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CURRENT MICROBIOLOGY Vol. 32 (1996), pp. 25–32 Study of the Denitrifying Enzymatic System of Comamonas sp.
Strain SGLY2 Under Various Aeration Conditions with a ParticularView on Nitrate and Nitrite Reductases Institut National de la Recherche Agronomique, Laboratoire de Biotechnologie de l’Environnement (LBE), Avenue des Etangs,11100 Narbonne, France Abstract. This paper studies the effect of oxygen on the denitrifying enzymatic system of
Comamonas sp. It is shown that nitrate respiration can take place in the presence of oxygen. Indeed,
even if a protein synthesis inhibitor is added in the medium, immediate nitrate consumption is
observed in an aerobic culture inoculated with cells that have never been subjected to nitrate.
Existence of a constitutive nitrate reductase could explain this phenomenon. Moreover the nitrate and
nitrite reductases are active and synthesized under aerobic conditions. The different levels of
inhibition of nitrate reductase activity by respiratory inhibitors and detergent, according to the
aerobic and anaerobic cultures, might suggest the existence of a double nitrate reductase enzymatic
Bacterial denitrification is an anaerobic respiration inhibition of denitrifying enzymes with Paracoccus where nitrate is reduced to nitrogen gas with nitrite, denitrificans [14] or Pseudomonas aeruginosa [12]; nitric oxide, and nitrous oxide as intermediates. This (ii) existence of a tolerance threshold towards dis- reaction is realized by facultative anaerobic microor- solved oxygen with Pseudomonas stutzeri; and (iii) ganisms, especially Pseudomonas sp. [39]. It is an co-respiration with Thiosphaera pantotropha, Alcalig- alternative way of energy production by transfer of enes faecalis [28, 30], Pseudomonas nautica [6] and electrons to four de novo-synthesized terminal oxido- reductases: nitrate reductase (NaR), nitrite reductase Berks and associates [4, 5] have characterized the (NiR), nitric oxide reductase (NoR), and nitrous oxide aerobic denitrifying pathway of Thiosphaera pantotro- reductase (N2OR). Synthesis and activity of these pha, even though Thomsen and colleagues [37] have enzymes are assumed to be completely repressed by assumed that aerobic denitrification with this strain is a oxygen and to be stimulated by one or the other of the utopic idea because of the existence of anaerobic microzones in the kind of reactor used. They have More recently, some authors have demonstrated purified a periplasmic nitrate reductase that is overex- that synthesis and activity of denitrifying enzymes pressed in a membrane-bound nitrate reductase mutant could occur under various aeration conditions. For strain [3]. They have explained the simultaneous use of example, Pseudomonas stutzeri exhibits its higher oxygen and nitrate by existence of this double nitrate nitrate reductase activity at 1 mg · L21 of dissolved reductase enzymatic system: the membrane-bound oxygen, but the threshold value for its nitrate reductase nitrate reductase, inhibited by oxygen, allows aerobic synthesis is 5 mg · L21 of dissolved oxygen [15].
expression of the periplasmic one [25].
Thiosphaera pantotropha denitrifies at a rate of 800 A strain named SGLY2, and identified as Comamo- nmol · min21 · mg21 of protein at 80% of air saturation nas sp., has been isolated in our laboratory from an [29]. Three different types of behavior can thus be upflow filter submitted to various aeration conditions.
distinguished in the presence of oxygen: (i) complete This strain was shown to denitrify in the presence ofhigh oxygen levels and to be able to co-respire the two electron acceptors [22]. In the present study, we characterized the enzymatic system of the strain (espe- protein synthesis. Cells were harvested, washed, and concentrated cially nitrate and nitrite reductases) by investigating in 0.9% NaCl solution. They were then used to inoculate the the influence of different respiratory protein synthesis different flasks (control and test flasks) in anaerobic, partial aerobic,and fully aerobic conditions to reach a concentration of 35 mg · L21 inhibitors and detergent (by modifying membrane of proteins. Flasks were shaken in a rotary shaker (200 rpm) at 35°C permeability) on the oxygen-nitrate respiration sys- tem. These experiments were carried out either in Assays of nitrate and nitrite reductase activities. Nitrate and
batch culture under various aeration conditions or in nitrite reductase activities were assayed on whole cells harvested enzymatic assays on whole cells and cell lysates.
from three different cultures: one 400-ml aerobic culture in a 1-LErlenmeyer flask filled with the medium described above except Materials and Methods
N-oxides; one 400-ml aerobic culture in a 1-L Erlenmeyer flaskfilled with the medium described above supplied with KNO Organisms and culture conditions. Isolation and characterization
one 400-ml anaerobic culture in Penicillin flasks filled with the of the strain SGLY2 used in this study have been described in detailelsewhere [22]. Cells were grown on a synthetic medium: phos- medium supplied with KNO3. These three cultures were inoculated (1/200) and were then grown for 16 h. After addition of chloram- M, pH 7.0; KNO3 (N-NO3 5 250 mg · L21) or phenicol (150 µg · ml21), cells were harvested, washed twice with (N-NO2 5 50 mg · L21); ethanol (C-C2H5OH 5 500 0.9% cold NaCl, and suspended in the same solution.
4 190 mg · L21; (NH4)2SO4 as nitrogen source Nitrate and nitrite reductase activities were measured accord- 4 5 58 mg · L21); yeast extract (Difco) 250 mg · L21; 1 ml · L21 of trace element solution [22]. To study the influence of ing to the combined methods of Brons and Zehnder [7] and Ko¨rner inhibitors or detergent on the oxygen-denitrifying respiration and Zumft [15]. A 4-ml mixture consisting of 2 ml 0.1 M phosphate system of the strain, batch cultures were performed under three buffer, pH 7.2; 1 ml 0.1 M KNO3 or 1.5 ml 0.1 M KNO2; 0.4 ml 0.5 M aeration conditions in 120-ml penicillin flasks (Poly Labo, Montpel- sodium acetate and demineralized water was pipetted into 15-ml lier), filled with the medium described above. Anaerobic conditions tubes. The tubes were then flushed with argon and hermetically were obtained by bubbling cultures with oxygen-nitrogen-free sealed with rubber stoppers for anaerobic tests. Different inhibitors were added to the reaction mixture: sodium azide to a final CO 1 CO2 , 0.1 ppm). To obtain partial aerobic conditions, we concentration of 100 µM and 10 mM, and erythromycin to a final first bubbled the cultures with argon. A known quantity of pure concentration of 200 µg · ml21. After equilibration at 35°C in a oxygen (oxygen C Alphagas, N2 , 5 ppm) was then added into the water bath, the reaction was started by injection of the equivalent of sealed bottle, with a syringe, until it reached a concentration of 20 2.4 mg of cell protein per tube. For the next hour, a sample was mmol · L21 of gas. This addition of a large quantity of oxygen withdrawn every 20 min. The reaction was stopped by eliminating pressurized the flasks. Since no oxygen was present in the liquid the cells by centrifugation at 4°C. Nitrate reductase activity was phase (because of sparging with argon), a part of the oxygen gas expressed as mmol of nitrate consumed per minute per milligram of was transferred to the liquid phase. At the beginning of the proteins. Nitrite reductase activity was expressed as nmol of nitrite experiment, this dissolved oxygen concentration corresponded to consumed per minute per milligram of proteins.
oxygen saturation (7.8 mg · L21 at sea level at 35°C). It subse- Nitrate reduction was also measured in cell-free extract, quently dropped owing to the bacterial oxygen consumption, obtained from a sonicated aerobic cell suspension, according to the compensated by redistribution between the gaseous and liquid modified procedure described by Krul and Veeningen [16]. The phase. Aerobic cultures were grown in cotton-wool plugs flasks assay mixture consisted of 2.5 ml 0.1 M phosphate buffer, pH 7.2; 1 filled with 50 ml of the medium described above, whereas in the ml 0.1 M KNO3; 1.25 ml benzylviologen 0.2 mg · L21; and 0.25 ml two other conditions the final volume was 100 ml. The smaller of demineralized water. The reaction vials were sparged with argon liquid volume in aerobic culture allowed better gas exchange.
and sealed with rubber stoppers to keep the anaerobic conditions.
Inhibitors and detergent tests. Two inhibitors of cytochrome
Thereafter, 0.5 ml of a mixed solution of 10 mg · L21 Na2S2O4 and 3) at two different concentrations were tested by 3 (vol/vol) was supplied. After 15 min of addition to the medium of sodium azide at 0.1 mM and 10 mM, shaking in a waterbath at 35°C, the equivalent of 100 mg · L21 of potassium cyanide at 10 and 100 µM. Diethyldithiocarbamic acid protein extract was injected. A sample was withdrawn every 10 min (DDC), a copper chelator, was used as an inhibitor of copper type in a period of 30 min. The reaction was stopped by aerating the nitrite reductase or other copper proteins of the respiratory chain samples in order to oxidize the residual electron donor.
(azurin) at a final concentration of 10 mM. Erythromycin andchloramphenicol were used at 200 µg · ml21 and 150 µg · ml21 Analysis of biomass, medium, and gas. Nitrate and nitrite were
respectively to inhibit protein synthesis. Effect of membrane measured by an exchange ion chromatography system with conduc- perturbations on reductases activities was observed based on triton tivity detection (DIONEX-100). Separation and elution of the anions were carried out on an IonPacAS4A Analytical Column with Inoculum was grown aerobically in a 15-ml cotton-wool plugs a carbonate-bicarbonate eluant and a sulfuric acid regenerant.
Erlenmeyer flask containing yeast extract (5 g · L21) and peptone Gas composition was analyzed by gas chromatography with a (15 g · L21). After overnight growth, this culture was used to Shimadzu GC-8A apparatus with argon carrier, by use of a inoculate two different precultures: one 500-ml Erlenmeyer flask katharometer detector. Carbon dioxide and nitrous oxide were filled with 200 ml of the medium described above except N-oxides separated on a Haye Sep Q column (80–100 mesh, 2.0 m 3 1/8 (this culture was named ‘‘nonadapted to N-oxides preculture’’) and inch). Oxygen and nitrogen were separated on a molecular sieve 5A one Penicillin flask filled with 200 ml of the complete medium and (20–100 mesh, 2.0 m 3 1/8 inch). Injector and detector temperature maintained under anaerobic conditions (this culture was named was 100°C; column temperature was 35°C. Nitric oxide was ‘‘adapted to N-oxides preculture’’). When the precultures were in measured on a Shimadzu-14A with helium carrier, by use of a the logarithmic growth phase, chloramphenicol was added to stop katharometer detector. The molecular sieve 5A (80–100 mesh, D. Patureau et al.: Denitrifying Enzymatic System of Comamonas sp.
Fig. 1. Influence of chloramphenicol (150 µg · ml21) on the nitriteproduction during anaerobic (M, N) and partial aerobic cultures(U, V) on nitrate with Comamonas sp. Cultures were inoculatedwith nonadapted preculture. Chloramphenicol was added at thebeginning of the culture. M, U, control cultures; N, V, cultureswith chloramphenicol.
Fig. 2. Consumption of nitrate during anaerobic (M), partial aerobic(U), and total aerobic (Q) growth conditions of Comamonas sp.
The arrow indicates the time at which oxygen has completelydisappeared in the partial aerobic culture. Cultures were inoculated 2.0 m 3 1/8 inch) column was maintained at 220°C, injector at Proteins were determined by Lowry’s procedure with bovine whereas 99 nmol were obtained in the control tube.
This measurement of residual activity might be owing to the existence of a nitrate reductase in the aerobicculture.
Existence of a constitutive nitrate reductase. Chlor-
Our experiments showed that a constitutive nitrate amphenicol (150 µg · ml21) was used to determine reductase is present in an aerobic culture nonadapted whether or not denitrifying enzymes were synthesized; to N-oxides. This enzyme could be active under nitrate reductase activities were measured in aerobic anaerobic as well as aerobic conditions at a small basal cultures inoculated with cells nonadapted to N-oxides.
It was added during cell harvesting by centrifugation(existence of anaerobic conditions in the pellet could Synthesis and activity of nitrate and nitrite reducta-
enhance synthesis of enzymes) and in the different ses under aerobic condition. The previous experi-
batch assays. Nitrate reduction was measured by ments showed that a higher nitrate reduction rate is production of nitrite (Fig. 1). Addition of chlorampheni- observed when protein synthesis is not inhibited (Fig.
col in the anaerobic and partial aerobic cultures 1). In the same way, nitrate consumption began resulted in a sharp decrease of the nitrate reduction immediately after inoculation of the medium with rate: 4 µg N-NO2 · h21 · mg21 of protein against 85.3 nonadapted cells, in both aerobic and anaerobic condi- µg · h21 · mg21 of protein in anaerobic conditions.
tions (Fig. 2). During aerobic culture, nitrate consump- However, an immediate production of nitrite was tion slowed down after 20 h because of a lack of noticed even though the cells used to inoculate the carbon source. Nitrate reductase activity resulted from culture were for the first time in the presence of nitrate.
the activity of the constitutive nitrate reductase and of Nitrate reductase activity was also measured on new enzyme synthesis. Thus, it seems that synthesis SGLY2 cell extracts obtained from a culture never and activity of nitrate reductase may occur in a wide subjected to nitrate. In this type of culture, an activity The same experiment was realized with nitrite as measured. In the same way, an enzymatic assay on the final electron acceptor (Fig. 3). Anaerobic nitrite whole cells from aerobic culture without nitrate was consumption started after a lag period of 1 day. In this realized by adding erythromycin (200 µg · ml21) and case, the use of nitrite is the only way to produce chloramphenicol (150 µg · ml21) in the test tube. These energy. However, according to the literature, no nitrite protein synthesis inhibitors decreased the nitrate reduc- reductase is synthesized in a nonadapted preculture.
tase activity: the activity, with chloramphenicol, was This lag phase corresponds to synthesis of a de novo nitrite reductase by using presumably residual energy Fig. 3. Consumption of nitrite during anaerobic (M), partial aerobic Fig. 4. Influence of triton (0.02%) on the consumption of nitrate (U), and total aerobic cultures (Q) of Comamonas sp. The arrow during anaerobic (M, N) and total aerobic (Q, S) cultures of indicates the time at which oxygen has completely disappeared in Comamonas sp. Cultures were inoculated with anaerobic cells. M, the partial aerobic culture. Cultures were inoculated with non- Q, control cultures; N, S; Cultures with triton; =, addition of present in the cells. In contrast, partial or fully aerobicconsumption of nitrite started after a smaller lag period(4 h). This consumption was correlated with nitrousoxide and nitrogen production (data not shown).
Oxygen consumption provided the energy necessary tosynthesize the enzyme. Addition of chloramphenicolresulted in inhibition of nitrite consumption no matterwhat culture conditions were used. These experimentsdemonstrated that nitrite reductase, which may not beconstitutive, is synthesized under aerobic conditionsand that nitrite and oxygen are consumed simulta-neously.
Effect of triton, sodium azide, and cyanide on
nitrate reductase activity.
Figure 4 shows the nitrate
consumption during anaerobic and aerobic batch cul-
Fig. 5. Influence of sodium azide (0.1 mM) on the consumption of tures of SGLY2 inoculated with adapted-to-N-oxide nitrate during anaerobic (M, N) and total aerobic (Q, S) cultures cells in the presence of triton. Addition of this deter- of Comamonas sp. Cultures were inoculated with nonadapted toN-oxides cells. M, Q, control cultures; N, S, cultures with azide.
gent to the culture medium resulted in a gentle decrease in the anaerobic nitrate consumption (0.111mg N-NO3 · h21 · mg21 of protein against 0.062 mgN-NO3 · h21 · mg21 of protein). It seems, however, to concentration tested (10 mM) neither denitrification have no effect on aerobic nitrate reduction. The same nor growth was observed. This means that the denitri- results are obtained with the nonadapted to N-oxides fying as well as the oxygen-respiring enzymatic sys- preculture. The presence of triton X-100 perturbated tem was completely inhibited at this high concentra- the permeability properties of the cytoplasmic mem- tion. In contrast, at 0.1 mM, the nitrate reduction rate of brane. This implies that under anaerobic conditions, the anaerobic culture, inoculated with the nonadapted the measurement of nitrate reductase activity is corre- cells (Fig. 5), fell to 7 µg N-NO3 · h21 · mg21 of lated with the membrane, whereas aerobic nitrate protein. This corresponds to a 97% inhibition of the reduction is independent of the membrane.
denitrifying enzyme activity measured under control The effect of azide on nitrate reductase activity anaerobic conditions. During aerobic culture at 0.1 during batch assay followed exactly the same pattern mM, no effect of azide on oxygen uptake was observed: observed with triton (Fig. 5). Whatever the aeration the disappearance of oxygen in the gaseous phase was conditions and the preculture used, at the highest correlated with protein synthesis. The only influence D. Patureau et al.: Denitrifying Enzymatic System of Comamonas sp.
Table 1. Influence of two concentrations (0.1 and 10 mM) ofsodium azide on nitrate reductase activity measured on whole cellsharvested from aerobic and anaerobic precultures (see Materialsand Methods for more details) Activity is expressed in nmol of disappeared nitrate per minute permg of protein.
of this respiratory inhibitor on denitrifying enzymeswas then noticed. Compared with the nitrate reductionrate measured under aerobic control conditions, therewas a 61% inhibition of the nitrate reductase activity(Fig. 5). The same conclusions were drawn from themeasurements of nitrate reductase activity in wholecells (Table 1). The presence of 0.1 mM of azide in thetest tube implied a 100% fall of the nitrate reductaseactivity of anaerobic whole cells, whereas a 53%decrease was observed on nitrate reductase activity ofaerobic whole cells. The pattern of inhibition of nitrate Fig. 6. Evolution of nitrate (M, N) and nitrite (Q, S) during reductase activity was similar to the results obtained in anaerobic (A) and partial aerobic (B) cultures of Comamonas sp. in batch assays with the highest concentration of azide.
the presence of cyanide (N, S) at a final concentration of 10 µM.
Cultures were inoculated with nonadapted to N-oxides cells.
Inhibition of 95% and 100% of the nitrate reductaseactivities was observed in aerobic and anaerobic wholecells respectively. This difference of azide inhibition chain between nitrate reductase and nitrite reductase.
level on the nitrate reductase activity may suggest that In turn, in the partial aerobic culture with cyanide, (i) two different nitrate reductases are active according nitrates were not consumed during the period of to the aeration conditions or (ii) two different electron oxygen consumption correlated with proteins synthe- donor pathways are used for the same nitrate reduc- sis (11 h). On the contrary, the concentration of nitrate sharply decreased without a lag phase in the control The inhibitory effect of cyanide on nitrate reduc- culture. Despite this phenomenon, after complete tion was another aspect contributing to these two disappearance of oxygen in the test culture, the nitrate previous hypotheses. It was demonstrated that a high consumption was similar to that observed under anaero- concentration of cyanide (100 µM) completely inhib- bic conditions. During fully aerobic conditions, at 10 ited denitrification under anaerobiosis as well as under µM, denitrification was not noticed, whereas the growth aerobiosis (data not shown). Figures 6a and 6b show rate was similar to that obtained in the control fully the evolution of anions during batch culture with and aerobic culture, implying no inhibition of oxidases.
without cyanide at 10 µM (added in the medium at time This suggests that 10 µM of cyanide directly inhibits 0) under anaerobic (a) and partial aerobic (b) condi- the aerobic denitrifying enzymes. Thus, cyanide influ- tions. Under anaerobic conditions, nitrate consump- ences denitrification under aerobic conditions by to- tion, with or without cyanide, followed the same tally inhibiting the nitrate reduction, whereas under pattern during 50 h. In contrast, a higher quantity of anaerobic conditions, no effect was observed on nitrate nitrite was accumulated in the presence of the respira- tory inhibitor compared with the control culture. Thus,it seems that a concentration of 10 µM of cyanide has Effect of triton X-100, azide, and DDC on nitrite
no effect on the nitrate reductase synthesized under reductase activity. Figure 4 shows that triton partially
anaerobic conditions. However, it partially inhibits the inhibited anaerobic nitrate consumption because of its nitrite reductase or an intermediate of the respiratory effect on membrane disorganization. In the same time, no nitrite was accumulated in the medium, whereas in served with Pseudomonas aeruginosa, a classical the control culture the nitrite concentration increased oxygen-sensitive denitrifier [12]. With Comamonas sharply to reach a peak of 80 mg N-NO2 · L21. By sp., the decrease in nitrate reduction rate was corre- disturbing the permeability of the cytoplasmic mem- lated with a decrease in nitrogen gas production and brane, triton could act on the antiport system nitrate/ partial nitrite, nitric oxide, and nitrous oxide accumula- nitrite by preventing the nitrate from joining the active tion. However, the presence of these denitrifying site of the enzyme and by holding the nitrite in the intermediates during continuous culture, at a dissolved cytoplasm. On the contrary, triton, when added in oxygen concentration of 100% of air saturation (data batch culture with nitrite as final electron acceptor, had not shown), implies that the four denitrifying enzymes no direct effect on nitrite reduction whatever the are active and synthesized under aerobic conditions.
culture and preculture conditions used. This may be The same conclusions are drawn, since higher aerobic owing to periplasmic localization of the nitrite reduc- nitrate–nitrite reduction rates were observed in culture without chloramphenicol compared with that obtained Azide and DDC were chosen to show the possible with the protein synthesis inhibitor (Figs. 1–3).
existence of a copper nitrite reductase or copper These aerobic nitrate and nitrite reductase activi- intermediates as pseudoazurin in the respiratory chain: ties could not be interpreted in terms of assimilation the former reacts with ferric centers, the latter links to because (i) (NH4)2SO4, used as the nitrogen source, copper centers. Nitrite reduction seems to be insensi- classically inhibits nitrate assimilation, (ii) ammonium tive to respiratory inhibitors during anaerobic batch disappearance was well correlated with biomass pro- assay with nitrate as the final electron acceptor: duction, and (iii) during continuous culture under addition of azide or DDC resulted on one hand in total oxygen-saturated conditions, nitrogen was produced, inhibition of nitrate reductase activity (Fig. 5) and, on which is the direct demonstration of denitrification the other hand, in a complete disappearance of nitrite reaction. Van Niel and associates [38], using nitrate accumulated during the previous hours. In the same labeled on nitrogen, have confirmed the idea of way, nitrate reductase activity measured by enzymatic Robertson and Kuenen [27] that a complete denitrify- assay on whole cells is expressed as the quantity of ing system is present under aerobic conditions in T. nitrate disappearing because no nitrite accumulated pantotropha. According to the experiments done with during the test. In the presence of the different chloramphenicol on cells never subjected to nitrate, inhibitors, batch assays with nitrite as the final electron the existence of a constitutive nitrate reductase was acceptor ended up with the same conclusion: whatever proposed to explain the possible aerobic denitrification the culture and preculture conditions, azide and DDC in Comamonas sp. An opposite conclusion resulted had no effect on nitrite reductase activity.
from the same experiments made with Paracoccusdenitrificans (NCIB 8944): nitrite, nitric oxide, nitrousoxide, and nitrogen gas were not produced during Discussion
anaerobic and partial aerobic cultures with chloram- Previous work had generally underlined that in many phenicol. Without the protein inhibitor, anaerobic bacteria, synthesis and activity of denitrifying en- denitrification started with a long lag period of 11 h, zymes could not occur under aerobiosis. Our work corresponding to the synthesis of a de novo nitrate leads to a modified conclusion, and the results allow us to propose a scheme to explain the nitrate-oxygen The close relation between the denitrifying en- co-respiration in Comamonas sp. strain SGLY2. The zymes and the electron transport pathway, the different first experiments done with the strain demonstrated its kinds of nitrate transport, the genetic and the regula- ability to use simultaneously the two electron accep- tory system of synthesis, and the activity of the tors [22]. Aeration of the culture resulted in a decrease enzymes are now well studied [11]. These enzymes are in the nitrate reduction rate: 1.85 µmol NO 2 shown to work in vitro in the presence of oxygen [1, 3, mg21 of protein under anaerobic culture against 0.287 24]. However, in vivo, other aspects have to be in aerated culture. These values lie close to that found considered to explain the possible aerobic denitrifica- with Thiosphaera pantotropha, in which the rate of tion. From a bioenergetic point of view, the idea of acetate-dependent nitrate reduction is around 1.6 µmol co-respiration seems illogical, because energy produc- · min21 · mg21 of protein at dissolved oxygen tion is higher with oxygen than nitrate and because the concentration less than 30%, and 0.8 at 30–80% of air main regulatory factor of denitrification is the redox saturation [29]. In contrast, at a concentration of potential of the respiratory chain [17]. For example, 0.25% of air saturation, no denitrification was ob- the presence of oxygen in a P. denitrificans culture D. Patureau et al.: Denitrifying Enzymatic System of Comamonas sp.
implies preferential diversion of electrons to oxygen tory effect of DDC was noticed on both anaerobic and owing to the modification of the redox potential of the aerobic nitrite reductase activities. On the other hand, a coupler ubiquinol/ubiquinone [9, 10]. Hernandez and large amount of nitrous oxide was accumulated in the colleagues [13] have also shown that, using Pseudomo- gaseous phase, perhaps owing to the blocking of a nas aeruginosa, oxygen indirectly inhibits enzyme multi-copper nitrous oxide reductase. Moreover, no activities by oxidizing the key molecules of the nitrite accumulation was observed during aerobic antiport nitrate-nitrite system. Moreover, it is known culture on nitrate with azide. These observations that oxygen regulates nitrate respiration by suppress- suggest the existence of a cdl-type nitrite reductase.
ing enzyme synthesis: a FnR-like binding site, re- According to the experiments done with and without quired for anaerobic gene expression in Escherichia protein inhibitor, this inducible enzyme seems to be coli, is present in cells of P. denitrificans [11, 33] and active and synthesized under aerobic conditions. At Pseudomonas stutzeri [8]. Bell et al. [2] have reported this point, the behavior of the strain differs from that of that T. pantotropha uses a periplasmic nitrate reductase T. pantotropha: using polyclonal antibodies, Moir [20] while denitrifying aerobically and employs another has shown that the cdl-type nitrite reductase was not membrane-bound reductase for anaerobic denitrifica- expressed under aerobic conditions. The status of tion. Thus, the oxygen inhibitory effect on nitrate- aerobic denitrifier of T. pantotropha is then not clear.
nitrite antiport system is evaded. The diversion of the In fact, it has been shown that there is a close electron flow to the denitrifying enzymes was ex- relationship between T. pantotropha and Paracoccus plained by the hypothesis of the ‘‘bottleneck’’ [26].
denitrificans [19, 35]. Moreover, although a periplas- Using sodium azide as respiratory inhibitor, Van Niel mic nitrate reductase seems to be synthesized in and coworkers [38] showed that nitrogen gas produc- Paracoccus denitrificans [32], no aerobic denitrifica- tion stopped immediately after the addition of 10 mM tion was noticed in this strain. Kuenen and Robertson azide to the aerobic cell suspension. Conversely, 0.02 [18], in their last experiments, observed that the mM azide is just enough to inhibit the nitrate reductase aerobic denitrification rate of T. pantotropha is now synthesized under anaerobic conditions [23]. In the equivalent to 5% of that found under anaerobic same way, the different levels of triton and azide conditions versus 50% at the beginning of their inhibition between aerobic and anaerobic cells suggest the existence of two nitrate reductases in SGLY2: one Physiological observations on Comamonas sp.
‘‘aerobic’’ enzyme insensitive to membrane damage strain SGLY2, using different respiratory or protein caused by triton and less sensitive to azide, and one synthesis inhibitors and detergent, explain the ability ‘‘anaerobic’’ enzyme partially inhibited by 0.02% of of the strain to denitrify under aerobic conditions.
From an ecological point of view, existence of this Cyanide effect is another factor contributing to the kind of aerobic denitrifier is interesting to explain hypothesis of existence of two nitrate reductases. The nitrogen losses of agronomic system. It has thus to be hypothesis of existence of two electron donor path- considered in agricultural practices, especially for the ways for one nitrate reductase was rejected after mode of application of nitrogen fertilizers.
comparison of the azide effect on nitrate and nitrite Literature Cited
reduction under anaerobic conditions. Since azide hadno effect on nitrite reductase activity, whereas it 1. Bell LC, Ferguson SJ (1991) Nitric and nitrous oxide reducta- completely inhibited nitrate reductase activity, we can ses are active under aerobic conditions in cells of Thiosphaerapantotropha. Biochem J 273:423–427 conclude that the nitrate reduction inhibition is a direct 2. Bell LC, Richardson DJ, Ferguson SJ (1990) Periplasmic and effect on the enzyme and not a consequence of membrane-bound respiratory nitrate reductases in Thiosphaera inhibition of an intermediate of the respiratory chain.
pantotropha. FEBS Microbiol Lett 265:85–87 Moreover, one enzyme could not be differently inhib- 3. Bell LC, Page MD, Berks BC, Richardson DJ, Ferguson SJ ited by the same inhibitor. This is why existence of two (1993) Insertion of transposon Tn5 into a structural gene of themembrane-bound nitrate reductase of Thiosphaera Pantotro- pha results in anaerobic overexpression of periplasmic nitrate No aerobic denitrification can occur with the reductase activity. J Gen Microbiol 139:3205–3214 existence of a copper-type nitrite reductase in the 4. Berks BC, Baratta D, Richardson DJ, Ferguson SJ (1993) denitrifying enzymatic system because of its proper- Purification and characterization of a nitrous oxide reductase ties to reduce oxygen to toxic peroxides. Moir and from Thiosphaera pantotropha. Implications for the mecha-nism of aerobic nitrous oxide reduction. Eur J Biochem associates [21] have purified a cdl-type nitrite reduc- tase from T. pantotropha and its presumably electron 5. Berks BC, Richardson DJ, Robinson C, Reilly A, Aplin RT, donor pseudoazurin. With Comamonas sp., no inhibi- Ferguson SJ (1994) Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.
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35. Stouthamer AH (1992) Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to phylog- 18. Kuenen JG, Robertson LA (1994) Combined nitrification and eny of prokariotes. Antonie van Leeuwenhoek 61:1–33 denitrification processes. FEMS Microbiol Rev 15:109–117 36. Thomas KL, Llyod D, Boddy L (1994) Effects of oxygen, pH 19. Ludwig W, Mittenhuber G, Friedrich CG (1993) Transfer of and nitrate concentration on denitrification by Pseudomonas Thiosphaera pantotropha to Paracoccus denitrificans. Int J species. FEMS Microbiol Lett 118:186 37. Thomsen JK, Iversen JJL, Cox RP (1993) Interactions between 20. Moir JWB (1993) Ph.D. thesis, University of Oxford, UK respiration and denitrification during growth of Thiosphaera 21. Moir JWB, Baratta D, Richardson DJ, Ferguson SJ (1993) The pantotropha in continuous culture. FEMS Microbiol Lett purification of a cdl-tyle nitrite reductase and the absence of a copper nitrite reductase from the aerobic denitrifier Thio- 38. Van Niel EWJ, Robertson LA, Cox RP, Kuenen JG (1992) sphaera pantotropha; the role of pseudoazurin as an electron Inhibition of denitrification and oxygen utilization by Thio- sphaera pantotropha. J Gen Appl Microbiol 38:553–558 22. Patureau D, Davison J, Bernet N, Moletta R (1994) Denitrifica- 39. Zumft WG (1992) The denitrifying procaryotes, p 554–582. In tion under various aeration conditions in Comamonas sp, strain Balows A, Tru¨per HG, Dworkin M, Harder W, Schleifer KH (eds.), The procaryotes, 2nd ed., Springer-Verlag, Berlin


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