink of electrons (Joshi and Tabita, 1996; Elsen et al., 2000, 2004; Emmerich et al., 2000). We consider it likely that the condition assumed in the second explanation continues as long as 3S-1 is viable for CH4 oxidation with nitrogenase activity impaired by O2. In any case, enhanced nif transcription may increase the synthesis of nitrogenase, which partly compensates for the loss caused by O2 damage. The unique pattern of nif expression in response to O2 may function as a strategy by this methanotroph to maintain N2-fixing activity on the background of aerobic CH4 oxidation within the same cell.
A proposed process in Methylosinus sp. 3S-1 to achieve mRNA levels of nif genes under high O2 conditions. The transcriptional regulator NifA is activated by nitrogen (N) depletion as in many proteobacteria. O2 builds up the signal of demand for fixed nitrogen by damaging nitrogenase under diazotrophic conditions. In the case where O2 does not inactivate NifA, as in other proteobacteria, the signal may be transduced to directly enhance nif transcription under conditions that are unfavorable for the maintenance of nitrogenase activity. Moreover, excess reducing equivalents (e–) due to a reduction in nitrogen fixation may serve to promote NifA activity. The autoactivation of nifA transcription also contributes to the increase in total NifA activity.
The present results establish the potential of 3S-1 for N2 fixation across a wide range of O2 concentrations. We suggested a difference between N2 fixation settings under high and low O2 conditions. NifH protein levels were lower at an initial O2 concentration of 20% than 2% despite the markedly up-regulated transcription (Fig. 2 and 4). The lower protein level is consistent with the low N2 fixation rate under the same condition. Moreover, we found that NifH was modified under 10% and 20% (v/v) O2 conditions, resulting in changes in its electrophoretic mobility. This NifH modification was also reported in M. trichosporium OB3b (Kim and Graham, 2001) and has yet to be characterized. It is important to note that there was a time lag before N2 fixation resumed at 20% O2 upon a shift from 2% O2; OD600 markedly increased, whereas cell growth stopped with no N2 fixation for this period (Table 1). This implies that the cellular and molecular settings for N2 fixation need to be remodeled to adapt to high O2 conditions. To elucidate the mechanisms enabling N2 fixation under high O2 conditions, the molecular events occurring during the time lag need to be examined. Unlike the shift to 20%, 3S-1 cells showed increases in both nif mRNA and NifH protein levels following the O2 shift from 2 to 10% (v/v) (Fig. 3 and 4). Since diazotrophic growth was slower at 10% O2 than at 2% O2 (Fig. 1), a fraction of nitrogenase may be inactive, thereby increasing NifA activity through the aforementioned process. Therefore, setting stepwise increases in the concentration of O2 in a turbidostat may be useful for investigating the mechanisms by which O2 affects each of the ordered steps in nitrogenase turnover, such as the transcription of nif genes, the maturation of nitrogenase complexes, inactivation due to metallocluster degradation, and proteolysis.
Abdela, A. A., Shinjo, R., Watanabe, T., Asakawa, S., Masuda, S., Shibata, A., et al. (2025) Transcription of Nitrogen Fixation Genes Is Enhanced at Unfavorably High Oxygen Concentrations for Diazotrophic Growth in a Methane-oxidizing Bacterium. Microbes Environ 40: ME25032.
https://doi.org/10.1264/jsme2.ME25032
This work was supported in part by the Advanced Technologies for Carbon-Neutral (ALCA-Next) program from the Japan Science and Technology Agency (JST), a project, JPNP18016, commissioned by the New Energy and Industrial Technology Development Organization (NEDO), JSPS KAKENHI Grant Number JP25K08874, and the RIKEN-TRIP initiative (fieldomics).
