Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia haasae.

In the present study, a comparative genome analysis of the novel alkaliphilic actinobacterial Nesterenkonia haasae with other members of the genus Nesterenkonia was performed. The genome size of Nesterenkonia members ranged from 2,188,008 to 3,676,111 bp. N. haasae and Nesterenkonia members of the present study encode the essential glycolysis and pentose phosphate pathway genes. In addition, some Nesterenkonia members encode the crucial genes for Entner-Doudoroff pathways. Some Nesterenkonia members possess the genes responsible for sulfate/thiosulfate transport system permease protein/ ATP-binding protein and conversion of sulfate to sulfite. Nesterenkonia members also encode the genes for assimilatory nitrate reduction, nitrite reductase, and the urea cycle. All Nesterenkonia members have the genes to overcome environmental stress and produce secondary metabolites. The present study helps to understand N. haasae and Nesterenkonia members' environmental adaptation and niches specificity based on their specific metabolic properties. Further, based on genome analysis, we propose reclassifying Nesterenkonia jeotgali as a later heterotypic synonym of Nesterenkonia sandarakina.

Myoglobin mutant with enhanced nitrite reductase activity regulates intracellular oxidative stress in human breast cancer cells.

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.

A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis.

Plant growth is the result of the coordinated photosynthesis-mediated assimilation of oxidized forms of C, N and S. Nitrate is the predominant N source in soils and its reductive assimilation requires the successive activities of soluble cytosolic NADH-nitrate reductases (NR) and plastid stroma ferredoxin-nitrite reductases (NiR) allowing the conversion of nitrate to nitrite and then to ammonium. However, nitrite, instead of being reduced to ammonium in plastids, can be reduced to nitric oxide (NO) in mitochondria, through a process that is relevant under hypoxic conditions, or in the cytoplasm, through a side-reaction catalyzed by NRs. We use a loss-of-function approach, based on CRISPR/Cas9-mediated genetic edition, and gain-of-function, using transgenic overexpressing HA-tagged Arabidopsis NiR1 to characterize the role of this enzyme in controlling plant growth, and to propose that the NO-related post-translational modifications, by S-nitrosylation of key C residues, might inactivate NiR1 under stress conditions. NiR1 seems to be a key target in regulating nitrogen assimilation and NO homeostasis, being relevant to the control of both plant growth and performance under stress conditions. Because most higher plants including crops have a single NiR, the modulation of its function might represent a relevant target for agrobiotechnological purposes.

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity.

Protein design is a useful strategy to interrogate the protein structure-function relationship. We demonstrate using a highly modular 3-stranded coiled coil (TRI-peptide system) that a functional type 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2 O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75-fold compared to previously reported systems. We find also that steric bulk can be used to enforce a three-coordinate CuI in a site, which tends toward two-coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metal-center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods.

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s-1) lower than that of wt SmNir (kcat=240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.

Molecular Mechanisms of Action of Gas Transmitters NO, CO and H2S in Smooth Muscle Cells and Effect of NO-generating Compounds (Nitrates and Nitrites) on Average Life Expectancy.

Gaseous signaling molecules (gas transmitters) take an especial position among the numerous signaling molecules involved in the regulation of both intracellular processes that occur in different types of cells and cell-cell interactions. At present time, gas transmitters include three molecules whose enzymatic systems of synthesis and degradation, physiological action and intracellular effectors, the change of which under the action of gas transmitters may result in physiological and/or pathophysiological effects are well- determined. These molecules include nitrogen oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S). They are involved in the regulation of functions of various organs and systems of the human body, including the circulatory system. Interaction of NO, CO and H2S with various enzymatic and structural components of endothelial and, especially, smooth muscle cells has a significant impact on vascular tone and blood pressure. Furthermore, the crossing of NO-, CO- and H2S-mediated signaling pathways at common effectors and interaction with each other can determine the end, resulting functional response of the cell. The knowledge of the molecular targets of gas transmitters' action, the structure of the binding centers for gas transmitters and their interaction with each other may be essential in the development of methods of regulation of these signaling systems by targeted, directed action. This review summarizes the molecular mechanisms of the NO, CO and H2S interaction with the main targets, which carry out their regulatory effect on vascular smooth muscle cells. Also we describe here different ways of cross-regulation of NO-, CO- and H2S-dependent signaling pathways. We analyzed NO-synthase and nitrite reductase systems of nitric oxide cycle and discuss the nitrate-nitrite background of the existence of modern man, which can substantially modify the signaling system, the metabolism of virtually all cell ultrastructure of neurons, neuron-neuron and neuron-glial interactions and exerts its influence on socially significant diseases that can affect the quality and the average life expectancy.

Characterization of the denitrifying bacterial community in a full-scale rockwool biofilter for compost waste-gas treatment.

The potential denitrification activity and the composition of the denitrifying bacterial community in a full-scale rockwool biofilter used for treating livestock manure composting emissions were analyzed. Packing material sampled from the rockwool biofilter was anoxically batch-incubated with 15N-labeled nitrate in the presence of different electron donors (compost extract, ammonium, hydrogen sulfide, propionate, and acetate), and responses were compared with those of activated sludge from a livestock wastewater treatment facility. Overnight batch-incubation showed that potential denitrification activity for the rockwool samples was higher with added compost extract than with other potential electron donors. The number of 16S rRNA and nosZ genes in the rockwool samples were in the range of 1.64-3.27 × 109 and 0.28-2.27 × 108 copies/g dry, respectively. Denaturing gradient gel electrophoresis analysis targeting nirK, nirS, and nosZ genes indicated that the distribution of nir genes was spread in a vertical direction and the distribution of nosZ genes was spread horizontally within the biofilter. The corresponding denitrifying enzymes were mainly related to those from Phyllobacteriaceae, Bradyrhizobiaceae, and Alcaligenaceae bacteria and to environmental clones retrieved from agricultural soil, activated sludge, freshwater environments, and guts of earthworms or other invertebrates. A nosZ gene fragment having 99% nucleotide sequence identity with that of Oligotropha carboxidovorans was also detected. Some nirK fragments were related to NirK from micro-aerobic environments. Thus, denitrification in this full-scale rockwool biofilter might be achieved by a consortium of denitrifying bacteria adapted to the intensely aerated ecosystem and utilizing mainly organic matter supplied by the livestock manure composting waste-gas stream.

PGPR-mediated expression of salt tolerance gene in soybean through volatiles under sodium nitroprusside.

Increasing evidence shows that nitric oxide (NO), a typical signaling molecule plays important role in development of plant and in bacteria-plant interaction. In the present study, we tested the effect of sodium nitroprusside (SNP)-a nitric oxide donor, on bacterial metabolism and its role in establishment of PGPR-plant interaction under salinity condition. In the present study, we adopted methods namely, biofilm formation assay, GC-MS analysis of bacterial volatiles, chemotaxis assay of root exudates (REs), measurement of electrolyte leakage and lipid peroxidation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for gene expression. GC-MS analysis revealed that three new volatile organic compounds (VOCs) were expressed after treatment with SNP. Two VOCs namely, 4-nitroguaiacol and quinoline were found to promote soybean seed germination under 100 mM NaCl stress. Chemotaxis assay revealed that SNP treatment, altered root exudates profiling (SS-RE), found more attracted to Pseudomonas simiae bacterial cells as compared to non-treated root exudates (S-RE) under salt stress. Expression of Peroxidase (POX), catalase (CAT), vegetative storage protein (VSP), and nitrite reductase (NR) genes were up-regulated in T6 treatment seedlings, whereas, high affinity K+ transporter (HKT1), lipoxygenase (LOX), polyphenol oxidase (PPO), and pyrroline-5-carboxylate synthase (P5CS) genes were down-regulated under salt stress. The findings suggest that NO improves the efficiency and establishment of PGPR strain in the plant environment during salt condition. This strategy may be applied on soybean plants to increase their growth during salinity stress.

Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.

Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

Soybean Fe-S cluster biosynthesis regulated by external iron or phosphate fluctuation.

KEY MESSAGE: Iron and phosphorus are essential for soybean nodulation. Our results suggested that the deficiency of Fe or P impairs nodulation by affecting the assembly of functional iron-sulfur cluster via different mechanisms. Iron (Fe) and phosphorus (P) are important mineral nutrients for soybean and are indispensable for nodulation. However, it remains elusive how the pathways of Fe metabolism respond to the fluctuation of external Fe or P. Iron is required for the iron-sulfur (Fe-S) cluster assembly in higher plant. Here, we investigated the expression pattern of Fe-S cluster biosynthesis genes in the nodulated soybean. Soybean genome encodes 42 putative Fe-S cluster biosynthesis genes, which were expressed differently in shoots and roots, suggesting of physiological relevance. Nodules initiated from roots of soybean after rhizobia inoculation. In comparison with that in shoots, iron concentration was three times higher in nodules. The Fe-S cluster biosynthesis genes were activated and several Fe-S protein activities were increased in nodules, indicating that a more effective Fe-S cluster biosynthesis is accompanied by nodulation. Fe-S cluster biosynthesis genes were massively repressed and some Fe-S protein activities were decreased in nodules by Fe deficiency, leading to tiny nodules. Notably, P deficiency induced a similar Fe-deficiency response in nodules, i.e, certain Fe-S enzyme activity loss and tiny nodules. However, distinct from Fe-deficient nodules, higher iron concentration was accumulated and the Fe-S cluster biosynthesis genes were not suppressed in the P-deficiency-treated nodules. Taken together, our results showed that both Fe deficiency and P deficiency impair nodulation, but they affect the assembly of Fe-S cluster maybe via different mechanisms. The data also suggested that Fe-S cluster biosynthesis likely links Fe metabolism and P metabolism in root and nodule cells of soybean.

Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

Crystal structure of NirD, the small subunit of the nitrite reductase NirbD from Mycobacterium tuberculosis at 2.0 Å resolution.

NirD is part of the nitrite reductase complex NirBD that catalyses the reduction of nitrite to NH(3) in nitrate assimilation and anaerobic respiration. The crystal structure analysis of NirD from Mycobacterium tuberculosis shows a double ß-sandwich fold. NirD is related in three-dimensional structure and sequence to the Rieske proteins; however, it does not contain any Fe-S cluster or other cofactors that might be involved in electron transfer. A cysteine residue at the protein surface, conserved in NirD homologues lacking the iron-sulfur cluster might be important for the interaction with NirB and possibly stabilize one of the Fe-S centers in this subunit.

High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

Orientational control over nitrite reductase on modified gold electrode and its effects on the interfacial electron transfer.

Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.

Human neuroglobin functions as a redox-regulated nitrite reductase.

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Contribution of cell elongation to the biofilm formation of Pseudomonas aeruginosa during anaerobic respiration.

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO(2) (-)) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process.

Denitrifying polyphosphate accumulating organisms population and nitrite reductase gene diversity shift in a DEPHANOX-type activated sludge system fed with municipal wastewater.

Enhanced biological phosphorus removal (EBPR) is a widely applied method for nutrients removal, although little is known about the key genes regulating the complex biochemical transformations occurring in activated sludge during phosphorus removal. In the present study, the nitrite reductase gene (nirS) diversity and the denitrifying polyphosphate accumulating organisms (DPAOs) population, grown in a bench scale, two-sludge, continuous flow plant, operating for biological anoxic phosphorus removal (DEPHANOX-type), fed with municipal wastewater, were examined by means of physicochemical analyses and the application of molecular techniques. The DEPHANOX configuration highly influenced biomass phosphorus as well as polyhydroxyalkanoates content and facilitated the enrichment of the DPAOs population. The application of double probe fluorescent in situ hybridization (double probe FISH) technique revealed that DPAOs comprised 20% of the total bacterial population. Based on clone libraries construction and nirS gene sequencing analysis, a pronounced shift in denitrifying bacteria diversity was identified during activated sludge acclimatization. Moreover, nirS gene sequences distinct from those detected in any known bacterial strain or environmental clone were identified. This is the first report studying the microbial properties of activated sludge in a DEPHANOX-type system using molecular techniques.

Thioredoxin reductase is essential for protection of Neisseria gonorrhoeae against killing by nitric oxide and for bacterial growth during interaction with cervical epithelial cells.

In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.