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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.

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 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.

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.

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.

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 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.