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PubMed2004

Expression profiles of polyhydroxyalkanoate synthesis-related genes in Paracoccus denitrificans.

A facultative methylotrophic bacterium, Paracoccus denitrificans can synthesize polyhydroxyalkanoate acids (PHA) from various alcohols. Recently, six genes, phaA, B, C, P, R, and Z, related to PHA synthesis have been cloned and characterized. PHA synthesis and the expression of phaA, B, C, P, R, and Z in P. denitrificans were examined at the transcriptional and translational levels under both nitrogen-sufficient and nitrogen-deficient conditions. The results showed that PHA synthesis is not regulated at the mRNA or protein level in phaA, B, and C. We also observed the condensation of acetyl-coenzyme A (acetyl-CoA) in the cells by high-performance liquid chromatography (HPLC). The results suggest that the amount of acetyl-CoA would regulate PHA synthesis. Finally, we discuss a possible regulation mechanism for PHA synthesis in P. denitrificans.

Journal Article↗
PubMed1992

Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Methanol and n-Amyl Alcohol by the Methylotrophic Bacteria Paracoccus denitrificans and Methylobacterium extorquens.

Strains of two types of methylotrophic bacteria, Paracoccus denitrificans and Methylobacterium extorquens, synthesized the copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when methanol and n-amyl alcohol were added together to nitrogen-limited medium. The composition of the copolyester differed considerably between the two strains: the copolyester from P. denitrificans was comparatively rich in 3-hydroxyvalerate (3HV). The 3HV content of the copolyester synthesized by this strain increased with increasing concentrations of n-amyl alcohol. Its maximum content was 91.5 mol% under the conditions used. In M. extorquens, the maximum 3HV content was limited to 38.2 mol%. Since n-amyl alcohol served as a substrate for a standard methanol dehydrogenase, the enzyme was proposed to oxidize both methanol and n-amyl alcohol in the first step of copolyester synthesis from these substrates by methanol-grown cells.

Journal Article↗
PubMed1993

Effect of Medium Composition on the Denitrification of Nitrate by Paracoccus denitrificans.

The course of denitrification of nitrate in static cultures of Paracoccus denitrificans was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by P. denitrificans is not stable and depends on the nutritional composition of the culture medium.

Journal Article↗
PubMed1994

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans.

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N(2)O and N(2) from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with N 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(2)O, and finally N(2)O 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.

Journal Article↗
PubMed1996

Growth-Associated Production of Poly(3-Hydroxyvalerate) from n-Pentanol by a Methylotrophic Bacterium, Paracoccus denitrificans.

Paracoccus denitrificans accumulated a polyester in its cells during growth on n-pentanol. The composition of the polyester varied during the cultivation: the level of the 3-hydroxyvalerate unit in the polyester increased, and eventually a homopolymeric poly(3-hydroxyvalerate) [P(3HV)] accumulated to an amount 22 to 24% of the cell dry weight. Growth-associated polyester synthesis was considerably affected by n-pentanol when its concentration was controlled at several levels. Maximum accumulation of the polyester was obtained at 0.02% (vol/vol). Physical and mechanical characteristics of the P(3HV) were determined and compared with those of other homo- and copolyesters. The P(3HV) was dextrorotatory and had number-averaged and weight-averaged molecular masses of 128,000 and 888,000 Da, respectively, with a rate of polydispersity of 6.93. The level of tensile strength of the P(3HV) was lower, and its extension to break was higher than that of the poly(3-hydroxybutyrate) homopolyester.

Journal Article↗
PubMed2000

Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel.

Tubular gel reactors containing Nitrosomonas europaea and Paracoccus denitrificans, which remove nitrogen from solutions through a process of nitrification and denitrification, require oxygen for ammonia oxidation, the first and rate-limiting step in the process. To accelerate ammonia oxidation, high concentrations of oxygen were applied to the reactors instead of air. Although a 50% O2:N2 gas mixture and pure oxygen were both toxic to free N. europaea cells, they actually accelerated ammonia oxidation by N. europaea immobilized within the tubular gel. Indeed, the rate of ammonia oxidation by a tube exposed to pure oxygen was twice that of one exposed to 20% O2. When the distribution of N. europaea cells within the tubes was investigated using a fluorescently-labeled antibody, colonies were found on the external surface of the tube exposed to 20% O2, but were located at a depth of 120-300 microm from the external surface in the case of the tube exposed to pure oxygen. The region between the external surface of the gel and the colonies apparently acted as a barrier, reducing the diffusion of oxygen and thus protecting the cells from oxygen cytotoxicity.

Journal Article↗
PubMed2001

Asymmetric reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate coupled with nitrate reduction by Paracoccus denitrificans.

We found that the asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate (EHB) by Paracoccus denitrificans could be induced by nitrate addition under anaerobic conditions. However, the addition of electron donors such as glucose, ethanol, and methanol together with nitrate did not stimulate EHB production from EAA. When the reaction was carried out under optimum conditions (cell concentration, 10 g-d.w.l(-1); EAA, 150 mM; NO3-, 100 mM), after 8 h of reaction 49 mM of EHB with an optical purity of 98.9% e.e. was produced. Furthermore, a fed-batch reaction was carried out with an intermittent addition of nitrate and EAA to reduce substrate inhibition. The linear reduction of EAA to EHB proceeded for 40 h after initiation of the reaction, and finally 124 mM of EHB with an optical purity of 88.7% e.e. was produced after 104 h.

Journal Article↗
PubMed1999

Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans.

The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S. (1997) Biochim. Biophys. Acta 1318, 202-216) does not involve the concentration of an electron donor. Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide. NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme. The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex.

Kinetics↗
PubMed1981

Removal of Paracoccus denitrificans outer membrane material by sodium chloride.

The effects of water washing and NaCl treatment on the cell surface of P. denitrificans were studied. Both treatments caused a release of material from cells. Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane. This treatment also increased the susceptibility of the organism to lysozyme. Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface. Both treatments were shown to alter the appearance of the cell surface. The disruptive effects of these procedures were found to be dependent upon the age of the culture.

Cell Membrane↗
PubMed1991

The three-subunit cytochrome bc1 complex of Paracoccus denitrificans. Its physiological function, structure, and mechanism of electron transfer and energy transduction.

The cytochrome bc1 complex purified from P. denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromes b, c1, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria. However, the bacterial bc1 complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary polypeptides, which have no obvious electron-transfer function. The P. denitrificans complex is a paradigm for the bc1 complexes of all gram-negative bacteria. In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, the P. denitrificans bc1 complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer.

Electron Transport↗
PubMed1989

Loss of penicillin resistance in Paracoccus denitrificans induced by mitomycin C and acridine orange.

In an attempt to eliminate the penicillin resistance gene of P. dentrificans by curing agents, such as acridine orange (AO) and mitomycin C, it was observed that AO treatment caused temporary phenotypic curing where development of sensitivity was a function of concentration of both the curing agent and benzylpenicillin. However, curing with mitomycin produced sensitive clones at a frequency of 6 X 10(-3) and two permanently cured clones were isolated. Heavy metal resistance and resistance to other drugs, however, remain unchanged in the mitomycin-cured isolate.

Acridine Orange↗