Biology subjects

Frostegard, A.

Publications and source records attributed to Frostegard, A..

9 recordsLinked to original sources

bioRxiv2025

Two Small Proteins Enable N2O Reduction in Acidic Environments

Denitrifying bacteria are major contributors to nitrous oxide (N2O) emissions, which rise with global soil acidification, likely because low pH impairs maturation of the periplasmic enzyme N2O reductase (NosZ) in most bacteria. While some bacteria can reduce N2O under acidic conditions, the underlying mechanism remain unclear. Here, we isolated bacteria from acidic soils and identified strains that efficiently reduce N2O at low pH. They carry two novel proteins, NosP and NosQ, along with NosZ clade I. Functional analyses of deletion mutants demonstrated that NosP and NosQ are essential for the maturation of NosZ at low pH, but are not required at circumneutral pH. Cryo-EM analysis indicates that NosPQ forms a protective complex with NosZ, safeguarding its maturation under acidic conditions. Metagenomic analysis shows that nosPQ genes are widely distributed across diverse bacterial taxa and soil types. Our findings reveal a key molecular adaptation mechanism for N2O reduction in acidic environments and provide valuable insights for developing efficient N2O mitigation strategies.

biochemistry↗

bioRxiv2025

Optimizing Carbon Sources to Promote Soil Denitrifiers: Lessons for Incubations, Enrichments, and Bioaugmentation

Oxygen concentrations fluctuate in soil across time and space. Under anoxic conditions, the three main microbial metabolic pathways - denitrification, fermentation, and DNRA - compete for the same carbon (C) sources. Studies on denitrification in complex soil communities often rely on incubation experiments to determine how various factors affect the regulatory biology of denitrifying organisms and their N2O emissions. These experiments typically require an exogenous C source to stimulate measurable activity, and the choice of C source is critical as it should support denitrification while minimizing competition from fermentation and DNRA. This consideration is equally important for the enrichment and isolation of diverse denitrifying organisms and for bioaugmentation-based N2O-mitigation strategies. Here, we compared twelve C sources, including glutamic acid, acetate, an artificial root exudate cocktail (eight compounds, individually and in combination), and a clover extract. By combining high-resolution denitrification gas kinetics, metagenomic sequencing, and 15N isotope labelling, we aimed to find a C source(s) that (1) supports a diverse soil-derived denitrifying community and (2) limits the competition for C from alternative anaerobic pathways. Among the tested substrates, only the clover extract supported denitrification and maintained a complex denitrifying community. Yet, it also promoted fermentation and DNRA, revealing that a trade-off must exist between fostering denitrifier diversity and limiting growth of organisms using competing anaerobic pathways. More broadly, our results highlight that C source is a methodological fulcrum in controlled soil microbiome studies. It shapes community composition, drives metabolic processes, and ultimately determines the ecological relevance of experimental outcomes and the success of enrichments and soil bioaugmentation approaches.

microbiology↗

bioRxiv2025

Distinct denitrification phenotypes in closely related bacteria: clues to understanding variations in nitrite accumulation among Stutzerimonas strains

Nitrite (NO2-) is a key denitrification intermediate, formed from nitrate (NO3-). Transient NO2- accumulation varies among denitrifiers, yet the underlying causes remain poorly understood, despite its potential toxicity and role in NO and N2O emissions. We profiled eighteen related Stutzerimonas strains, including the model S. perfectomarina ZoBell, and identified three phenotypic clusters (full, partial and low nitrite accumulators; FNA, PNA and LNA) based on the fraction of NO3--N transiently accumulated as NO2-. LNA strains lack or express the membrane-bound nitrate reductase (NarG) late, relying on periplasmic NapA for NO3- reduction, possibly explaining their balanced NO2- production/reduction. FNA and PNA strains possess NapA and NarG but differ in their nitrite reductase (NirS) clades. Delayed nirS transcription as FNA strains transition to NO3- respiration likely accounts for some NO2- accumulation. However, addition of NO3- halted NO2- reduction in FNA strains, suggesting additional metabolic control. This may require the cytochromes NirTB, which are only found in FNA strains. The regulator DnrE was also unique to NO2--accumulators, likely having a role in finetuning NO2- regulation. Our findings reveal diverse NO2--handling phenotypes among denitrifiers and provide insights for optimizing wastewater nitrogen removal and soil bioaugmentation strategies to mitigate N2O emissions.

microbiology↗

bioRxiv2023

Suggested role of NosZ in preventing N2O inhibition of dissimilatory nitrite reduction to ammonium

Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess clade II nosZ genes and the ability to respire N2O. However, the selective advantages that may favor these nosZ-harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N2O on Nrf-mediated DNRA was examined in a recently isolated soil bacterium, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C2H2, a NosZ inhibitor, with or without N2O, and the results were compared with C2H2-free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N2O. The NO2--to-NH4+ reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C2H2-amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possible involvement of C2H2 was dismissed since the cultures continuously flushed with C2H2/N2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N2O-containing gas. The findings provide novel ecological and evolutionary insights into the oft-observed presence of nosZ genes in DNRA-catalyzing microorganisms. ImportanceDissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3- and/or NO2- to NH4+. Interestingly, many DNRA-catalyzing microorganisms possessing nrfA genes harbor nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes that had probably been acquired via horizontal gene transfers: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.

microbiology↗

bioRxiv2022

Denitrifying bradyrhizobia retain strong N2O reduction during periods of starvation

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also have to survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2 tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well-known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O-over NO3 --reduction in bradyrhizobia is retained under carbon limitation. Here we show that starved cultures of a Bradyrhizobium strain with respiration rates 1-18% of well-fed cultures, reduced all available N2O before touching provided NO3-. Proteomics showed similar abundance of Nap (periplasmic NO3 - reductase) and NosZ (N2O reductase), suggesting that competition between electron pathways to Nap and NosZ favoured N2O reduction also in starved cells, similar to well-fed cultures. This contrasts the general notion that NosZ activity diminishes under carbon limitation. The results suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions and that this criterion should be considered in the development of biofertilizers. ImportanceLegume cropped farmlands account for substantial N2O emissions globally. Legumes are commonly inoculated with N2-fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging to Bradyrhizobium, the micro-symbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N2O reductase and will be N2O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N2O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here we demonstrate that bradyrhizobia retain their strong preference for N2O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N2-fixation and minimize N2O emission.

microbiology↗

bioRxiv2021

A novel dual enrichment strategy provides soil- and digestate- competent N2O-respiring bacteria for mitigating climate forcing in agriculture

Manipulating soil metabolism by heavy inoculation with microbes is deemed realistic if waste from anaerobic digestion (digestate) is utilized as substrate and vector, but requires organisms that can grow both in digestate and soil (=generalist). We designed a strategy to enrich and isolate such generalist N2O-respiring bacteria (NRB) in soil and digestate, to provide inoculum for reducing N2O-emissions from agricultural soil. Sequential anaerobic enrichment cultures were provided with a small dose of O2 and unlimited N2O, alternating between sterilized digestate and soil as substrates. The cultures were monitored for gas kinetics and community composition (16SrDNA), and cluster-analysis identified generalist-OTUs which became dominant, digestate/soil-specialists which did not, and a majority that were diluted out. Several NRBs circumscribed by generalist-OTUs were isolated, genome sequenced to screen for catabolic capacity, and phenotyped, to assess their capacity as N2O-sinks in soil. The two isolates Cloacibacterium sp., carrying only N2O-reductase (Clade-II) and Pseudomonas sp., with full-fledged denitrification-pathway, were both very effective N2O-sinks in soil, with Pseudomonas sp., showing a long-lasting sink effect, suggesting better survival in soil. This avenue for utilizing waste to bioengineer the soil microbiota holds promise to effectively combat N2O-emissions but could also be utilized for enhancing other metabolic functions in soil. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2020

Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil

Denitrifier community phenotypes often result in transient accumulation of denitrification (NO3-[->]NO2-[->]NO[->]N2O[->]N2) intermediates. Consequently, anoxic spells drive NO-, N2O- and possibly HONO-emissions to the atmosphere, affecting both climate and tropospheric chemistry. Soil pH is a key controller of intermediate levels, and while there is a clear negative correlation between pH and emission of N2O, NO2- concentrations instead increase with pH. These divergent trends are probably a combination of direct effects of pH on the expression/activity of denitrification enzymes, and an indirect effect via altered community composition. This was studied by analyzing metagenomics/transcriptomics and phenomics of two soil denitrifier communities, one of pH 3.8 (Soil3.8) and the other 6.8 (Soil6.8). Soil3.8 had severely delayed N2O reduction despite early transcription of nosZ, encoding N2O reductase, by diverse denitrifiers, and of several nosZ accessory genes. This lends support to a post-transcriptional, pH-dependent mechanism acting on the NosZ apo-protein or on enzymes involved in its maturation. Metagenome/metatranscriptome reads of nosZ were almost exclusively clade I in Soil3.8 while clade II dominated in Soil6.8. Reads of genes and transcripts for NO2--reductase were dominated by nirK over nirS in both soils, while qPCR-based determinations showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK community. The -omics results suggested that low NO2- concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to enzymatic activity. The NO reductase gene qnor was strongly expressed in Soil3.8, suggesting an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2- accumulating soil, was estimated to be ten times higher from Soil3.8 than from Soil6.8. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

microbiology↗

bioRxiv2020

Bacteria in biogas digestates for reduced climate forcing

Inoculating agricultural soils with N2O-respiring bacteria (NRB) can reduce N2O-emissions, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB's, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB's were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

microbiology↗

bioRxiv2020

Competition for electrons favors N2O reduction in denitrifying Bradyrhizobium isolates

Bradyrhizobia are common members of soil microbiomes and known as N2-fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2O. Inoculation with compatible rhizobia is often needed for optimal N2-fixation, but the choice of inoculant may also have consequences for N2O emission. Here, we analyzed the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. All were dinitrifiers, but only ~1/3 could reduce N2O while most others were net N2O producers. The N2O-reducing isolates showed strong preference for N2O- over NO3--reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3- reductase) and Nos (N2O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3- reductase (Nar), reduced N2O and NO3- simultaneously. We propose that the control at the metabolic level, favoring N2O reduction over NO3- reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar. Originality-Significance StatementThis study extends the current knowledge on denitrification in bradyrhizobia, which mostly originates from studies of one model strain, by investigating the denitrification phenotypes of a diverse collection of Bradyrhizobium isolates. Only 1/3 of them could reduce N2O while the others were net sources for this potent greenhouse gas. All N2O-reducers showed strong preference for N2O over NO3-. We revealed by proteomics that this was not explained by differences in the abundances of Nap (periplasmic nitrate reductase) and Nos (N2O reductase), which strengthens our hypothesis (Mania et al., 2020) of a metabolic control mechanism by which Nos competes efficiently with Nap for electrons, making these organisms strong sinks for N2O. The findings highlight the potential importance of these organisms as N2O sinks in natural and agricultural ecosystems and pinpoint the need to take N2O reduction into account, along with N2-fixation effectiveness, when searching for strains suitable for production of inoculants.

microbiology↗