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1.
A heterotrophic nitrifying Alcaligenes sp. from soil was grown as a denitrifier on nitrate and subjected to oxidant pulse experiments to ascertain the apparent effeciencies of proton translocations during O 2 and nitrogen-oxide respirations. With endogenous substrate as the reducing agent the H +/2e – ratios, extrapolated to zero amount of oxidant per pulse, were 9.4, 3.7, 4.3 and 3.5 for O 2, nitrate, nitrite and N 2O, respectively. The value for O 2 and those for the N-oxides are, respectively, somewhat larger and smaller than corresponding values for Paracoccus denitrificans. None of the three permeant ions employed with the Alcaligenes sp. (valinomycin-K +, thiocyanate and triphenylmethylphosphonium) was ideal for all purposes. Thiocyanate provided highest ratios for O 2 but abolished the oxidant pulse response for nitrate and N 2O. Valinomycin was slow to penetrate to the cytoplasmic membrane and relatively high concentrations were required for optimal performance. Triphenylmethylphosphonium enhanced passive proton permeability and diminished proton translocation at concentrations required to realize the maximal oxidant pulse response. 相似文献
2.
The molar yields (g cell/mol) for Alcaligenes faecalis, Pseudomonas stutzeri, Paracoccus denitrificans and Pseudomonas perfectomarinus batch cultures, under nitrous oxide (N 2O) as the electron acceptor, were 11.2, 8.2, 6.1 and 4.4, respectively. Paracoccus denitrificans and Pseudomonas perfectomarinus, which had the slowest growth rates, gave the lowest yields. Large maintenance energy costs may be partially responsible for this. The growth efficiencies of A. faecalis and Ps. perfectomarinus on N 2O indicate that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N 2O reduction are about 49% and 39% of those in the electron transport system associated with O 2 respiration, respectively. 相似文献
3.
Limited aeration of cell suspension in growth medium was used to study the kinetics of formation of nitrite reductase and
nitrous-oxide reductase and their physiological electron donor, cytochrome c-550, during the anaerobic adaptation of Paracoccus denitrificans. The crucial step in the regulation of synthesis of these components is the repressive effect of oxygen while nitrogenous
acceptors (NO 3
−, NO 2
−, N 2O) probably play no role as inducers. The time course of the enzyme activites was analogous (after a lag phase a sharp increase
with a maximum after 3 h) and differed from the kinetics of synthesis of cytochrome c-550 (gradual rise throughout the 8-h experiment). 相似文献
4.
Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO 3
-, NO, and N 2O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA
Thenoyltrifluoroacetone
- NHQNO
2-n-nonyl-4-hydroxyquinoline N-oxide 相似文献
5.
When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO
3
–
quantitatively to NO
2
–
in NO
3
–
-respiration. In the absence of nitrate, NO
2
–
was immediately reduced to NO or N 2O but not to N 2 indicating that NO
2
–
-reductase but not N 2O-reductase was active. The formation of the products NO or N 2O depended on the pH in the medium and the concentration of NO
2
–
present. When P. aeruginosa was grown anaerobically for at least three davs N 2O-reductase was also active. Such cells reduced NO to N 2 via N 2O. The new strain generated a H +-gradient and grew by reducing N 2O to N 2 but not by converting NO to N 2O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H +/NO during the reduction of NO to N 2O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed. 相似文献
6.
We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N 2O and N 2 from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with 15N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N 2O, and finally N 2O was converted to N 2. At pH 8.5, nitrate was converted directly to N 2, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant. 相似文献
7.
Rhizobium hedysari strain HCNT 1 rapidly reduced nitrite to N 2O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O 2 uptake, to non-toxic N 2O. Concentrations of nitrite as small as 3 M diminished O 2 uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O 2 concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O 2-limited conditions, may account for the toxicity of nitrite in strain HCNT 1. 相似文献
9.
For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous
oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H +-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth
yields (Y
s
max
-values) were essentially the same with O 2 or N 2O but were 1/3 and 2/3 lower with NO
2
-
or NO
3
-
, respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients
were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N 2-fixing conditions drastically reduced the Y
s
max
-values in the N 2O and O 2-respiring cells. In the H +-translocation measurements, the
/oxidant ratios were 5.6 for O 2→H 2O, 2.5–2.8 for NO
3
-
→NO
2
-
, 2.2 for NO
2
-
→N 2O and 3.1 for N 2O→N 2 respirations when the cells were preincubated with valinomycin and K +. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP +) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H +-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore,
nitrate and nitrite respirations were accompanied by a H +-uptake
. Any significant H +-translocation could not be detected in N 2O- and O 2-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas
nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal
molar growth yield determinations and the absence of any H +-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of
respiration. The low H +-extrusion values for N 2O-respiration compared to O 2-respiration in cells treated with valinomycin plus TPMP + are, however, not in accord with such an interpretation. 相似文献
10.
Proton translocation during the reduction of NO
3
-
, NO
2
-
, N 2O and O 2, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO
2
-
to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO
3
-
, N 2O or O 2 was added to cells in the dark or with these compounds and NO
2
-
in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO
3
-
, NO
2
-
, N 2O or O 2. The proton extrusion stoichiometry (
ratios) in illuminated cells were as follows: NO
3
-
0.5N 2, 4.82; NO
2
-
0.5N 2, 5.43; N 2ON 2, 6.20; and O 2H 2O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher
values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO
3
-
, NO
2
-
or N 2O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption,
ratios without a permeant ion were NO
3
-
NO
2
-
,-1.95; NO
2
-
0.5 N 2O,-3.03 and N 2ON 2,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH
reduced benzyl viologen
- CCCP
carbonyl cyanide m-chlorophenyl hydrazone
- DIECA
N, N-diethyl-dithiocarbamate
- HOQNO
2-n-heptyl-4-hydroxyquinoline-N-oxide
- NEM
N-ethylmaleimide 相似文献
11.
Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO 2-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO 2-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO 2-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X
prosthetic group of methanol dehydrogenase
-
q
substrate
specific rate of consumption of substrate (mol/g biomass. h.)
-
Y
substrate, Y
substrate
MAX
are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol)
-
m
substrate
maintenance requirement (mol substrate/g biomass)
-
specific growth rate (h -1)
-
M
[methanol]/[mannitol] ratio in the nutrient
- N
part of mannitol that is assimilated when M=o
-
R
m
amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent
-
P/O
N
, P/O
F
, P/O
X
is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H +, FADH 2 and XH 2 to oxygen 相似文献
12.
Nitrate, nitrite and nitrous oxide were denitrified to N 2 gas by washed cells of Rhizobium japonicum CC706 as well as by bacteroids prepared from root nodules of Glycine max (L.) Merr. (CV. Clark 63). Radiolabelled N 2 was produced from either K 15NO 3 or Na 15NO 2 by washed cells of Rh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N 2O. Similar results were obtained with bacteroids of G. max. The stoichiometry for the utilization of 15NO
3
-
or 15NO
2
-
and the produciton of 15N 2 was 2:1 and for N 2O utilization and N 2 production it was 1:1. Some of the 15N 2 gas produced by denitrification of 15NO
3
-
in bacteroids was recycled via nitrogenase into cell nitrogen. 相似文献
13.
An almost stoichiometric conversion of nitrite to nitrous oxide was observed during the nitrite reduction by Paracoccus denitrificans cells in a medium of pH 6.4. The N 2O accumulated in the reaction medium and was decomposed only after nitrite had been consumed; when the pH of the medium was higher than 7.3–7.4, nitrous oxide did not accumulate. The activity of N 2O reductase was, in the whole range of pH 6.4–9.2, higher than the activity of NO −2 reductase, both activities showing the maximum at the pH higher than 8.0. Using an artificial donor, TMPD plus ascorbate, the maximum activity of NO −2 reductase, but not N 2O reductase was shifted by about two pH units to acidic region. The activity of nitrite reductase declined in the presence of N 2O only at higher pH values. Cytochrome c, as a common electron donor for both N 2O and NO −2 reductase, was more oxidized at pH < 7.3 in the presence of NO −2 than in the presence of N 2O, the opposite being true at pH > 7.3. The increased flux of electrons to cytochrome c has for a constant pH value (6.4) no effect on their distribution over NO −2 and N 2O. The results indicate that the distribution of electrons in the terminal part is determined by the different pH optima for NO −2 reductase and N 2O reductase, and by a mutual dependence of activities of the two reductases due to the competition for redox equivalents from a substrate. 相似文献
14.
Despite its ecological importance, essential aspects of microbial N2O reduction—such as the effect of O2 availability on the N2O sink capacity of a community—remain unclear. We studied N2O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N2O and O2 can occur, (ii) the mechanism governing the competition for N2O and O2, and (iii) how the N2O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N2O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O2. When supplied simultaneously, N2O reduction to N2 was only detected when the O2 concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N2O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (KS) for O2 was found to be two orders of magnitude lower than the value for N2O, explaining the preferential use of O2 over N2O under most environmentally relevant conditions. 相似文献
15.
Summary Experiments with T. denitrificans have shown that this bacterium cannot develop in media devoid of ammonium salts, and that iron is required for growth. Pure cultures have been isolated by means of a modified medium which permits rapid growth in serial transfers. The organism has been characterized as an obligatory chemoautotroph which can oxidize a number of inorganic sulfur compounds (elementary sulfur, thiosulfate, tetrathionate) either aerobically with O 2, or anaerobically with nitrate as oxidant.Nitrite was found to be highly toxic to the nitrate-reducing enzyme system; concentrations as low as 3.5 · 10 -4 m inhibit denitrification in the presence of sulfur about 40%. In the presence of thiosulfate, nitrite is rapidly decomposed to NO; this gas can subsequently be used as an oxidant, and is reduced to N 2. The formation of NO depends on the presence of both thiosulfate and nitrite.With resting cell suspensions CO 2 assimilation has been established; the assimilation products are not merely carboxylation products, as demonstrated in experiments with C 14O 2. 相似文献
16.
Escherichia coli K12 reduces nitrous oxide stoichiometrically to molecular nitrogen with rates of 1.9 mol/h x mg protein. The activity is induced by anaerobiosis and nitrate. N 2+formation from N 2O is inhibited by C 2H 2 ( K
i 0.03 mM in the medium) and nitrite ( K
i=0.3 mM) but not by azide. A mutant defective in FNR synthesis is unable to reduce N 2O to N 2. The reaction in the wild type could routinely be followed by gas chromatography and alternatively by mass spectrometry measuring the formation of 15N 2 from 15N 2O. The enzyme catalyzing N 2O-reduction in E. coli could not be identified; it is probably neither nitrate reductase nor nitrogenase. E. coli does not grow with N 2O as sole respiratory electron acceptor. N 2O-reduction might not have a physiological role in E. coli, and the enzyme involved might catalyze something else in nature, as it has a low affinity for the substrate N 2O (apparent K
m3.0 mM). The capability for N 2O-reduction to N 2 is not restricted to E. coli but is also demonstrable in Yersinia kristensenii and Buttiauxella agrestis of the Enterobacteriaceae. E. coli is able to produce NO and N 2O from nitrite by nitrate reductase, depending on the assay conditions. In such experiments NO
inf2
sup-
is not reduced to N 2 because of the high demand for N 2O of N 2O-reduction and the inhibitory effect of NO
inf2
sup-
on this reaction.Dedicated to Professor L. Jaenicke, Köln, on the occassion of his 70th birthday 相似文献
17.
Nitrosomonas europaea and Nitrosovibrio sp. produced NO and N 2O during nitrification of ammonium. Less then 15% of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N 2O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 M) inhibited the production of NO and N 2, but not of nitrite indicating that NO and N 2O were not produced during the oxidative conversion of ammonium to nitrite. NO and N 2O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N 2O. 相似文献
18.
Campylobacter sputorum subspecies bubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate and L-lactate are used as hydrogen donors. Cells of C. sputorum grown with nitrate or nitrite contain cytochromes of the b-and c-type and a carbon monoxide-binding cytochrome c. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate or L-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H +/O values and H +/NO
2
-
values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) and L-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H +/2e value of 2.0. The H +/O and H +/NO
2
-
values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP
carbonylcyanide-m-chlorophenylhydrazone
- HQNO
2-n-heptyl-4-hydroxyquinoline-N-oxide
- MTPP +
methyltriphenylphosphonium cation
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine; H +/O (H +/NO
2
-
), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO
2
-
); H +/2e (q +/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor 相似文献
19.
- The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
- Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
- Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
- Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
- From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.
相似文献
20.
Denitrification potentials of epilithic microbial populations were assessed using the acetylene inhibition method, in which acetylene is used to block the reduction of nitrous oxide (N 2O) to nitrogen (N 2). Samples of the epilithic community were incubated in filtered river water containing modified Bushnell-Haas salts, glycerol, and yeast extract—under aerobic (0.2 atm O 2) and anaerobic (0.2 atm He) acetylene atmospheres. N 2O was produced under both atmospheres only if exogenous nitrate of nitrite was added. Denitrification potentials were typically higher when nitrite was the added electron acceptor. The rates of denitrification were temperature-and carbon-dependent and the maximum rate, 8.53 g N 2O–N per cm 2 per day occurred at 23°C when nitrite was the electron acceptor. 相似文献
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