Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium

A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO₂. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Received: 6 November 1998 / Accepted: 19 January 1999     

Anaerobic degradation of cresols by denitrifying bacteria

The initial reactions in anaerobic metablism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o-or p-cresol as sole organic substrate with a generation time of 11 h; o-or p-cresol was completely oxidized to CO₂ with nitrate being reduced to N₂. A denitrifying Pseudomonas-like strain oxidized m-or p-cresol as the sole organic growth substrate completely to CO₂ with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps:(1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol 4-OH-benzaldehyde 4-OH-benzoate 4-OH-benzoly-CoA benzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating).(2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH₃-benzoyl-CoA as central intermediate as follows: o-cresol 4-OH-3-CH₃-benzoate 4-OH-3-CH₃-benzoyl-CoA 3-CH₃-benzoyl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between ¹⁴CO₂ and the carboxyl of 4-OH-3-CH₃-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH₃-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH₃-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH₃-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH₃-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH₃-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH₃-benzoate and further to 3-CH₃-benzoyl-CoA.(3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4-and 3,4-dimethylphenol were oxidized to 4-OH-3-CH₃-and 4-OH-2-CH₃-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.The hydroxyl and methyl groups are abbreviated as OH-and CH₃-, respectively     

Siderophore Production by Pseudomonas stutzeri under Aerobic and Anaerobic Conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.     

Studies on anaerobic biphasic growth of a denitrifying bacterium, Pseudomonas stutzeri

Growth of Pseudomonas stutzeri(VAN NIEL strain) in the presenceof a limiting amount of nitrate under anaerobic conditions ischaracterized by 2 logarithmic phases separated distinctly byan intermediate phase where the growth rate is very low. Inthe first logarithmic phase nitrate is reduced stoichiometricallyto nitrite stage, and in the second phase nitrite is reducedto nitrogen gas. The nitrite reducing activity of cells in the second growthphase is 3–4 times higher than that of cells in the firstphase. The rise in nitrite reducing activity is correlated witha remarkable increase in the content of cytochromes a₂ and c-552. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan. ²Present address: Institute of Molecular Biology, Faculty ofScience, Nagoya University, Nagoya, Japan. (Received June 16, 1969; )     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

Anaerobic degradation of phenylacetate and 4-hydroxyphenylacetate by denitrifying bacteria

From various oxic or anoxic habitats anaerobic enrichment cultures were set up which completely oxidized aromatic amino acids to CO₂ with nitrate as electron acceptor. Tyrosine and tryptophan at first were degraded to phenol and indole, respectively, prior to utilization of the aromatic ring; with phenylalanine no intermediates were detected. Attempts to isolate denitrifying bacteria able to completely degrade aromatic amino acids were unsuccessful. Starting with these enrichments several strains of denitrifying bacteria were anaerobically enriched and isolated with known fermentation products of amino acids (phenylacetate, 4-OH-phenylacetate, 2-OH-benzoate) plus nitrate as sole sources of carbon and energy.Three strains were characterized further. They grew well in defined mineral salts medium, were gram-negative and facultatively anaerobic with strictly oxidative metabolism; molecular oxygen, nitrate or nitrite served as electron acceptors. The isolates were tentatively identified as pseudomonads, but could not be aligned to known species. They oxidized a variety of aromatic compounds completely to CO₂ anaerobically and, with some exceptions, also aerobically. The substrates included among others: (4-OH)-phenylacetate, (4-OH)-phenylglyoxylate, benzoate, 2-aminobenzoate, phenol, OH-benzoates, indole and notably toluene. Reduced alicyclic compounds were not utilized. During anaerobic degradation of (4-OH)-phenylacetate transient accumulation of (4-OH)-phenylglyoxylate was observed.It is proposed that anaerobic -oxidation of the-CH₂–COOH side chain to -CO–COOH initiates anaerobic degradation of (4-OH)-phenylacetate. This implies a novel type of anaerobic -hydroxylation with water as the oxygen donor. Abbreviation. Hydroxyl groups were abbreviated as OH     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria.

Three strains of anaerobically benzoate-degrading, denitrifying bacteria of the genus Pseudomonas were able to grow on 2-fluorobenzoate as the sole carbon and energy source. Fluoride ion release was stoichiometric, and the reduction of dissolved organic carbon indicated total degradation. Cells grown anaerobically with benzoate were adapted for immediate growth with 2-fluorobenzoate, and both compounds were substrates for an inducible benzoyl-coenzyme A synthetase, the initial enzyme of anaerobic degradation. It is proposed that fluoride is eliminated gratuitously by a regioselective reaction in a sequence common to both carbon sources. Benzoate, but not 2-fluorobenzoate, was oxidized by aerobically grown cells.     

Anaerobic degradation ofm-cresol via methyl oxidation to 3-hydroxybenzoate by a denitrifying bacterium

The anaerobic degradation of m-cresol was studied in a denitrifying bacterium. In the initial studies, hypothetical intermediates of m-cresol degradation were tested in growth experiments and in adaptation studies with dense cell suspensions. Results suggested a degradation of m-cresol via 3-hydroxybenzoate. To verify this, the degradation of m-cresol was followed in concentrated cell suspensions in the presence of metabolic inhibitors. Fluoroacetate treatment resulted in the transient accumulation of substantial amounts of 3-hydroxybenzoate. In the presence of iodoacetamide, not only was 3-hydroxybenzoate transiently formed, but benzoate was also accumulated. These findings support a degradation of m-cresol via initial anaerobic methyl oxidation to 3-hydroxybenzoate, followed by reductive dehydroxylation to benzoate or benzoyl-CoA. Studies with extracts of m-cresol-grown cells showed the presence of several enzyme activities to be postulated for this pathway. No evidence was found for a carboxylation, hydroxylation of the aromatic ring, or direct ring reduction as the initial step in m-cresol metabolism. Received: 29 November 1994 / Accepted: 7 March 1995     

Competition for nitrate between denitrifying Pseudomonas stutzeri and nitrate ammonifying enterobacteria

Abstract Competition for nitrate between nitrate ammonifying enterobacteria and a denitrifying pseudomonad was studied in electron acceptor-limited chenostats. In pure cultures, using different carbon and energy sources, the C/N-ratio needed for denitrification is far lower than that required for nitrate ammonification. In mixed cultures of Citrobacter freundii and Pseudomonas stutzeri , competing for nitrate with l -lactate as electron donor, the nitrate ammonifying organism dominated at dilution rates of D ≤ 0.14 h⁻¹. Competition for both nitrate and l -lactate at a dilution rate of D = 0.05 h⁻¹ always resulted in the coexistence of both species. Using glucose as additional carbon source, the final ratio of nitrate ammonifying and denitrifying organism depended on the C/N-ratio as well as on the dilution rate. The results of the study are discussed with respect to field data.     

Anaerobic degradation of p-cresol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of p-cresol under denitrifying conditions by a bacterial consortium was studied in batch and continuous cultures. Concentrations up to 3 mm were degraded within 5–6 days with 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4-hydroxybenzoate as intermediates. Steady states could be maintained at only one dilution rate, D=0.04 h^–1. A further increase in the dilution rate to 0.0 8 h^–1 resulted in culture wash-out. An estimation of the Saturation constant was made (<1 mg/l), taking the maximum specific growth rate as 0.045 h^–1, thus yielding a value of 0.125 mg p-cresol/l. Correspondence to: N. Khoury     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Anaerobic degradation of 2-aminobenzoate (anthranilic acid) by denitrifying bacteria

In the presence of oxygen many aminoaromatic compounds polymerize to form recalcitrant macromolecules. To circumvent undesirable oxidation reactions, the anaerobic biodegradation of a simple member of this class of compounds was investigated. Two strains of bacteria were isolated which degrade 2-aminobenzoate anaerobically under denitrifying conditions, with nitrate as the terminal electron acceptor. Both organisms, which were assigned to the genus Pseudomonas, oxidized 2-aminobenzoate completely to CO2 and NH4+. Nitrate was reduced to nitrite. When nitrate was deplete from the growth medium the accumulated nitrite was reduced to nitrogen. The results establish a model system for the anaerobic, rapid, and complete oxidation of an aminoaromatic compound.     

Anaerobic degradation of 4-methylbenzoate by a newly isolated denitrifying bacterium, strain pMbN1

A novel alphaproteobacterium isolated from freshwater sediments, strain pMbN1, degrades 4-methylbenzoate to CO(2) under nitrate-reducing conditions. While strain pMbN1 utilizes several benzoate derivatives and other polar aromatic compounds, it cannot degrade p-xylene or other hydrocarbons. Based on 16S rRNA gene sequence analysis, strain pMbN1 is affiliated with the genus Magnetospirillum.     

Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1

Tetrachloroethylene (PCE) is thought to have no natural source, so it is one of the most difficult contaminants to degrade biologically. This common groundwater pollutant was thought completely nonbiodegradable in the presence of oxygen. Here we report that the wastewater bacterium Pseudomonas stutzeri OX1 degrades aerobically 0. 56 micromol of 2.0 micromol PCE in 21 h (Vmax approximately 2.5 nmol min(-1) mg(-1) protein and KM approximately 34 microM). These results were corroborated by the generation of 0.48 micromol of the degradation product, chloride ions. This degradation was confirmed to be a result of expression of toluene-o-xylene monooxygenase (ToMO) by P. stutzeri OX1, since cloning and expressing this enzyme in Escherichia coli led to the aerobic degradation of 0.19 micromol of 2.0 micromol PCE and the generation of stoichiometric amounts of chloride. In addition, PCE induces formation of ToMO, which leads to its own degradation in P. stutzeri OX1. Degradation intermediates reduce the growth rate of this strain by 27%.     

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N₂O reductase and nitrite reductase (cytochrome cd₁) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N₂O reductase in a mutant strain deficient in the chromophore of N₂O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.     

Anaerobic oxidation of p-cresol by a denitrifying bacterium

Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07. The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions. The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation. Nitrate is required as the external electron acceptor and is reduced to molecular N2. Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor. In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates. The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation. The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments.