Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

Growth of Pseudomonas aeruginosa on nitrous oxide

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of Pseudomonas aeruginosa on nitrous oxide.

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

土壤中反硝化酶活性变化与N2O排放的关系

研究施肥条件下,土壤反硝化酶活性硝酸还原酶(NR)活性、亚硝酸还原酶(NiR)活性及羟胺还原酶(HyR)活性在玉米生长季节中的变化及其与土壤含水量、硝态氮含量、N₂O排放之间的关系。结果表明,3种还原酶都有明显的季节变化规律并受土壤水分含量及施肥的影响。通过研究3种反硝化酶活性与土壤含水量及N₂O排放量之间的关系后指出,反硝化酶活性变化可作为一个区分旱田N₂O产生途径的指标.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.     

硫化物抑制潮土反硝化过程中氧化亚氮还原的菌群机制

【背景】土壤中的反硝化作用形成气态产物N_2O和N_2,会导致氮素的气态损失,并造成温室效应。硫化物对土壤的N_2O还原具有抑制作用,但其对菌群和功能基因的影响机制还不清楚。【目的】研究有无外加碳源情况下,硫化物对反硝化作用中间产物(NO、N_2O)的积累、反硝化功能基因(narG、nirS、nirK和nosZ)表达量以及菌群结构的影响。【方法】分别设置不同量葡萄糖(0和1000mg-C/kg干重土壤)和硫化钠(0和150mg-S/kg干重土壤)添加的交叉处理,进行室内微宇宙培养实验,利用自动化培养与实时气体检测系统检测培养过程中NO、N_2O和N_2的积累量,通过反转录定量PCR测定反硝化功能基因表达量,利用MiSeq技术平台基于16S rRNA基因序列的高通量测序分析样品的菌群结构。【结果】硫化钠的添加显著抑制N_2O还原,但是其对于N_2O积累量没有显著影响,却显著降低了NO的积累量。硫化钠的添加短时间内在转录水平上显著抑制N_2O还原酶的活性,并且抑制固氮弧菌属(Azoarcus)、微枝形杆菌属(Microvirga)、剑菌属(Ensifer)、氮氢单胞菌属(Azohydromonas)、芽孢杆菌属(Bacillus)、斯科曼氏球菌属(Skermanella)、申氏杆菌属(Shinella)和西索恩氏菌属(Chthoniobacter)的基因转录,降低它们的转录本丰度,结合Kyoto Encyclopedia of Genes andGenomes(KEGG)数据库的查询结果,发现硫化钠的添加抑制了不产生N_2O的N_2O还原反硝化细菌的生长。【结论】堆肥或其他原因引起的土壤硫化物增加,导致反硝化过程N_2O还原被抑制的原因是由于其对氧化亚氮基因转录的抑制和对不同反硝化菌的选择作用,研究结果有助于认识硫化物对氮代谢影响的微生物机制。     

Loss of nitrous oxide reductase in Pseudomonas aeruginosa cultured under N2O as determined by rocket immunoelectrophoresis

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N(2)O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N(2)O.     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.     

Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite

The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa.     

NosX function connects to nitrous oxide (N2O) reduction by affecting the Cu(Z) center of NosZ and its activity in vivo

The effect of loss of the 34-kDa periplasmic NosX protein on the properties of N2O reductase was investigated with an N2O-respiration negative, double mutant of the paralogous genes nosX and nirX of Paracoccus denitrificans. In spite of absence of whole-cell N2O-reducing activity, the purified reductase was catalytically active, which attributes NosX a physiological role in sustaining the reaction cycle. N2O reductase exhibited the spectroscopic features of Cu(A) and the redox-inert, paramagnetic state, Cu(Z)*, of the catalytic center. Cu(Z)*, hitherto considered the result of spontaneous reaction of the reductase with dioxygen, attains cellular significance.     

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.     

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2(-) and O2 concentrations.

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at 相似文献   

Detergent inhibition of nitric-oxide reductase activity

Gas chromatography revealed that exposure of extracts of the denitrifiers 'Achromobacter cycloclastes', Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N2O, and thus concomitantly inhibited reduction of NO2- to N2O. After exposure of extracts to Triton X-100, the ratio of H+ consumed to NO2- added decreased from approx. 2.0 (for untreated extracts) to approx. 1.5, which indicated that NO2- was reduced to NO by the treated extracts. Addition of a CHAPS-soluble extract (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P. denitrificans restored capacity for reduction of NO2- on to N2O. Exposure to either the NO that accumulated from reduction of NO2- or to enthetic NO transiently inhibited rates of NO2- reduction in Triton X-100-treated extracts. Use of an Oxides of Nitrogen analyzer indicated that only 5-33% of NO2- reduced by untreated extracts appeared in the stripping gas as NO, whereas 80-95% of NO2- reduced by Triton X-100-treated extracts was recovered as NO.     

15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. Evidence for direct production of N2O without free NO as an intermediate

Nitrite reductase (cytochrome c,d1) was purified from Pseudomonas aeruginosa. In the presence of the reducing system, ascorbate-N,N,N',N'-tetramethylphenyl-enediamine, which alone had no ability to reduce nitrite or NO at pH 7.5, the enzyme catalyzed the reduction of nitrite to NO and N2O as major and minor products, respectively, as determined by gas chromatography-mass spectrometry. The rate of reduction of NO to N2O was considerably lower than the rate of reduction of nitrite to N2O and might be zero. The N2O produced in a system containing [15N]nitrite and natural NO was more highly enriched in 15N than was the NO pool and, in this regard, closely resembled the enrichment of the nitrite pool. The amount of 14N in the NO pool changed little, if any, as the result of enzymatic processes. For the enzyme, free NO seems not to be an intermediate between nitrite and N2O, just as was found by this laboratory for certain intact denitrifying bacteria. The results are consistent with reduction of nitrite to enzyme-bound NO, which can partition between release and further reduction.     

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.     

Heterotrophic nitrification among denitrifiers.

Twelve denitrifying bacteria representing six genera were tested for an ability to nitrify pyruvic oxime heterotrophically. Six of these bacteria exhibited appreciable nitrification activity, yielding as much as 5.8 mM nitrite and little or no nitrate when grown in a mineral salts medium containing 7 mM pyruvic oxime and 0.05% yeast extract. Of the six active bacteria, four (Pseudomonas denitrificans, Pseudomonas aeruginosa, and two strains of Pseudomonas fluorescens) could grow on yeast extract but not pyruvic oxime, one (Pseudomonas aureofaciens) could grow slowly on pyruvic oxime, and one (Alcaligenes faecalis) could apparently grow on pyruvic oxime in the presence of yeast extract but not in its absence. Eight of the twelve bacteria in the resting state could oxidize hydroxylamine to nitrite, and P. aureofaciens was remarkably active in this regard. In general, those denitrifiers active in the nitrification of pyruvic oxime or hydroxylamine or both are abundant in soils. A possible advantage of having nitrification and denitrification capabilities in the same organism is discussed.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.