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Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain, nnrV, under control of a previously identified NnrR site, was also identified, paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase, paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome [c.sub.2] together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions. doi: 10.1128/JB.01026-09

Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure. Pseudomonas aeruginosa is a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least two N-acyl-L-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification in P. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification in P. aeruginosa was repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitrate-respiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific to P. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.

Growth of Moraxella catarrhalis in a biofilm resulted in marked upregulation of two open reading frames (ORFs), aniA and norB, predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated nsrR, which was located between aniA and norB, was shown to encode a predicted transcriptional regulator. Inactivation of nsrR resulted in increased expression of aniA and norB in three different M. catarrhalis strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type nsrR gene in trans in an nsrR mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an nsrR mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an nsrR mutant than with wild-type cells. However, growth of nsrR mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an aniA mutation into the nsrR mutant and was completely reversed by the presence of a wild-type nsrR gene in trans. NsrR regulation of the expression of aniA was sensitive to nitrite, whereas NsrR regulation of norB was sensitive to nitric oxide.

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-L-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the/as quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious IdhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

Researchers have discovered that a strain of Rhodococcus can completely degrade nitroglycerin, removing all three nitrogen atoms. Pseudomonas putida, Arthrobacter sp., and Klebsiella sp. were also able to use nitroglycerin as their sole nitrogen source, but only Rhodococcus could remove all three nitrogen atoms.

Researchers estimated denitrification and nitrogen fixation rates in sediments from a Texas sea grass bed using an isotope-pairing technique and a membrane inlet mass spectrometer. Denitrification rates were an average of 20 microgram-atoms of N per square meter per hour, and nitrogen fixation rates were an average of 60 microgram-atoms of N per square meter per hour.

It may not be necessary to recycle sludge between anaerobic and aerobic zones when using Paracoccus denitrificans as the polyphosphate-accumulating organism. This bacterium is capable of removing phosphate and nitrate without the need for alternating anaerobic/aerobic switches.`

Results points out to the involvement of cobK and cobJ genes in the anaerobic synthesis of cobalamin in Paracoccus denitrificans. This unusual anerobic behavior is mediated by the cobalamin-dependent ribonucleotide reductase.

Research was conducted to examine the occurrence of anaerobic processes in aerated activated sludge using a combination of various techniques. Microsensor calculations were taken for the different parameters in 250 individual activated sludge flocs. Results indicate that the respiration capacity of activated sludge is simply not sufficient to create anoxia under air saturation. A likely explanation for the absence of anoxia may be that oxygen transport is not only diffusional but enhanced by advection.

Denitrifying bacteria in the gut of earthworms are responsible for the emission of nitrous oxide by the earthworm. This emission accounts for about one-third of the nitrous oxide emitted by garden soil.

Chloramphenicol should not be used in denitrifying enzyme activity (DEA) assays in concentrations greater than 0.1 g/liter. The antibiotic is used in these assays to inhibit the synthesis of new denitrifying enzymes, but it inhibits existing enzymes as well.

Researchers report five denitrifying strains of bacteria that can degrade compounds with a quaternary carbon atom in the absence of oxygen. Two strains belong to the alpha-Proteobacteria and three belong to the beta-Proteobacteria.

Research was conducted to show the requirement of periplasmic nitrate reductase in the first step of denitrification in Pseudomonas sp. strain G-179 using both biochemical and genetic proof. It was proposed that two systems are responsible for dissimilatory nitrate reduction in denitrifiers. Results demonstrate that the periplasmic nitrate reductase provides one of the significant mechanisms for aerobic nitrate reduction. Also, aerobic nitrate reduction and aerobic denitrification may have potential applications in water and industrial water treatment.

The diversity of nitrous oxide reductase (nosZ) genes in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves has been investigated using the polymerase chain reaction assaying technique. An estimated 1,100 bp of the nosZ gene were amplified using nosZ gene-specific primers, with 37 copies characterized. The conservation of important structural motifs was confirmed by alignment of the derived amino acid sequences. In addition, three major clusters of nosZ genes have been identified by phylogenetic analysis, with little overlap between culture-based and environmental groups.

Two sets of PCR primers have been designed to amplify fragments of the gene coding for nitric reductase, a key enzyme involved in bacterial denitrification. Consensus regions in gene sequences encoding the two forms of nitrite reductase, cd(sub 1)-nir and Cu-nir were used in the preparation of the primers. Results show that the primers were able to amplify the correct gene, although they failed to amplify genes of nondenitrifying strains.

Research was conducted to examine the kinetics of nirS expression (cytochrome cd1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification. The signal and regulator requirements for the transcriptional activation of nirS, norCB and nosZ were examined after a shifting of the respiratory metabolism from oxygen to nitrate or nitrite. Results provide evidence that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is required for the activation of denitrification genes.

Research was conducted to examine the potential of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with a nitrifying sludge as the biocatalyst. Results demonstrated that with low oxygen concentrations, nitrite oxidizers were strongly inhibited. Findings indicated that autotrophic conversion of NH4+-N to N2 in the OLAND system may be influenced largely by ammonium oxidizers and not by nitrite oxidizers.

Research was conducted to examine the vertical and temporal distribution of NO3- in diatom-covered sediment during light-dark cycles using a novel microscale NO3- biosensor characterized by absence of interference from chemical species other than NO2- and N2O. Vertical distribution of NO3- assimilation, nitrification and denitrification was measured from the calculated steady-state NO3- profiles by diffusion reaction models. Results show that the stimulatory effect on nitrification by illumination was caused by algal production of O2.NH4+.

Acetylene blockage method was used to examine impact of nitrate concentration on the denitrification rate and N2O production in intact sediment cores from a river continuum. An increase in denitrification rate was observed after the addition of nitrate in the overlying water. A Michaelis-Menten-type curve was shown and a strong response to nitrate addition was observed in the denitrification rate of intact sediment.

The isolation and identification of three new phenol-degrading denitrifying microorganisms of the genera Azoarcus from sediments of various geographical locations are described. The strains have the ability to use phenol as a source of carbon and energy under denitrifying conditions. The strains are short gram-negative rods and, although a polar flagellum is present in the exponential phase, motility is lost in older cultures. Moreover, the isolates are only able to degrade phenol under denitrifying states and not in aerobic conditions.

Photoinhibition of nitrite reduction caused light-mediated nitrite accumulation of Pseudomonas sp. strain JR12. This was gleaned in an examination of the effect of light on the denitrifying capacity of this nonphotosynthetic denitrifier. Inhibition of nitrite reduction in cells illuminated with green light was the highest, as against inhibition upon exposure to blue and red light. Exposure of the cells to light caused a photooxidation of cytochrome c, affirming that light exposure prevents electron flow from cytochrome c to nitrite reductase.

The effect of several monoterpenes on methane oxidation by pure cultures of the methanotrophs Methylosinus trichosporium OB3b and Methylobacter luteus, denitrification by six environmental isolates and aerobic metabolism by heterotrophic laboratory cultures were investigated. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 nM (-)-alpha-pinene lasted for more than two days. Inhibition was greater in conditions under which particulate methane monooxygenase was expressed.

A study was conducted on the isolation of two species of Pseudomonas from nitroglycerin (NG)-contaminated soils using enrichment culture. One species uses NG as a sole source of nitrogen while the other tolerates high concentrations of NG in the growth medium. The results indicate that the two flavoenzymes exhibit different selectivities for removal of the first nitro group from NG. Moreover, the selectivity of each enzyme is partially maintained during removal of the second nitro group from the dinitroglycerol isomers.

A study was conducted on the structural genes of nitric oxide reductases in Alcaligenes eutrophus H16. The results indicate that inactivation of both norB and norZ was lethal to the cells under anaerobic living conditions and that anaerobic growth was restored through the introduction of either norB or norZ on a broad-host-range plasmid. Thus, the norB and norZ gene products are instrumental and isofunctional in denitrification.

A study was conducted on the variation of nitrifying and denitrifying bacteria culture aeration in relation to NO and N2O emissions. NO and N2O production were examined in the denitrifiers Pseudomonas stutzeri and Alcaligenes eutrophus and the nitrifier Nitrosomonas europaea cultured at different aeration levels. Results reveal that nitrifiers can only dominate soil NO and N2O emissions when nitrite is present.

The physiology and enzymology involved in denitrification by Shewanella putrefaciens was tested for coupling of the reduction of nitrate and/or nitrite. The study also examined the ability of S. putrefaciens to respire nitrate under aerobic growth conditions. Results reveal that anaerobic growth with nitrate led to rapid and complete reduction of nitrate to nitrite in all of the S. putrefaciens strains tested.

The anaerobic and aerobic degradation of a pyridine by a newly isolated denitrifying bacterium were studied. The research also described the isolation of a new bacteria that grow on pyridine as the sole carbon source under denitrifying conditions. Results suggest that strain pF6, a newly-isolated denitrifying bacterium, had considerable advantages over strictly aerobic bacteria in field applications.

Cultures enriched from contaminated aquifer material degraded pristane under nitrate-reducing conditions. Analysis of slow pristane degradation in enrichment cultures containing denitrifying microcosms indicated the complete mineralization of the compound within 102 days of incubation in nitrate-reducing conditions. Furthermore, low-molecular-weight polar compounds such as acetate and propionate were not detectable at the end of the incubation period.

A biosensor for nitrate can be constructed by inserting a N2O microsensor into a 30- to 70-micrometer wide capillary with immobilized denitrifying bacteria. The bacteria reduces O2 so that anaerobic denitrification is possible at lower levels of the capillary while the bacteria at the tip of the capillary are exposed to O2. Acetylene is added to block nitrous oxide reductase. The reduction of NO3- or NO2- by the bacteria to N2O is measured by the sensor. The N2O evolved is proportional to NO3- in the culture.

The formation of acetate in forest soils in the absence of oxygen may be part of a trophic connection between aerobic and anaerobic processes in the soil. Under anaerobic conditions acetate was produced and remained stable, though was later used in the formation of methane and reduce sulfate. However, when oxygen and nitrate were present, acetate that was produced was quickly consumed, probably in aerobic respiration and denitrification.

Analysis of the denitrification system of the fungus Cylindrocarpon tonkinense reveals the occurrence of distinct cell growth along with nitrite-dependent N2O formation. Respiration inhibitors suppress the N2O formation and/or cell growth, while ammonium ions exert no influence on these processes. Examination of purified Nor activity indicates that the activity depends on two isozymes of cytochrome P-405nor. Characteristics of these enzymes are compared.

The denitrification process, a primary source of NO and N20 gases in the atmosphere converts combined nitrogen utilized by eukaryotic organisms to dinitrogen gas. The production of more NO and N20 gases destroy the atmospheric ozone and increase global warming. The biogeochemical nitrogen cycle includes the process of denitrification.

Cell suspensions of Paracoccus denitrificans reveal that nitrate directly converts to N2 at pH 8.5, while intermediates accumulate at low steady-state concentrations. Quadrupole mass spectrometer is used to study the effect of pH on denitrification intermediate production from nitrate and nitrite, when exposed to anoxic conditions. Denitrification is due to the bacterial conversion of nitrite and nitrate to N2O and N2 resepectively.

The effect on the outcome of competition trials between two isolates of denitrifying bacteria with the difference in their ability to carryout denitrification is discussed. This involves an evaluation of how the presence of indigenous microflora affects the ability of introduced denitrifierto colonize and grow in soil. Both isolates were found to persist in soil underboth conditions, even though their inability were disparate.

The principle of the acetylene block technique, which is used to assess denitrifying activity, involves the prevention of nitrous oxide oxidation, thereby allowing the accumulation of the intermediate to facilitate the quantitation of rates and activity. An investigation was conducted to verify whether the inclusion of acetylene can prevent the biodegradation of certain alkylbenzenes under denitrifying conditions. The results confirmed the inhibitory effect of acetylene, even in acclimated samples of subsurface aquifer material. Therefore, the results demonstrate a potential for underestimation of biodegradation rates using the acetylene block technique.

A sensitive bioassay to determine nitrite and nitrate levels in rhizosphere soil samples was devised using denitrifying Pseudomonas aeruginosa. The ON12 strain was grown in specific media, producing cells which had specific capabilities for converting nitrates and nitrites to nitrous oxide. Nitrous oxide was the parameter measured. Nitrites were more abundant in the rhizosphere than in bulk soil for both wet and dry soil. Nitrates were more abundant in the rhizosphere under dry conditions, but not in the wet.

Anaerobic enrichment cultures yielded a bacterium tentatively identified as Thiobacillus denitrificans subsp ACN20. Batch cultures under denitrifying conditions showed a stepwise oxidation of hydrogen sulfide, with the formation of elemental sulfur, which was further oxidized to sulfate. Gradient cultures oxidized hydrogen sulfide directly to sulfate. The presence of ethylene in both batch and gradient cultures inhibited the oxidation of elemental sulfur, which was explained by inhibition of sulfate reductase. Reduction of dinitrogen oxide was detected both in the presence and absence of ethylene.

Tn5 was used to generate five nitrite reduction- deficient (Nir-) Pseudomonas fluorescens mutants. Three Nir- mutants with Tn5 inserted into the nirS genesresulted in absence of nitrite reductase on Westernimmunoblot. At least three genes are involved in nitrite reductase assembly since insertion of Tn5 in the nirC genes and two other segments also produceddefective nitrite reductase. The five Nir- mutants also failed to reduce nitrous oxide (NO) with a reduced extent of labelled oxygen exchange with tagged water during reduction of NO to nitric oxide. These suggest that dissimilatory nitrite and NO reduction are related in P. fluorescens.

The mechanisms of nitrogen and oxygen transformations in a bioturbated estuarine sediment were lucidated. The method involved using 15N isotopes, specific inhibitor and microsensor techniques in a continuous-flow core system. Results showed that NO3- reduction to NH4+ was insignificant and denitrification was the only NO3- reducing pathway. The study also indicated that most denitrification process occured in the faunal-rich sediments.

The denitrification mechanism in actinomycetes was determined through the purification of nitrite reductase and azurin from the Streptomyces thioluteus JCM 4844. Denitrification is a biological mechanism important in the nitrogen global cycle since it is involved in the nitrogen cycle that counteracts nitrogen fixation. It was revealed that actinomycetes transformed nitrite or nitrate to nitric oxide as a denitrification product. The greenhouse effect of nitric oxide was believed to be 200 times greater than that of carbon dioxide on a per-molecule basis.

The rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA) and anammox (AN) are determined at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom. The log-log relationships have shown an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are controlled at cellular and protein levels.

The activity and diversity of denitryfying bacterial populations in developing Hole-in-the-Donut (HID) soils are characterized in order to understand changes in nitrogen (N) cycling during short-term primary succession. The results have shown that nitrile reductase gene nirK communities are more sensitive than nitrile reductase nirS communities to environmental gradients in these soils.

A study was conducted to determine the physiological and proteomic responses of the denitrifying bacterium Aromatoleum aromaticum strain EbN1 to semi-inhibitory concentrations of its aromatic growth substrates. It is found that under all applied growth conditions, the potential electron supply provided by the organic substrate exceeded the electron-accepting capacity of nitrate.

The influence of eight nonleguminous grassland plant species belonging to two functional groups on the composition of soil denitrifier communities is analyzed. The results have shown that individual species of nonleguminous plants have directly affected the composition of nitrifier communities in soil, but environmental conditions are shown to have additional significant effects.

A study was conducted to examine the community dynamics of two dispersed-growth denitrifying reactors during period of functional stability and instability. The results reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was employed to study denitrifiers across a glacier foreland. Statistical analyses showed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important triggers changing the denitrifying community along the chronosequence.

A study was conducted on river biofilms that were composed of polycarbonate strips complemented by combined nutrients with or without hexadecane. It was observed that there was cyclic response of the anaerobic heterotrophic biofilm community and the effective interaction between nutrients and the contaminant hexadecane.

Genetic signatures from denitrifying bacteria capable of benzoate utilization in coastal sediments were discerned using DNA-stable isotope probing (SIP) and (super 13)C-carrier DNA for the physical separation of (super 13)C-DNA. Results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader in a bacterial population and to document the cross-feeding microbial community.