A role of nitrite reductase (NirBD) for NO homeostatic regulation in Streptomyces coelicolor A3(2).

Although nitric oxide (NO) is an important signaling molecule in bacteria and higher organisms, excessive intracellular NO is highly reactive and dangerous. Therefore, living cells need strict regulation systems for cellular NO homeostasis. Recently, we discovered that Streptomyces coelicolor A3(2) retains the nitrogen oxide cycle (NO3-→NO2-→NO→NO3-) and nitrite removal system. The nitrogen oxide cycle regulates cellular NO levels, thereby controlling secondary metabolism initiation (red-pigmented antibiotic, RED production) and morphological differentiation. Nitrite induces gene expression in neighboring cells, suggesting another role for this cycle as a producer of transmittable intercellular communication molecules. Here, we demonstrated that ammonium-producing nitrite reductase (NirBD) is involved in regulating NO homeostasis in S. coelicolor A3(2). NirBD was constitutively produced in culture independently of GlnR, a known transcriptional factor. NirBD cleared the accumulated nitrite from the medium. Nir deletion mutants showed increased NO-dependent gene expression at later culture stages, whereas the wild-type M145 showed decreased expression, suggesting that high NO concentration was maintained in the mutant. Moreover, the nir deletion mutant produced more RED than that produced by the wild-type M145. These results suggest that NO2- removal by NirBD is important to regulate NO homeostasis and to complete NO signaling in S. coelicolor.

Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling.

Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule.

Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.

Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 µM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family.

Nitrite formation from organic nitrogen by Streptomyces antibioticus supporting bacterial cell growth and possible involvement of nitric oxide as an intermediate.

The actinomycete Streptomyces antibioticus was shown to produce nitrite (NO-(2)) and ammonium (NH+(4)]) when aerobically incubated in an organic nitrogen-rich medium. The production of NO-(2) was synchronized with rapid cell growth, whereas most NH+(4)] was produced after cell proliferation had ceased. Intracellular formation of nitric oxide (NO) was also observed during the incubation. The production of these inorganic nitrogen compounds along with cell growth was prevented by several enzyme inhibitors (of nitric oxide synthase or nitrate reductase) or glucose. Distinct, membrane-bound nitrate reductase was induced in the NO-(2)-producing cells. Tungstate (a potent inhibitor of this enzyme) prevented the NO-(2) production and cell growth, whereas it did not prevent the NO formation. These results revealed the occurrence of novel nitrogen metabolic pathway in S. antibioticus forming NO-(2) from organic nitrogen by which rapid cell growth is possible. NO synthase, NO dioxygenase (flavohemoglobin), and dissimilatory nitrate reductase are possible enzymes responsible for the NO-(2) formation.

New insights into the nature of observable reaction intermediates in cytochrome P450 NO reductase by using a combination of spectroscopy and quantum mechanics/molecular mechanics calculations.

Cytochrome P450 NO reductase is an unusual member of the cytochrome P450 superfamily. It catalyzes the reduction of nitric oxide to nitrous oxide. The reaction intermediates were studied in detail by a combination of experimental and computational methods. They have been characterized experimentally by UV/Vis, EPR, Mössbauer, and MCD spectroscopy. In conjunction with quantum mechanics/molecular mechanics (QM/MM) calculations, we sought to characterize the resting state and the two detectable intermediates in detail and to elucidate the nature of the key intermediate I of the reaction. Six possible candidates were taken into account for the unknown key intermediate in the computational study, differing in protonation state and electronic structure. Two out of the six candidates could be identified as putative intermediates I with the help of the spectroscopic data: singlet diradicals Fe(III)-NHO(·)(-) and Fe(III)-NHOH(.). In a companion publication (C. Riplinger, F. Neese, ChemPhysChem- 2011, 12, 3192) we have used QM/MM models based on these structures and performed a kinetic simulation. The combination of these two studies shows the nature of the key intermediate to be the singlet diradical, Fe(III)-NHOH(·).

Brevibacillus fulvus sp. nov., isolated from a compost pile.

Two strains, designated K2814(T) and K282, were isolated from a compost pile in Japan. These strains were Gram-stain-variable, aerobic, motile and endospore-forming rods. The strains produced a characteristic brown non-diffusible pigment. The 16S rRNA gene sequences of the strains were 100% identical and had high similarity to that of Brevibacillus levickii LMG 22481(T) (97.3%). Phylogenetic analyses based on 16S rRNA gene sequences revealed that these strains belong to the genus Brevibacillus. Strains K2814(T) and K282 contained meso-diaminopimelic acid in their cell walls. Strains K2814(T) and K282 contained MK-7 (96.0 and 97.2%, respectively) and MK-8 (4.0 and 2.8%, respectively) as the major and minor menaquinones, respectively. Their major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(15 : 0) and iso-C(17 : 0). The DNA G+C contents of strains K2814(T) and K282 were 48.8 and 49.8 mol%, respectively. Polar lipids of strain K2814(T) were composed of phosphatidyl-N-methylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, three unidentified polar lipids, an unidentified aminophospholipid and an unidentified aminolipid. The level of DNA-DNA relatedness between strains K2814(T) and K282 was 99 or 100%, and levels between strain K2814(T) and the type strains of seven related species of the genus Brevibacillus, including Brevibacillus levickii LMG 22481(T), were below 59%. From the chemotaxonomic and physiological data and the levels of DNA-DNA relatedness, these two strains should be classified as representing a novel species of the genus Brevibacillus, for which the name Brevibacillus fulvus sp. nov. (type strain K2814(T) = JCM 18162(T) = ATCC BAA-2417(T) = DSM 25523(T)) is proposed.

Effectiveness of heat treatment to protect introduced denitrifying bacteria from eukaryotic predatory microorganisms in a pilot-scale bioreactor.

Bioaugmentation of bioreactor systems with pre-cultured bacteria has proven difficult because inoculated bacteria are easily eliminated by predatory eukaryotic-microorganisms. Here, we demonstrated an intermediate thermal treatment was effective for protecting introduced denitrifying bacteria from eukaryotic predators and consequently allowed the inoculated bacteria to survive longer in a denitrification reactor.

Bioaugmentation of a wastewater bioreactor system with the nitrous oxide-reducing denitrifier Pseudomonas stutzeri strain TR2.

In bioaugmentation technology, survival of inoculant in the treatment system is prerequisite but remains to be a crucial hurdle. In this study, we bioaugmented the denitrification tank of a piggery wastewater treatment system with the denitrifying bacterium Pseudomonas stutzeri strain TR2 in two pilot-scale experiments, with the aim of reducing nitrous oxide (N(2)O), a gas of environmental concern. In the laboratory, strain TR2 grew well and survived with high concentrations of nitrite (5-10 mM) at a wide range of temperatures (28-40°C). In the first augmentation of the pilot-scale experiment, strain TR2 inoculated into the denitrification tank with conditions (30°C, ~0.1 mM nitrite) survived only 2-5 days. In contrast, in the second augmentation with conditions determined to be favorable for the growth of the bacterium in the laboratory (40-45°C, 2-5 mM nitrite), strain TR2 survived longer than 32 days. During the time when the presence of strain TR2 was confirmed by quantitative real-time PCR, N(2)O emission was maintained at a low level even under nitrite-accumulating conditions in the denitrification and nitrification tanks, which provided indirect evidence that strain TR2 can reduce N(2)O in the pilot-scale system. Our results documented the effective application of growth conditions favorable for strain TR2 determined in the laboratory to maintain growth and performance of this strain in the pilot-scale reactor system and the decrease of N(2)O emission as the consequence.

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.

Fungal denitrification and nitric oxide reductase cytochrome P450nor.

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.

Advenella faeciporci sp. nov., a nitrite-denitrifying bacterium isolated from nitrifying-denitrifying activated sludge collected from a laboratory-scale bioreactor treating piggery wastewater.

Strain M-07(T) was isolated from nitrifying-denitrifying activated sludge treating piggery wastewater. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain M-07(T) belonged to the genus Advenella. 16S rRNA gene sequence similarity between M-07(T) and Advenella incenata CCUG 45225(T), Advenella mimigardefordensis DPN7(T) and Advenella kashmirensis WT001(T) was 96.5, 97.3 and 96.9%, respectively. The DNA G+C content of strain M-07(T) was 49.5 mol%, which was approximately 5 mol% lower than the range for the genus Advenella (53.5-58.0 mol%). The predominant cellular fatty acids of strain M-07(T) were C(16:0), summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(17:0) cyclo and summed feature 2 (comprising one or more of C(14:0) 3-OH, iso-C(16:1) I, an unidentified fatty acid with an equivalent chain-length of 10.928 and C(12:0) alde). The isoprenoid quinone was Q-8. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness, strain M-07(T) should be classified as a novel species of the genus Advenella, for which the name Advenella faeciporci sp. nov. is proposed. The type strain is M-07(T) ( = JCM 17746(T)  = KCTC 23732(T)).

Generation of gaseous sulfur-containing compounds in tumour tissue and suppression of gas diffusion as an antitumour treatment.

BACKGROUND AND AIMS: The mechanisms of cancer cell growth and metastasis are still not entirely understood, especially from the viewpoint of chemical reactions in tumours. Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid), which can non-enzymatically react and form carcinogenic substances. There is speculation that this reaction produces gaseous sulfur-containing compounds in tumour tissue. The aims of this study were to clarify the products in tumour and to investigate their effect on tumour proliferation. METHODS: Products formed in the reaction between glucose and methionine or its metabolites were analysed in vitro using gas chromatography. Flatus samples from patients with colon cancer and exhaled air samples from patients with lung cancer were analysed using near-edge x-ray fine adsorption structure spectroscopy and compared with those from healthy individuals. The tumour proliferation rates of mice into which HT29 human colon cancer cells had been implanted were compared with those of mice in which the cancer cells were surrounded by sodium hyaluronate gel to prevent diffusion of gaseous material into the healthy cells. RESULTS: Gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide were produced when glucose was allowed to react with methionine or its metabolites homocysteine or cysteine. Near-edge x-ray fine adsorption structure spectroscopy showed that the concentrations of sulfur-containing compounds in the samples of flatus from patients with colon cancer and in the samples of exhaled air from patients with lung cancer were significantly higher than in those from healthy individuals. Animal experiments showed that preventing the diffusion of sulfur-containing compounds had a pronounced antitumour effect. CONCLUSIONS: Gaseous sulfur-containing compounds are the main products in tumours and preventing the diffusion of these compounds reduces the tumour proliferation rate, which suggests the possibility of a new approach to cancer treatment.

Conserved residues in membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon.

A membrane-intrinsic acid pyrophosphatase (ST2226) from Sulfolobus tokodaii, a thermoacidophilic archaeon, is possibly involved in glycoprotein biosynthesis and belongs to the phosphatidic acid phosphatase class 2 superfamily, including both membrane-intrinsic and soluble enzymes with divergent functions ranging from dephosphorylation of undecaprenylpyrophosphate and phospho-monoesters such as glucose-6-phosphate to vanadium-containing chloroperoxidation. ST2226 is an archaeal ortholog of these enzymes sharing a common phosphatase motif. Through site-directed mutagenesis as to each of the conserved residues, the catalytic roles of the latter were deduced, as well as the transmembrane topology with all the conserved residues in close proximity to the outside of the membrane.

Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae.

Multiple flavohemoglobin (FHb) homolog genes are found in the genomes of eukaryotic microorganisms, but their functions remain unknown. In this study, two distinct types of FHbs (predictive cytosolic FHb1 and predictive mitochondrial FHb2) from the fungus Aspergillus oryzae were investigated to elucidate the physiological roles of these FHbs. The fhb1 gene responded to external nitric oxide (NO) stress at the transcriptional level, whereas the fhb2 gene did not. Disrupting fhb1 increased cell hypersensitivity to NO stress, whereas deficiency of the fhb2 gene had no effect on phenotype compared to the wild-type strain. By fusing GFP protein to FHbs, we determined that FHb1 and FHb2 are located in the cytosol and mitochondria, respectively. In the wild-type strain, the transcriptional level of the fhb2 gene was too low to be detected, but its expression was detectable in the NirK (mitochondrial copper-containing dissimilatory nitrite reductase) overexpression strain (AoHnirK), which showed a significantly higher denitrification capability than that shown by the wild-type strain. The induction of the fhb2 gene in the AoHnirK strain may be due to the abundance of NO produced by overexpressed NirK in the mitochondria. These results suggest that FHb1 and FHb2 may play a role in protecting cells from external and internal NO stress, respectively.

Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis.

Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462∷ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462∷ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.

Multi-energy metabolic mechanisms of the fungus Fusarium oxysporum in low oxygen environments.

The fungus Fusarium oxysporum produces energy under hypoxic and anoxic conditions by denitrification (nitrate respiration) and ammonia fermentation respectively. Here we found that glucose repressed both of these metabolisms, whereas it supported another anoxic metabolism, hetero-lactic acid fermentation. Ammonia fermentation occurred only after the glucose present in the medium was metabolized to ethanol via alcohol fermentation. During this transition, clear diauxic growth was observed. Glucose regulated the activity of the enzymes involved in ammonia fermentation, hetero-lactic acid fermentation, and denitrification. Highest cell growth was supported by hetero-lactic acid fermentation, followed by denitrification and ammonia fermentation. These results indicate that the energy metabolisms of F. oxysporum are dependent not only on environmental O(2) tension but also on the carbon source, and that ammonia fermentation is an adaptative mechanism acting physiologically as a secondary fermentative mechanism replacing the primary hetero-lactic acid fermentation.

Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve.

The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a=b=174.8, c=163.8 A, and diffracted to beyond 1.7 A resolution.

Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,ß-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

The possible involvement of copper-containing nitrite reductase (NirK) and flavohemoglobin in denitrification by the fungus Cylindrocarpon tonkinense.

The occurrence of denitrification and nitrate respiration among eukaryotes has been established during the last few decades. However, denitrification-related eukaryotic genes have been isolated from only a few fungi, and eukaryotic denitrification (or nitrate respiration) is still inadequately understood. In this study, we identified genes that were up-regulated under denitrifying conditions in the fungus Cylindrocarpon tonkinense using the suppression subtraction hybridization technique, and the expression patterns of these genes were characterized by Northern analysis. We identified copper-containing nitrite reductase, cytochrome P450 nitric oxide reductase, flavohemoglobin (Fhb), and formate/nitrite transporter homolog genes as possibly involved in fungal denitrification. Our results concerning the involvement of Fhb and formate/nitrite transporter perhaps provide new insight into the fungal denitrification system.

A eukaryotic copper-containing nitrite reductase derived from a NirK homolog gene of Aspergillus oryzae.

We cloned a bacterial copper-containing nitrite reductase (NirK) homolog gene of Aspergillus oryzae (AonirK). Alignment showed that amino acid residues crucial for copper binding are conserved in the deduced sequence of the fungal protein. The recombinant protein exhibited distinct nitrite reductase activity, and its absorption and EPR spectra showed the presence of type 1 and type 2 copper atoms in the molecule. AonirK transcriptionally responded to denitrification conditions. Although the denitrifying activity of A. oryzae was weak under the conditions employed, high expression of the gene in the fungal cells enhanced the denitrifying activity 6-fold, accompanied by distinct cell growth. Furthermore, the highly expressed AoNirK was subcellularly localized to the mitochondria. The results demonstrated that AoNirK is responsible for fungal denitrification. Discussion is added on the novel insight concerning the origin and evolution of the mitochondrion provided by the findings for eukaryotic NirKs.