Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.
ABSTRACT: Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.
Project description:High input of organic carbon and/or slowly renewing bottom waters frequently create periods with low dissolved oxygen concentrations on continental shelves and in coastal areas; such events can have strong impacts on benthic ecosystems. Among the meiofauna living in these environments, benthic foraminifera are often the most tolerant to low oxygen levels. Indeed, some species are able to survive complete anoxia for weeks to months. One known mechanism for this, observed in several species, is denitrification. For other species, a state of highly reduced metabolism, essentially a state of dormancy, has been proposed but never demonstrated. Here, we combined a 4 weeks feeding experiment, using 13C-enriched diatom biofilm, with correlated TEM and NanoSIMS imaging, plus bulk analysis of concentration and stable carbon isotopic composition of total organic matter and individual fatty acids, to study metabolic differences in the intertidal species Ammonia tepida exposed to oxic and anoxic conditions. Strongly contrasting cellular-level dynamics of ingestion and transfer of the ingested biofilm components were observed between the two conditions. Under oxic conditions, within a few days, intact diatoms were ingested, degraded, and their components assimilated, in part for biosynthesis of different cellular components: 13C-labeled lipid droplets formed after a few days and were subsequently lost (partially) through respiration. In contrast, in anoxia, fewer diatoms were initially ingested and these were not assimilated or metabolized further, but remained visible within the foraminiferal cytoplasm even after 4 weeks. Under oxic conditions, compound specific 13C analyses showed substantial de novo synthesis by the foraminifera of specific polyunsaturated fatty acids (PUFAs), such as 20:4(n-6). Very limited PUFA synthesis was observed under anoxia. Together, our results show that anoxia induced a greatly reduced rate of heterotrophic metabolism in Ammonia tepida on a time scale of less than 24 hours, these observations are consistent with a state of dormancy.
Project description:Microorganisms can influence inorganic phosphate (Pi) in pore waters, and thus the saturation state of phosphatic minerals, by accumulating and hydrolyzing intracellular polyphosphate (poly-P). Here we used comparative metatranscriptomics to explore microbial poly-P utilization in marine sediments. Sulfidic marine sediments from methane seeps near Barbados and from the Santa Barbara Basin (SBB) oxygen minimum zone were incubated under oxic and anoxic sulfidic conditions. Pi was sequestered under oxic conditions and liberated under anoxic conditions. Transcripts homologous to poly-P kinase type 2 (ppk2) were 6-22 × more abundant in metatranscriptomes from the anoxic incubations, suggesting that reversible poly-P degradation by Ppk2 may be an important metabolic response to anoxia by marine microorganisms. Overall, diverse taxa differentially expressed homologues of genes for poly-P degradation (ppk2 and exopolyphosphatase) under different incubation conditions. Sulfur-oxidizing microorganisms appeared to preferentially express genes for poly-P degradation under anoxic conditions, which may impact phosphorus cycling in a wide range of oxygen-depleted marine settings.
Project description:Freshwater reservoirs are important sites of organic carbon (OC) burial, but the extent to which reservoir OC burial is a new anthropogenic carbon sink is currently unclear. While burial of aquatic OC (by, e.g., phytoplankton) in reservoirs may count as a new C sink, the burial of terrestrial OC in reservoirs constitutes a new C sink only if the burial is more efficient in reservoirs than in other depositional environments. We carried out incubation experiments that mimicked the environmental conditions of different depositional environments along the land-sea continuum (oxic and anoxic freshwater, oxic and anoxic seawater, oxic river bedload, and atmosphere-exposed floodplain) to investigate whether reservoirs bury OC more efficiently compared to other depositional environments. For sediment OC predominantly of terrestrial origin, OC degradation rates were significantly lower, by a factor of 2, at anoxic freshwater and saltwater conditions compared to oxic freshwater and saltwater, river, and floodplain conditions. However, the transformation of predominantly terrestrial OC to methane was one order of magnitude higher in anoxic freshwater than at other conditions. For sediment OC predominantly of aquatic origin, OC degradation rates were uniformly high at all conditions, implying equally low burial efficiency of aquatic OC (76% C loss in 57 days). Since anoxia is more common in reservoirs than in the coastal ocean, these results suggest that reservoirs are a depositional environment in which terrestrial OC is prone to become buried at higher efficiency than in the ocean but where also the terrestrial OC most efficiently is transformed to methane.
Project description:Foraminifera are single-celled eukaryotes (protists) of large ecological importance, as well as environmental and paleoenvironmental indicators and biostratigraphic tools. In addition, they are capable of surviving in anoxic marine environments where they represent a major component of the benthic community. However, the cellular adaptations of Foraminifera to the anoxic environment remain poorly constrained. We sampled an oxic-anoxic transition zone in marine sediments from the Namibian shelf, where the genera Bolivina and Stainforthia dominated the Foraminifera community, and use metatranscriptomics to characterize Foraminifera metabolism across the different geochemical conditions. Relative Foraminifera gene expression in anoxic sediment increased an order of magnitude, which was confirmed in a 10-day incubation experiment where the development of anoxia coincided with a 20-40-fold increase in the relative abundance of Foraminifera protein encoding transcripts, attributed primarily to those involved in protein synthesis, intracellular protein trafficking, and modification of the cytoskeleton. This indicated that many Foraminifera were not only surviving but thriving, under the anoxic conditions. The anaerobic energy metabolism of these active Foraminifera was characterized by fermentation of sugars and amino acids, fumarate reduction, and potentially dissimilatory nitrate reduction. Moreover, the gene expression data indicate that under anoxia Foraminifera use the phosphogen creatine phosphate as an ATP store, allowing reserves of high-energy phosphate pool to be maintained for sudden demands of increased energy during anaerobic metabolism. This was co-expressed alongside genes involved in phagocytosis and clathrin-mediated endocytosis (CME). Foraminifera may use CME to utilize dissolved organic matter as a carbon and energy source, in addition to ingestion of prey cells via phagocytosis. These anaerobic metabolic mechanisms help to explain the ecological success of Foraminifera documented in the fossil record since the Cambrian period more than 500 million years ago.
Project description:In the present study we have investigated the influence of light and anoxia on the energetic state of the aerobic anoxygenic phototroph (AAP) Dinoroseobacter shibae. Respiration, chemiosmotic proton translocation and the adenylate energy charge (AEC) of the cells were measured comparing light versus dark and oxic versus anoxic conditions. Light caused a decrease of the respiration rates of washed cells. This might be a substitution rather than a direct inhibitory effect, because both photosynthesis and respiration contribute to the proton-motive force. As known from other AAPs, light alone did not induce proton translocation if applied to anoxic cell suspensions. However, additions of small oxygen pulses to anoxic cell suspensions caused two times more proton translocation in the light than in the dark. The AEC of the cells was measured by means of a modified luciferin-luciferase method. Growing cells of D. shibae kept an AEC of 0.93, indicating that the adenylate pool was highly phosphorylated. After harvesting and storing the cells under anoxic conditions for 2 h, the AEC dropped to 0.12. However, the cells remained reactive. Upon addition of oxygen, the AEC increased to its original value within 40 s by the formation of about 12 mM of intracellular ATP. There were no differences whether this recovery experiment was carried out in the dark or in the light. We conclude that D. shibae is able to change its energetic state not only in response to the light regime but also during oxic-anoxic transitions. Both responses appear suited to save in situ organic substrates and endogenous electron donors, thus enhancing the role of photosynthetic energy conservation.
Project description:Ferrous iron has been known to function as an electron source for iron-oxidizing microorganisms in both anoxic and oxic environments. A diversity of bacteria has been known to oxidize both soluble and solid-phase Fe(II) forms coupled to the reduction of nitrate. Here, we show for the first time Fe(II) oxidation by Sphaerotilus natans strain DSM 6575(T) under mixotrophic condition. Sphaerotilus natans has been known to form a sheath structure enclosing long chains of rod-shaped cells, resulting in a thick biofilm formation under oxic conditions. Here, we also demonstrate that strain DSM 6575(T) grows mixotrophically with pyruvate, Fe(II) as electron donors and nitrate as an electron acceptor and single cells of strain DSM 6575(T) are dominant under anoxic conditions. Furthermore, strain DSM 6575(T) forms nanoball-shaped amorphous Fe(III) oxide minerals encrusting on the cell surfaces through the mixotrophic iron oxidation reaction under anoxic conditions. We propose that cell encrustation results from the indirect Fe(II) oxidation by biogenic nitrite during nitrate reduction and that causes the bacterial morphological change to individual rod-shaped single cells from filamentous sheath structures. This study extends the group of existing microorganisms capable of mixotrophic Fe(II) oxidation by a new strain, S. natans strain DSM 6575(T) , and could contribute to biogeochemical cycles of Fe and N in the environment.
Project description:Anoxia induces several heat shock proteins and a heat pre-treatment can acclimatize Arabidopsis seedlings to a subsequent anoxic treatment. In this work we analyzed the response of Arabidopsis seedlings to anoxia, heat and a combined heat+anoxia stress. A significant overlapping between the anoxic and heat shock responses has been observed by whole-genome microarray analysis. Overall design: We treated Arabidopsis seedling, 4-days old, dark germinated with: -Control (23°C, dark, liquid Murashige-Skoog medium containing 30mM sucrose). -Heat-treated (38°C for 90 minutes, dark, liquid Murashige-Skoog medium containing 30mM sucrose). -Anoxia-treated (23°C, under anoxia for 6h, dark, liquid Murashige-Skoog medium containing 30mM sucrose). -combined heat+Anoxia-treatment (23°C, treated at 38°C for 90 min and thereafter under anoxia for 6h, dark, liquid Murashige-Skoog medium containing 30mM sucrose). Two biological replicates for each condition.
Project description:Our aim was to elucidate how plant tissues under a severe energy crisis cope with imposition of high NaCl, which greatly increases ion fluxes and hence energy demands. The energy requirements for ion regulation during combined salinity and anoxia were assessed to gain insights into ion transport processes in the anoxia-tolerant coleoptile of rice.We studied the combined effects of anoxia plus 50 or 100 mm NaCl on tissue ions and growth of submerged rice (Oryza sativa) seedlings. Excised coleoptiles allowed measurements in aerated or anoxic conditions of ion net fluxes and O2 consumption or ethanol formation and by inference energy production.Over 80?h of anoxia, coleoptiles of submerged intact seedlings grew at 100 mm NaCl, but excised coleoptiles, with 50 mm exogenous glucose, survived only at 50 mm NaCl, possibly due to lower energy production with glucose than for intact coleoptiles with sucrose as substrate. Rates of net uptake of Na+ and Cl- by coleoptiles in anoxia were about half those in aerated solution. Ethanol formation in anoxia and O2 uptake in aerobic solution were each increased by 13-15 % at 50 mm NaCl, i.e. ATP formation was stimulated. For acclimation to 50 mm NaCl, the anoxic tissues used only 25 % of the energy that was expended by aerobic tissues. Following return of coleoptiles to aerated non-saline solution, rates of net K+ uptake recovered to those in continuously aerated solution, demonstrating there was little injury during anoxia with 50 mm NaCl.Rice seedlings survive anoxia, without the coleoptile incurring significant injury, even with the additional energy demands imposed by NaCl (100 mm when intact, 50 mm when excised). Energy savings were achieved in saline anoxia by less coleoptile growth, reduced ion fluxes as compared to aerobic coleoptiles and apparent energy-economic ion transport systems.
Project description:Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO[Formula: see text], NO[Formula: see text], Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.
Project description:The transcriptional response of Staphylococcus aureus strain Newman to sunlight exposure was investigated under both oxic and anoxic conditions using RNA sequencing to gain insight into potential mechanisms of inactivation. S. aureus is a pathogenic bacterium detected at recreational beaches which can cause gastrointestinal illness and skin infections, and is of increasing public health concern. To investigate the S. aureus photostress response in oligotrophic seawater, S. aureus cultures were suspended in seawater and exposed to full spectrum simulated sunlight. Experiments were performed under oxic or anoxic conditions to gain insight into the effects of oxygen-mediated and non-oxygen-mediated inactivation mechanisms. Transcript abundance was measured after 6 h of sunlight exposure using RNA sequencing and was compared to transcript abundance in paired dark control experiments. Culturable S. aureus decayed following biphasic inactivation kinetics with initial decay rate constants of 0.1 and 0.03 m2 kJ-1 in oxic and anoxic conditions, respectively. RNA sequencing revealed that 71 genes had different transcript abundance in the oxic sunlit experiments compared to dark controls, and 18 genes had different transcript abundance in the anoxic sunlit experiments compared to dark controls. The majority of genes showed reduced transcript abundance in the sunlit experiments under both conditions. Three genes (ebpS, NWMN_0867, and NWMN_1608) were found to have the same transcriptional response to sunlight between both oxic and anoxic conditions. In the oxic condition, transcripts associated with porphyrin metabolism, nitrate metabolism, and membrane transport functions were increased in abundance during sunlight exposure. Results suggest that S. aureus responds differently to oxygen-dependent and oxygen-independent photostress, and that endogenous photosensitizers play an important role during oxygen-dependent indirect photoinactivation.