Nitrite reduction to nitrous oxide by propionibacteria: Detoxication mechanism

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N₂O. Recovery of added nitrite-N as N₂O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N₂O production were 3 to 6 orders of magnitude lower than specific rates of N₂ production by common denitrifiers. Oxygen but not acetylene inhibited N₂O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N₂O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Inhibition of nitrous oxide reduction by a component of Hamilton Harbour sediment

Abstract: A component of Hamilton Harbour sediment prevented nitrous oxide (N₂O) reduction in denitrification assays with a mixed population of endogenous bacteria and a pure culture (HH1) isolated from the sediment. A 5% (v/v) concentration of sediment in nutrient broth caused near maximum inhibition of N₂O reduction. Sediment taken from a site closer to pollution sources (Site 906) was twice as inhibitory (as measured by N₂O accumulation) as sediment from Site 910, further from pollution sources. N₂O persistence was associated with the particulate sediment fraction only. Several heavy metals were tested at in situ concentrations, and ionic cadmium (Cd) and chromium (Cr) caused N₂O accumulation. Ashed sediment did not cause N₂O accumulation, but did decrease initial nitrate reduction rates with HH1.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.     

Effect of copper dosing on sulfide inhibited reduction of nitric and nitrous oxide

The stimulating effect of copper addition on the reduction rate of nitrous oxide (N(2)O) to dinitrogen (N(2)) in the presence of sulfide was investigated in batch experiments (pH 7.0; 55 degrees C). N(2)O was dosed either directly as a gas to the headspace of the bottles or formed as intermediate during the denitrification of nitrite in Fe(II)EDTA(2-)-containing medium and nitrate in Fe(II)EDTA(2-)-free medium. Sulfide was either dosed externally or generated from endogenous sulfur sources during anaerobic incubation of the sludge. In the presence of sulfide (from 15 microM to 1mM), heterotrophic denitrification using ethanol as electron donor was incomplete, i.e., N(2)O accumulated instead of N(2) or was transiently formed. Copper addition (60 microM) rapidly stimulated the reduction of N(2)O to N(2). Zinc addition (60 microM) did not have a similar strong stimulating effect as observed for copper and the N(2)O reduction rate was not stimulated at all upon supply of FeCl(3) (2 mM). Thus, a copper deficiency for N(2)O reduction is most likely developed in the presence of sulfide. It is suggested that sulfide induces this deficiency as it readily precipitates as copper sulfide and thus scavenges copper in the medium or that sulfide inactivates the N(2)OR reductase as it sequesters the copper of this metalloenzyme.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd 1) among denitrifying pseudomonads

The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzeri-group (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd ₁) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.     

Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Mutations affecting pore formation by haemolysin from Escherichia coli

Summary By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9–37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI–DIII) located between amino acids 238–410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498–830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670–830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli

Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.     

Identification of a copper protein as part of the nitrous oxide-reducing system in nitrite-respiring (denitrifying) pseudomonads

Copper is the essential transition element for nitrous oxide respiration in Pseudomonas perfectomarinus. Two novel kinds of copper proteins were detected in this organism. Their distribution was studied under different growth conditions and in other pseudomonads, as well as their association with N₂O reduction of intact cells. A low molecular mass copper protein (M _r 38,000) with a single absorption band at 340 nm (oxidized form), was found only in P. perfectomarinus and was not required for N₂O reduction. N₂O respiration was consistently associated with a high molecular mass copper protein (M _r 120,000) in P. perfectomarinus, Pseudomonas stutzeri, and in strains of Pseudomonas fluorescens that were capable of this type of respiration. The oxidized protein was violet to pink with absorption bands at 350, 480, 530, 620, and 780 nm. Pseudomonas chlororaphis and Pseudomonas aureofaciens which did not respire with N₂O as electron acceptor, did not contain the novel type of copper protein. Cytochrome patterns were compared in these denitrifying pseudomonads to search for the physiological electron carrier to N₂O reductase. The content and nature of the soluble c-type cytochromes depended strongly on the species and the particular growth condition.Abbreviations M _r relative molecular mass     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Gene expression analysis of the response by Escherichia coli to seawater

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3000) of the 4228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth. This revised version was published online in August 2006 with corrections to the Cover Date.