cbb3-Type Cytochrome c Oxidases,Aerobic Respiratory Enzymes,Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

For bacteria, many studies have focused on the role of respiratory enzymes in energy conservation; however, their effect on cell behavior is poorly understood. Pseudomonas aeruginosa can perform both aerobic respiration and denitrification. Previous studies demonstrated that cbb₃-type cytochrome c oxidases that support aerobic respiration are more highly expressed in P. aeruginosa under anoxic conditions than are other aerobic respiratory enzymes. However, little is known about their role under such conditions. In this study, it was shown that cbb₃ oxidases of P. aeruginosa PAO1 alter anaerobic growth, the denitrification process, and cell morphology under anoxic conditions. Furthermore, biofilm formation was promoted by the cbb₃ oxidases under anoxic conditions. cbb₃ oxidases led to the accumulation of nitric oxide (NO), which is produced during denitrification. Cell elongation induced by NO accumulation was reported to be required for robust biofilm formation of P. aeruginosa PAO1 under anoxic conditions. Our data show that cbb₃ oxidases promote cell elongation by inducing NO accumulation during the denitrification process, which further leads to robust biofilms. Our findings show that cbb₃ oxidases, which have been well studied as aerobic respiratory enzymes, are also involved in denitrification and influence the lifestyle of P. aeruginosa PAO1 under anoxic conditions.     

A Network Biology Approach to Denitrification in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO₂), nitric oxide (NO) and nitrous oxide (N₂O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O₂), nitrate (NO₃), and phosphate (PO₄) suggests that PO₄ concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO₄ on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N₂O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.     

The complex effects of the invasive polychaetes Marenzelleria spp. on benthic nutrient dynamics

The effects of the polychaetes Marenzelleria sp. (Polychaeta, Spionidae), nonindigenous, rapidly increasing species in the Baltic Sea, on benthic nutrient fluxes, denitrification and sediment pore water nutrient concentration were studied in laboratory experiments using a flow-through setup with muddy sediment from coastal regions of the Gulf of Finland. In addition, different forms of sediment phosphorus (P), separated by chemical fractionation, were studied in three sediment layers. At a population density corresponding to about half the highest measured in the northern Baltic Sea, Marenzelleria sp. increased the fluxes of P and ammonium to the water column. No effect could be recorded for denitrification. Since the previously dominant species of the area, Monoporeia affinis, can enhance denitrification and lower the amount of dissolved P in the pore water, the replacement of M. affinis with Marenzelleria spp. may lead to increased P flux to the water column and decreased denitrification, further increasing the ammonium flux to the water column. However, sediment reworking by Marenzelleria spp. also oxidizes the surface sediment in the long run, improving its ability to retain P and support nitrification. Therefore, the impact of Marenzelleria spp. on sediment nutrient release may not be as drastic as the initial reactions seen in our experiments suggest.     

Simulation Model of the Coupling between Nitrification and Denitrification in a Freshwater Sediment

A model was constructed to simulate the results of experiments which investigated nitrification and denitrification in the freshwater sediment of Lake Vilhelmsborg, Denmark (K. Jensen, N. P. Sloth, N. Risgaard-Petersen, S. Rysgaard, and N. P. Revsbech, Appl. Environ. Microbiol. 60:2094-2100, 1994). The model output faithfully represented the profiles of O₂ and NO₃^- and rates of nitrification, denitrification, and O₂ consumption as the O₂ concentration in the overlying water was increased from 10 to 600 μM. The model also accurately predicted the response, to increasing O₂ concentrations, of the integrated (micromoles per square meter per hour) rates of nitrification and denitrification. The simulated rates of denitrification of NO₃^- diffusing from the overlying water (D_w) and of NO₃^- generated by nitrification within the sediment (D_n) corresponded to the experimental rates as the O₂ concentration in the overlying water was altered. The predicted D_w and D_n rates, as NO₃^- concentration in the overlying water was changed, closely resembled those determined experimentally. The model was composed of 41 layers 0.1 mm thick, of which 3 represented the diffusive boundary layer in the water. Large first-order rate constants for nitrification and denitrification were required to completely oxidize all NH₄⁺ diffusing from the lower sediment layers and to remove much of the NO₃^- produced. In addition to the flux of NH₄⁺ from below, the model required a flux of an electron donor, possibly methane. Close coupling between nitrification and denitrification, achieved by allowing denitrification to tolerate some O₂ (~10 μM), was necessary to reproduce the real data. Spatial separation of the two processes (no toleration by denitrification of O₂) resulted in too high NO₃^- concentrations and too low rates of denitrification.     

Nitrous Oxide Production in Sputum from Cystic Fibrosis Patients with Chronic Pseudomonas aeruginosa Lung Infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O₂ for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N₂O), an intermediate in the denitrification pathway. We measured N₂O and O₂ with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO₃ ⁻ and NO₂ ⁻ in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N₂O (range 1.4–157.9 µM N₂O). The concentration of N₂O in the sputum was higher below the oxygenated layers. In 4 samples the N₂O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO₃ ⁻ decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N₂O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

Autotrophic denitrification using elemental sulfur

Experimental studies were conducted to determine the feasibility of autotrophic denitrification with Thiobacillus denitrificans as a nitrate removal process for wastewater. S⁰-acclimated activated sludge, which can carry out autotrophic denitrification using S⁰ as electron donors, was prepared by the fill and draw cultivation method. The kinetic constants for the S⁰-denitrification reaction using S⁰-acclimated activated sludge under NO₃-N limiting growth conditions were determined to be Y = 0.33 mg-NO₃-N and b = 0.058 d⁻¹. High percentages of denitrification (over 95%) and rates of denitrification ranging from 0.19 to 0.24 mg-NO₃-N/mg-TOC·d were obtained in the continuous denitrification experiments using S⁰-acclimated activated sludge.     

Influence of Potamogeton pectinatus and microphytobenthos on benthic metabolism,nutrient fluxes and denitrification in a freshwater littoral sediment in an agricultural landscape: N assimilation versus N removal

The influence of Potamogeton pectinatus colonisation on benthic nitrogen dynamics was studied in the littoral zone of a lowland pit lake with high nitrate concentration (~200 μM). Our hypothesis was that in aquatic environments where nitrogen availability is not limiting, colonisation by rooted macrophytes changes the dynamics of the benthic nitrogen cycle, stimulating N assimilation and denitrification and increasing the system capacity to take up external nitrogen loads. To test this hypothesis, we quantified and compared seasonal variations of light and dark benthic metabolism, dissolved inorganic nitrogen (DIN) fluxes, denitrification and N assimilation rates in an area colonised by P. pectinatus and a reference site colonised by microphytobenthos. In both areas, the benthic system was net autotrophic and a sink for DIN (2,241–2,644 mmol m^?2 y^?1). Plant colonisation increased nitrogen losses via denitrification by 30% compared to the unvegetated area. In contrast to what is generally observed in coastal marine systems, where the presence of rooted macrophytes limits denitrification rates, under the very high nitrate concentrations in the studied lake, both denitrification (1,237–1,570 mmol m^?2 y^?1) and N assimilation (1,039–1,095 mmol m^?2 y^?1) played important and comparable roles in the removal of DIN from the water column.     

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans,Hyphomicrobium denitrificans,and Hyphomicrobium zavarzinii

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.     

Effects of macrophyte-associated nitrogen cycling bacteria on denitrification in the sediments of the eutrophic Gonghu Bay, Taihu Lake

Integrated Elodea nuttallii-immobilized nitrogen cycling bacteria (INCB) technology was used for ecological restoration in the eutrophic Gonghu Bay, Taihu Lake. Sediment denitrification was investigated through microcosm incubations with four different treatments: bare sediment core as control without restoration, sediment + E. nuttallii, sediment + E. nuttallii + INCB, and sediment + INCB. The sediments with E. nuttallii-INCB assemblage (E-INCB) had the highest denitrification rates among all the treatments, and the E-INCB increased the denitrification rate by 162% in the sediments. The presence of macrophytes yielded a penetration depth of O₂ to more than 20 mm below the sediment–water interface (SWI), while the depth was only 4 mm in the sediments without macrophytes. The quantity of denitrifier in E-INCB sediments (within ~2 cm below the SWI) showed a significant increasing trend during one-month incubation, which was one order of magnitudes higher than that in the sediments without INCB. Macrophytes caused deeper O₂ penetration and increased oxic-anoxic interface, which could stimulate the coupled nitrification–denitrification. The high denitrification rate of the E-INCB treatment may result from the increased inorganic nitrogen content in the vicinity of the SWI, causing more nitrate to reach the anoxic denitrification zone. The results showed that E-INCB assemblage could increase benthic N removal by stimulating denitrification via combined O₂ penetration and enhanced microbial N cycling processes. E-INCB might be used as a potential restoration method for controlling fresh water system eutrophication.     

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.     

Role of norEF in Denitrification,Elucidated by Physiological Experiments with Rhodobacter sphaeroides

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (V_max and K_s), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in V_max but no change in apparent K_s. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.     

Effect of the Spartina alterniflora Root-Rhizome System on Salt Marsh Soil Denitrifying Bacteria

Nitrous oxide (N₂O) reductase activity was used as an index of the denitrification potential in salt marsh soils. In a short Spartina alterniflora marsh, the seasonal distribution of N₂O reductase activity indicated a causal relationship between S. alterniflora root-rhizome production and the denitrification potential of the soil system. The relationship was not discerned in samples from a tall S. alterniflora marsh. To further examine the in situ plant-denitrifier interaction in the short S. alterniflora marsh, plots with and without living S. alterniflora were established and analyzed for N₂O reductase activity 5 and 18 months later. In the plots without living Spartina there was a significant reduction in the soil denitrification potential after 18 months, indicating that in the SS marsh the denitrifiers are tightly coupled to the seasonal production of below-ground Spartina macroorganic matter. In plots with intact Spartina, the soil denitrification potential was not altered by NH₄NO₃ or glucose enrichment. However, in plots without living Spartina, there were significant changes in soil N₂O reductase activity, thus indicating that the plants can serve as a “buffer” against this form of pulse perturbation.     

Characterization and comparison of potential denitrifiers in microbial mats from King George Island,Maritime Antarctica

Cyanobacterial microbial mats are highly structured communities commonly found in Antarctic inland waters including melt streams. These benthic microbial associations comprise a large number of microorganisms with different metabolic capacities, impacting nutrient dynamics where established. The denitrification process is a feasible nitrogen loss pathway and a biological source of nitrous oxide, a potent greenhouse gas that also promotes ozone depletion. Potential denitrifiers from five microbial mats were characterized using a PCR-DGGE (denaturing gradient gel electrophoresis) approach. Molecular markers encoding for key enzymes in the denitrification process (nirK, nirS and nosZ) were used. Fingerprints were obtained for the five sampled mats and compared for two successive years. Distance analysis showed that despite the sampled year, the denitrifying genetic potential was similar between most of the sites when represented in Euclidean space. The number of dominant denitrifiers detected for each sample ranged between 6 and 18 for nirK, 4–10 for nirS and 6–17 for nosZ. The seventy-two sequenced phylotypes showed 80–98 % identity to previously reported environmental sequences from water column, sediments and soil samples. These results suggest that Antarctic microbial mats have a large denitrification potential, previously uncharacterized and composed by both site-specific and common phylotypes belonging mainly to Alpha-, Beta- and Gammaproteobacteria.     

Genome Sequence of a Highly Efficient Aerobic Denitrifying Bacterium,Pseudomonas stutzeri T13

Pseudomonas stutzeri T13 is a highly efficient aerobic denitrifying bacterium. Information about the genome of this aerobic denitrifying bacterium has been limited until now. We present the draft genome of P. stutzeri T13. The results could provide further insight into the aerobic denitrification mechanism in strain T13.     

Growth Yields in Bacterial Denitrification and Nitrate Ammonification

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H₂ plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.