Anammox bacteria drive fixed nitrogen loss in hadal trench sediments

Benthic N2 production by microbial denitrification and anammox is the largest sink for fixed nitrogen in the oceans. Most N2 production occurs on the continental shelves, where a high flux of reactive organic matter fuels the depletion of nitrate close to the sediment surface. By contrast, N2 production rates in abyssal sediments are low due to low inputs of reactive organics, and nitrogen transformations are dominated by aerobic nitrification and the release of nitrate to the bottom water. Here, we demonstrate that this trend is reversed in the deepest parts of the oceans, the hadal trenches, where focusing of reactive organic matter enhances benthic microbial activity. Thus, at ∼8-km depth in the Atacama Trench, underlying productive surface waters, nitrate is depleted within a few centimeters of the sediment surface, N2 production rates reach those reported from some continental margin sites, and fixed nitrogen loss is mainly conveyed by anammox bacteria. These bacteria are closely related to those known from shallow oxygen minimum zone waters, and comparison of activities measured in the laboratory and in situ suggest they are piezotolerant. Even the Kermadec Trench, underlying oligotrophic surface waters, exhibits substantial fixed N removal. Our results underline the role of hadal sediments as hot spots of deep-sea biological activity, revealing a fully functional benthic nitrogen cycle at high hydrostatic pressure and pointing to hadal sediments as a previously unexplored niche for anaerobic microbial ecology and diagenesis.

Diversity and evolution of nitric oxide reduction

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- → NO2- → NO → N2O → N2). Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we propose that a number of uncharacterized HCO families perform nitric oxide reduction and demonstrate that an enzyme from Rhodothermus marinus, belonging to one of these families does perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. They also exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans.

Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

Testing Whether Pre-Pod-Fill Symbiotic Nitrogen Fixation in Soybean Is Subject to Drift or Selection Over 100 Years of Soybean Breeding

Soybean [Glycine max (L.) Merr.] is the world's leading legume crop and the largest oilseed crop. It forms a symbiotic relationship with rhizobia bacteria residing in root nodules that provide fixed nitrogen to host plants through symbiotic nitrogen fixation (SNF). In soybean, it has been widely reported that the highest SNF occurs at the pod-filling stage, associated with the peak demand for nitrogen. However, the majority of seed nitrogen is derived from remobilizing root/shoot nitrogen, representing cumulative SNF from the seedling stage to the pre-pod-fill stage. Therefore, the question arises as to whether there has also been selection for improved SNF at these earlier stages, or whether pre-pod-fill SNF traits have drifted. To test this hypothesis, in this study, pre-pod SNF-related traits were evaluated in soybean cultivars that span 100 years of breeding selection in the Canadian Province of Ontario. Specifically, we evaluated SNF traits in 19 pedigree-related historical cultivars and 25 modern cultivars derived from the University of Guelph soybean breeding program. Field trials were conducted at Woodstock, Ontario, Canada in 2016 and 2017, and various SNF-related traits were measured at pre-pod-fill stages (R1-R3), including nitrogen fixation capacity. Considerable variation was observed among Canadian soybean cultivars released over the past 100 years for pre-pod-fill nitrogen fixation. The modern soybean cultivars had similar or moderately higher pre-pod-fill SNF compared to the historical lines in terms of the percentage of nitrogen derived from the atmosphere (%Ndfa) and total shoot fixed nitrogen. These findings suggest that, despite no direct selection by breeders, pre-pod-fill nitrogen fixation, and associated SNF traits have been maintained and possibly improved in modern soybean breeding. However, the low level of pre-pod-fill SNF in some modern cultivars, and generally wide variation observed in SNF between them, suggest some level of genetic drift for this trait in some pedigrees. Specific historical and modern soybean cultivars were identified as potential parents to enable targeted breeding for improved pre-pod-fill SNF. This retrospective study sheds light on our understanding of the impact of decades of recent selective breeding on pre-pod-fill nitrogen fixation traits in soybean in a temperate environment.

UCYN-A/haptophyte symbioses dominate N2 fixation in the Southern California Current System

The availability of fixed nitrogen (N) is an important factor limiting biological productivity in the oceans. In coastal waters, high dissolved inorganic N concentrations were historically thought to inhibit dinitrogen (N2) fixation, however, recent N2 fixation measurements and the presence of the N2-fixing UCYN-A/haptophyte symbiosis in nearshore waters challenge this paradigm. We characterized the contribution of UCYN-A symbioses to nearshore N2 fixation in the Southern California Current System (SCCS) by measuring bulk community and single-cell N2 fixation rates, as well as diazotroph community composition and abundance. UCYN-A1 and UCYN-A2 symbioses dominated diazotroph communities throughout the region during upwelling and oceanic seasons. Bulk N2 fixation was detected in most surface samples, with rates up to 23.0 ± 3.8 nmol N l−1 d−1, and was often detected at the deep chlorophyll maximum in the presence of nitrate (>1 µM). UCYN-A2 symbiosis N2 fixation rates were higher (151.1 ± 112.7 fmol N cell−1 d−1) than the UCYN-A1 symbiosis (6.6 ± 8.8 fmol N cell−1 d−1). N2 fixation by the UCYN-A1 symbiosis accounted for a majority of the measured bulk rates at two offshore stations, while the UCYN-A2 symbiosis was an important contributor in three nearshore stations. This report of active UCYN-A symbioses and broad mesoscale distribution patterns establishes UCYN-A symbioses as the dominant diazotrophs in the SCCS, where heterocyst-forming and unicellular cyanobacteria are less prevalent, and provides evidence that the two dominant UCYN-A sublineages are separate ecotypes.

Crocosphaera as a major consumer of fixed nitrogen despite its capability of nitrogen fixation

Crocosphaera watsonii (hereafter Crocosphaera) is a key nitrogen (N) fixer in the ocean, but its ability to consume combined N sources is still unclear. Using in situ microcosm incubations with an ecological model, we show that Crocosphaera has high competitive capability both under low and moderately high combined N concentrations. In field incubations, Crocosphaera accounted for the highest consumption of ammonium and nitrate, followed by pico-eukaryotes. The model analysis shows that cells have a high ammonium uptake rate (~7 mol N (mol N)-1 d-1 at the maximum), which allows them to compete against pico-eukaryotes and non-diazotrophic cyanobacteria when combined N is sufficiently available. Even when combined N is depleted, their capability of nitrogen fixation allows higher growth rates compared to potential competitors. These results suggest the high fitness of Crocosphaera in combined N limiting, oligotrophic oceans, and thus heightens its potential significance in its ecosystem and in biogeochemical cycling.

Hydrogen Peroxide Can Be a Plausible Biomarker in Cyanobacterial Bloom Treatment

The effect of combined stresses, photoinhibition and nutrient depletion, on the oxidative stress of cyanobacteria was measured in laboratory experiments, to develop the biomass prediction model. Phormidium ambiguum was exposed to various photosynthetically active radiation (PAR) intensities and phosphorous concentrations with fixed nitrogen concentration. The samples were subjected to stress assays by detecting hydrogen peroxide (H2O2) concentration and antioxidant activities of catalase (CAT) and superoxide dismutase (SOD). H2O2 concentration decreased to 30 µmolm-2s-1 of PAR, then increased further with higher PAR intensity. Regarding phosphorus concentration, H2O2 concentration generally decreased with increasing phosphorus concentration. SOD and CAT activities were proportionate to the H2O2 protein-1. No H2O2 concentration detected outside of cells indicated the biological production of H2O2, and the accumulated H2O2 concentration inside cells was parameterized with H2O2 concentration protein-1. Over 30 µmolm-2s-1 of PAR, H2O2 concentration protein-1 had a similar increasing trend with PAR intensity, independently of phosphorous concentration. Meanwhile, with increasing phosphorous concentration, H2O2 protein-1 decreased in a similar pattern regardless of PAR intensity. Protein content decreased with increasing H2O2 gradually up to 4nmol H2O2 mg-1protein, which provides a threshold to restrict the growth of cyanobacteria. With these results. an empirical formula was developed to obtain the cyanobacteria biomass.

Nitrogen Fixation and Diazotrophic Community in Plastic-Eating Mealworms Tenebrio Molitor L.

Mealworms, the larvae of a coleopteran insect Tenebrio molitor L., are capable of eating, living on and degrading the non-hydrolyzable vinyl plastics as sole diet. However, vinyl plastics are carbon-rich but nitrogen-deficient. It remains puzzling how plastic-eating mealworms overcome the nutritional obstacle of nitrogen limitation. Here, we provide the evidence for nitrogen-fixation activity within plastic-eating mealworms. Acetylene reduction assays illustrate that the nitrogen-fixing activity ranges from 12.3 ± 0.7 to 32.9 ± 9.3 nmol ethylene·h− 1·gut− 1 and the corresponding fixed nitrogen equivalents of protein are estimated as 8.6 to 23.0 µg per day per mealworm. Nature nitrogen isotopic analyses of plastic-eating mealworms provide further evidence for the importance of nitrogen fixation as a new nitrogen source. Eliminating the gut microbial microbiota with antibiotics impairs the mealworm’s ability to fix nitrogen from atmosphere, indicating the contribution of gut microbiota to nitrogen fixation. By using the traditional culture-dependent technique, PCR and RT-PCR of nifH gene, nitrogen-fixing bacteria diversity within the gut was detected and the genus Klebsiella was demonstrated to be an important nitrogen-fixing symbiont. These findings first build the relationship between the plastic degradation (carbon metabolism) and nitrogen fixation (nitrogen metabolism) within mealworms. Combined with previously reported plastic-degrading capability and nitrogen-fixing activity, mealworms may be potential candidates for up-recycling of plastic waste to produce protein sources.

Disrupting hierarchical control of nitrogen fixation enables carbon-dependent regulation of ammonia excretion in soil diazotrophs

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.