Cells Of Paracoccus Denitrificans Research Articles

Removal of nitrate from water by cells of Paracoccus denitrificans in a membrane flow reactor.

A new type of flow bioreactor designed to remove nitrate from water was developed. Denitrification activity of native Paracoccus denitrificans cells was used, the cells being separated from the refined medium by a semipermeable membrane. Relationships between the degree of nitrate conversion and the denitrification rate, on the one hand, and the volume flow rate and the amount of biomass, on the other, together with the results concerning denitrification during closed-circuit recirculation of the medium are discussed.

Cytochrome o ( bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild-type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc 1 complex by myxothiazol. Coupled to this activity, which involves only b-type cytochromes, there is translocation of 1.5–1.9 H +/e − across the cytoplasmic membrane. Similar H + translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa 3 to which it is structurally related.

The release of nitric oxide from denitrifying cells of Paracoccus denitrificans by an uncoupler is the basis for a new oscillator

Micromolar concentrations of the uncoupler 3-chlorophenylhydrazonepropanedinitrile caused the release of nitric oxide from the cells of Paracoccus denitrificans respiring nitrite. At the same time, a new component with an absorption maximum at 573 nm could be observed in the difference spectra. Damped oscillations of the concentrations of the cytochrome c-NO complex were observed during the trapping of released NO by added mammalian cytochrome c; reduced cytochrome c accumulated simultaneously in the reaction mixture.

Insights into the cell envelope of Paracoccus denitrificans, a member of the α-subdivision of purple bacteria, through studies of its lysozyme susceptibility

Exponential-phase Paracoccus denitrificans cells grown in a complex medium are known to be lysozyme susceptible without pretreatment with outer membrane perturbing agents. Stationary-phase cells are more lysozyme resistant, but simple washing of these cells with moderate concentrations (154 mM) of monovalent inorganic cations (NaCl, KCl, or LiCl), or 100 mM Tris(hydroxymethyl)aminomethane-HCl (Tris–HCl) rendered them lysozyme susceptible. Subsequent washing with divalent cations reversed the enhanced lysozyme susceptibility. Cells grown in succinate–salts medium were not rendered lysozyme susceptible by NaCl washing. Supplementation of complex medium with various salts used in succinate–salts medium showed that Mg2+ and Ca2+ supplementation resulted in increased growth yields and in cells that were not rendered lysozyme susceptible by NaCl washing. Measurement of the Mg2+ and Ca2+ content of peptone and yeast extract revealed Mg2+ and Ca2+ were present at 50–80 and 15–30 μM concentrations, respectively, in complex medium. Omission of Ca2+ or reduction of Mg2+ from 810 to 50 μM in succinate–salts medium resulted in cells that became lysozyme susceptible after NaCl washing. Incubation of cells grown in succinate–salts medium with Tris–HCl – ethylenediaminetetraacetate – sucrose caused them to become lysozyme susceptible, indicating that their lack of lysozyme susceptibility was due to a more effective outer membrane barrier than that of complex medium grown cells, rather than a lysozyme-resistant peptidoglycan. The protein composition of outer membranes from complex and complex + Mg2+ + Ca2+ grown cells was similar, suggesting that divalent cations did not have a major influence on protein production related to outer membrane stability. The results indicate that certain complex bacteriological media may be deficient in divalent cations for maximum cell envelope stability and growth yield. This might be particularly significant for soil bacteria such as P. denitrificans, which may have evolved to have outer membranes more dependent on divalent cations for structural integrity than animal host adapted bacteria such as enteric bacteria. The results also draw attention to NaCl as an outer membrane perturbing agent.

Denitrifikace vody imobilizovanými buňkami Paracoccus denitrificans.

Denitrifikace vody imobilizovanými buňkami Paracoccus denitrificans.

Aerobic dissimilatory reduction of nitrite by cells of Paracoccus denitrificans: the role of nitric oxide

With anaerobically grown cells of Paracoccus denitrificans it was previously found (Kučera, I. and Dadák, V. (1983) Biochem. Biophys. Res. Commun. 117, 252–258) that, in the presence of an uncoupler, nitrite as terminal acceptor was preferred to oxygen, the consumption of which was simultaneously inhibited. In the present study it is shown that besides an increased inhibition of terminal oxidases brought about by NO 2 − anion another potent inhibitor originating in the course of nitrite reductase reaction affects the division of electron flow between oxygen and nitrite. The inhibitor, the creation of which is accompanied by the aerobic nitrite reduction, is formed, even in the absence of an uncoupler, only as a result of a slight inhibition of oxygen respiration exhibited by hydroxylamine addition. From the comparison of the inhibitory effect of the intermediate on aerobically grown cells and membrane vesicles derived from them, it was proved that at neutral pH this substance does not carry an electric charge. A complex absorbing at 563 nm was formed due to the interaction of the inhibitor (generated from nitrite in the presence of uncoupler) with ferricytochrome c from bovine heart. From these findings we were led to conclude that it was most probably nitric oxide (NO).

Isolation and characterization of ubiquinol oxidase complexes from Paracoccus denitrificans cells cultured under various limiting growth conditions in the chemostat.

To obtain more information about the composition of the respiratory chain under different growth conditions and about the regulation of electron-transfer to several oxidases and reductases, ubiquinol oxidase complexes were partially purified from membranes of Paracoccus denitrificans cells grown in carbon-source-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. The isolated enzymes consisted of cytochromes bc1, c552 and aa3. In comparison with the aerobic ubiquinol oxidase complex, the oxygen- and nitrate-limited ones contained, respectively, less and far less of the cytochrome aa3 subunits and the anaerobic complex also contained lower amounts of cytochrome c552. In addition, extra haem-containing polypeptides were present with apparent Mr of 14,000, 30,000 and 45,000, the former one only in the anaerobic and the latter two in both the anaerobic and oxygen-limited preparations. This is the first report describing four different membrane-bound c-type cytochromes. The potentiometric and spectral characteristics of the redox components in membrane particles and isolated ubiquinol oxidase fractions were determined by combined potentiometric analysis and spectrum deconvolution. Membranes of nitrate- and oxygen-limited cells contained extra high-potential cytochrome b in comparison with the membranes of aerobically grown cells. No difference was detected between the three isolated ubiquinol oxidase complexes. Aberrances with already published values of redox potentials are discussed.

Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat.

Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials. (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV. The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550. In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones. Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present. Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions.

Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans

An NADH dehydrogenase complex was isolated from the plasma membranes of aerobically grown Paracoccus denitrificans cells by extraction with NaBr and purification on an NAD-agarose column. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase complex was about 10 times higher than that of the NaBr extract. The preparation was composed of 10 (6 major and 4 minor) unlike polypeptides, and lacked identifiable components and activities characteristic of other enzyme complexes of the oxidative phosphorylation system. The purified enzyme contained noncovalently bound FMN, nonheme iron, and acid-labile sulfide. The ratio of FMN to nonheme iron to acid-labile sulfide was 1:13~14:11~12, suggestive of the presence of multiple iron-sulfur clusters. The isolated NADH dehydrogenase complex cross-reacted with antisera to beef heart mitochondrial complex I and protein fraction derived therefrom, indicating the presence in the Paracoccus enzyme of antigenic sites similar to those in the intact complex I and its iron-sulfur protein and possibly hydrophobic protein fractions.

The inhibitory effect of nitrite on the oxidase activity of cells of Paracoccus denitrificans

It was observed with anaerobically grown cells of Paracoccus denitrificans which possess dissimilatory nitrite reductase that on increasing the flux of electrons into the terminal part of the respiratory chain by means of N, N, N', N-tetramethyl- p-phenylenediamine (TMPD) plus ascorbate the sensitivity of the oxidase activity towards the inhibitory action of nitrite markedly increased. It was shown that under these conditions the reduction of nitrite gave rise to the formation of an agent strongly inhibiting the terminal oxidases, the presence of which was detected using aerobically grown cells or membrane vesicles derived from them. The strong inhibition of TMPD-oxidase activity of membranes was transient in time and could also be brought about by addition of nitric oxide (NO) but not by nitrous oxide (N 2O).

Anaerobic adaptation ofParacoccus denitrificans: Sequential formation of denitrification pathway and changes in activity of 5-aminolevulinate synthase and catalase

Aerobically grown cells ofParacoccus denitrificans exhibited a marked increase in the activity of 5-aminolevulinate synthase in the early stage of adaptation to the decreased aeration of the growth medium. The effect was dependent on the functional proteosynthesis; it was not influenced by the presence of nitrate in the medium. An enhancement of nitrate reductase activity could be observed simultaneously; the rise of soluble cytochromec content was somewhat retarded. A significant lag phase of about 1 h appeared in the formation of nitrite reductase, which coincided with the shap drop in the catalase activity of the bacterium.

The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

(1) The apparent K m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K m (NO 3 −) is obtained. It is suggested that the very low K m (NO 3 −) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN − > NO 3 −, NO 2 −, pyruvate, acetate > CI − > SO 4 2−. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO 2 and CH 3COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO −) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.

The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans

Mucidin similar to antimycin inhibits the electron flow to cytochrome c and the enzyme activities dependent on cytochrome c reduction in the cells of Paracoccus denitrificans, but it does not inhibit the electron flow to nitrate reductase and cytochrome o. Unlike antimycin mucidin does not permit a residual electron flow through the cytochrome bc 1 region. In the presence of antimycin the electron flow to nitrate is lower than in using mucidin in contrast with a higher extent of cytochrome b reduction. This result is in contradiction to the participation of the constitutive cytochrome b as an electron donor in the nitrate reduction.

The puzzle of high phosphorylation potential and low protonmotive force generated by oxidative phosphorylation in Paracoccus denitrificans membrane vesicles in the presence of nitrate

The puzzle of high phosphorylation potential and low protonmotive force generated by oxidative phosphorylation in <i>Paracoccus denitrificans</i> membrane vesicles in the presence of nitrate

The effect of uncoupler on the distribution of the electron flow between the terminal acceptors oxygen and nitrite in the cells of Paracoccus denitrificans

The preferential utilization of oxygen, the terminal acceptor, in anaerobically grown cells of Paracoccus denitrificans was abolished in the presence of uncoupler (3 μM carbonyl cyanide m-chlorophenylhydrazone) which brought about a switch to the reduction of nitrite. It has been proved by measuring the redox state of cytochromes that this effect is due to the inhibition of the electron flow to oxygen caused by nitrite, which attains the site of its inhibitory action when the membrane potential is lowered.

Effects of electron transport inhibitors and uncouplers on denitrification inParacoccus denitrificans

Gaschromatographic analysis shows that whole cells of Paracoccus denitrificans produce dinitrogen in the absence and nitrous oxide in the presence of thiocyanate during nitrate reduction. NADH nitrate reductase activity in vesicles is much more sensitive to thiocyanate than either NADH oxidase activity in vesicles or reduction of nitrate by endogenous substrates in whole cells. NADH nitrate reductase activity is not inhibited and NADH oxidase activity is partially inhibited by antimycin A in vesicles. Production of nitrous oxide from nitrate in cells is completely inhibited by the simultaneous presence of thiocyanate and Triton X-100. Carbonylcyanide m-chlorophenylhydrazone does not cause a lag phase in reduction of nitrate by NADH in vesicles, in contrast to the situation in cells.

The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors.

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite. Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50 = 15 microM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.

Electron pathways from H2 to nitrate in Paracoccus denitrificans. Effects of inhibitors of the UQ-cytochrome b region

Electron flow from hydrogenase to nitrate reductase, in membranes of Paracoccus denitrificans cells grown autotrophically with nitrate as terminal electron acceptor, is inhibited by 2,5-dibromothymoquinone (DBMIB), 2,4-dinitrophenylether of iodonitrothymol (DNP-INT), 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), and trifluraline, but is insensitive to antimycin A. A scheme of anaerobic electron flow from H2 to nitrate is proposed with inhibition sites for various compounds known to act in the ubiquinone-cytochrome b region.

Energetic aspects of growth of Paracoccus denitrificans: oxygen-limitation and shift from anaerobic nitrate-limination to aerobic succinate-limitation

1. Growth yields and efficiency of energy conservation were the same for aerobic succinate-limited and oxygen-limited cells of Paracoccus denitrificans. 2. A shift from anaerobic nitrate-limitation to aerobic succinatelimitation showed that before and after the shift cells grew with the same capacity of energy conservation. 3. Respiration-driven proton translocation showed the presence of H+-translocating sites 1 and 2, which translocate respectively 2–3 and 4 protons per 2 electrons in oxygen-, anaerobic nitrate-and aerobic succinate-limited cells. 4. Cytochrome spectra and flash-photolysis spectra of oxygen- and nitrate-limited cells gave evidence for the presence of an alternative oxidase, cytochrome a1, never before recognized in Paracoccus denitrificans. 5. Only a-type cytochromes liganded with CO could be flash-photolysed. No evidence for a functional cytochrome o was found in photolysis experiments. 6. Fast oxidation, before photolysis, of the bc-pool after introduction of oxygen in a CO-liganded sample at-20° to-30° C, indicated the presence of a cytochrome oxidase other than cytochrome a1 with a very high affinity for oxygen and a low affinity for CO. 7. In photochemical action spectra, light released CO-inhibition of respiration, but the release was independent of the wavelength used (560–610 nm).

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa 3 and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.