Biology subjects

Shapleigh, J. P.

Publications and source records attributed to Shapleigh, J. P..

5 recordsLinked to original sources

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↗

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

Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil

Plant-derived phenolic acids are metabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria would enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (PHB). We further investigated whether PHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. The activity of Paraburkholderia and Caballeronia dominated PHB degradation in all soils regardless of tree species or soil type. We isolated the principal PHB-degrading phylotype (Paraburkholderia madseniana RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, aryl-alcohol oxidase and DyP-type peroxidase, and confirmed its ability to degrade phenolics. The addition of PHB to soil led to significant enrichment (23-fold) of the RP11T phylotype (RP11ASV), as well as enrichment of other phylotypes of Paraburkholderia and Caballeronia. Metabolism of PHB primed significant loss of SOC (3 to 13 {micro}mols C g-1 dry wt soil over 7 days). In contrast, glucose addition reduced SOC mineralization (-3 to -8 {micro}mols C g-1 dry wt soil over 7 days). RP11ASV abundance and the expression of PHB monooxygenase (pobA) correlated with PHB respiration and were inversely proportional to SOC content in the field. We propose that plant-derived phenolics stimulate the activity of phenolic acid-degrading bacteria thereby causing soil priming and SOC loss. We show that Burkholderiaceae dominate soil priming in diverse forest soils and this observation counters the prevailing view that priming phenomena are a generalized non-specific response of community metabolism.

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↗