Igor Kučera

Unraveling an FNR based regulatory circuit in Paracoccus denitrificans using a proteomics-based approach.

The switch from aerobic to anaerobic respiration in the bacterium Paracoccus denitrificans is orchestrated by the action of three FNR-type transcription regulators FnrP, NNR and NarR, which are sensors for oxygen, nitric oxide and nitrite, respectively. In this work, we analyzed the protein composition of four strains (wild type, FnrP-, NNR- and NarR-mutant strains) grown aerobically, semiaerobically and semiaerobically in the presence of nitrate to discover the global role of FNR-family transcription regulators using proteomics, with data validation at the transcript and genome levels. Expression profiles were acquired using two-dimensional gel electrophoresis for 737 protein spots, in which 640 proteins were identified using mass spectrometry. The annotated 2-D proteome map provided the most comprehensive coverage of P. denitrificans proteome available to-date and can be accessed on-line at http://www.mpiib-berlin.mpg.de/2D-PAGE/. Our results revealed several types of regulation under the conditions tested: (1) FnrP-controlled regulation of nitrous oxide reductase, UspA and OmpW as confirmed at protein, transcript and DNA level (position of FNR boxes). (2) Proteins regulated via additional regulators, including proteins involved in NNR and NarR regulons: nitrate reductase beta-subunit, TonB-dependent receptors, nitrite reductase, a TenA-type transcription regulator, and an unknown protein with an alpha/beta hydrolase fold. (3) Proteins whose expression was affected mainly by the growth condition. This group contains SSU ribosomal protein S305 / sigma(54) modulation protein, and two short-chain reductase-dehydrogenase proteins.

A STUDY ON THE TRANSPORT AND DISSIMILATORY REDUCTION OF NITRATE IN PARACOCCUS DENITRIFICANS USING VIOLOGEN DYES AS ELECTRON DONORS

Abstract The controversial subject of nitrate transport into the denitrifying cells of Paracoccus denitrificans was studied employing methyl or benzyl viologen cation radicals as electron donors for the respiratory nitrate reductase. In intact cells, the oxidation of methyl viologen by nitrate was sensitive to low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone ( I 50 = 0.8 μ M). Treatment of cells with 0.1%v Triton X-100 (1) completely relieved the inhibition by the uncoupler, (2) raised the values of K m (NO 3 − ) and V m from 23 ± 5 μ M and 6.4 ± 0.3 nmol MV s −1 mg dw −1 to 120 ± 10 μ M and 13.5 ± 0.5 nmol MV s −1 mg dw −1 , respectively, and (3) changed the type of inhibition by azide from noncompetitive ( K i = 0.18 ± 0.01 μ M) to competitive ( K i = 0.18 ± 0.02 μ M) with respect to nitrate. On the other hand, permeabilisation had little effects on the activation energy ( ≈ 57 kJ mol −1 ) and on the maximal degree of inhibition by 2-heptyl-4-hydroxyquinoline- N -oxide (≈ 50%). Observations (1) and (2) suggest an active transport mechanism for nitrate, whereas (3) can be best explained by a simultaneous leakage of the accumulayed nitrate from the cells. Results from a HPLC analysis of cellular nitrate levels and their changes elicited by azide (an increase) or uncoupler (a decrease) are compatible with the occurrence of a cytoplasmic nitrate pool linking up nitrate transporter and nitrate reductase.

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

SummaryUbiquinone-10 (Q10) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O2 in the sparging gas). Whereas the cellular level of Q10 decreased monotonically from 1.2 to 0.5 μmol/g d.w., the total yield of Q10 was maximal at 2.5 % O2 and amounted to 350 nmol (0.3 mg) per L of culture.

Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c550

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1 ) has been purified from the anoxically grown cytochrome c550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N‐terminal amino acid sequence). Under our assay conditions, the half‐saturation of electron transport occurred at about 10 μM pseudoazurin; the reaction was retarded by increasing ionic strength.

The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans

Abstract 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 2 O 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 2 O 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 2 O reductase was shifted by about two pH units to acidic region. The activity of nitrite reductase declined in the presence of N 2 O only at higher pH values. Cytochrome c , as a common electron donor for both N 2 O and NO − 2 reductase, was more oxidized at pH − 2 than in the presence of N 2 O, 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 2 O. 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 2 O reductase, and by a mutual dependence of activities of the two reductases due to the competition for redox equivalents from a substrate.

The role of ubiquinone in linking nitrate reductase and cytochrome o to the respiratory chain of Paracoccus denitrificans

Abstract Three alternatives of the mode of branching in the ubiquinone-cytochrome b region of the anaerobic respiratory chain of Paracoccus denitrificans were experimentally tested. It was found that the view that the constitutive cytochrome b -560 or b -566 serves as an electron donor for the nitrate reductase is incompatible with the proposed scheme of the cyclic electron flow in the bc 1 segment. By means of the extraction procedure, the extent of reduction of ubiquinone was determined in cells utilizing oxygen and nitrate in the presence of antimycin. It was found that the redox response of ubiquinone was consistent with what had been predicted by the pool model of Kroger and Klingenberg, extended for more than one terminal acceptor. Our results are in support of the assumption that in cells of P. denitrificans ubiquinol (QH 2 ) has a function of an electron donor both for nitrate reductase and cytochrome o .

Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans

The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.

Detection, with a pH indicator, of bacterial mutants unable to denitrify.

In order to facilitate isolation of mutants with alterations in the denitrification pathway, a new screening procedure using phenol red incorporated into agar overlay has been defined. Alkalinization in the neighbourhood of denitrifying colonies respiring nitrate or nitrite gives rise to a red circular halo. Antimycin blocked these colour changes, which suggests their association with the periplasmic reduction of nitrite. Inhibition of nitrous oxide reductase by acetylene had no significant effect on alkalinization elicited by nitrate or nitrite. Several mutants negative by the phenol red staining test were generated by transposon Tn5 mutagenesis of Paracoccus denitrificans. All these mutants were defective in the activities of nitrite and nitric oxide reductases while the other denitrification activities were present at the wild-type level.

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.

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 microM 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.