Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of kcat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.
- MeSH
- flavinadenindinukleotid metabolismus MeSH
- flavinmononukleotid metabolismus MeSH
- flaviny metabolismus MeSH
- FMN-reduktasa genetika metabolismus MeSH
- NADP MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans genetika metabolismus MeSH
- sekvence aminokyselin genetika MeSH
- terciární struktura proteinů MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC-MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe-4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.
- MeSH
- bakteriální proteiny biosyntéza genetika MeSH
- flavoproteiny genetika metabolismus MeSH
- mutace * MeSH
- oxidační stres účinky léků genetika MeSH
- Paracoccus denitrificans genetika metabolismus MeSH
- paraquat farmakologie MeSH
- proteomika MeSH
- regulace genové exprese u bakterií účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this work was to compare denitrification activity of three types of encapsulated biomass containing pure culture of Paracoccus denitrificans or Pseudomonas fluorescens or mixed culture of psychrophilic denitrifiers cultivated at 5 °C from activated sludge. The experiments were held with synthetic wastewater containing 50 mg L(-1) N-NO(3)(-) under the temperature 15, 10, 8 and 5 °C. Specific denitrification rates related to the weight of pellets and to the protein content were calculated and the temperature coefficients describing the dependence of denitrification rate on the temperature were determined. Although the mixed culture showed the highest denitrification rate at the temperatures below 10 °C, using of pellets containing pure culture is recommended as the mixed culture has slow growth rate and its activity at temperatures above 10 °C is very low.
- MeSH
- čištění vody metody MeSH
- denitrifikace fyziologie MeSH
- měření biologické spotřeby kyslíku MeSH
- nízká teplota MeSH
- odpad tekutý - odstraňování metody MeSH
- Paracoccus denitrificans metabolismus MeSH
- Pseudomonas fluorescens metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
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.
Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc(1) complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c(1) (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc(1) content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.
- MeSH
- antibakteriální látky farmakologie MeSH
- antimycin A farmakologie MeSH
- buněčná membrána enzymologie metabolismus MeSH
- elektrony MeSH
- fyziologický stres MeSH
- koncentrace vodíkových iontů MeSH
- mutace MeSH
- operon MeSH
- osmotický tlak MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans metabolismus MeSH
- respirační komplex III MeSH
- sukcinát: cytochrom c oxidoreduktasa MeSH
- teplota MeSH
The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.
- MeSH
- antibakteriální látky metabolismus MeSH
- antimycin A metabolismus MeSH
- bakteriální proteiny genetika chemie metabolismus MeSH
- cytochromy c genetika chemie metabolismus MeSH
- financování organizované MeSH
- inhibitory enzymů metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans chemie metabolismus MeSH
- respirační komplex III genetika chemie metabolismus MeSH
- statická elektřina MeSH
Enzymatic activity (denitrification) of Paracoccus denitrificans was estimated electrochemically by reduction of duroquinone (DQ). Graphite electrodes covered with whole bacterial cells behind a dialysis membrane were used for measurement. P. denitrificans reduce nitrate and/or nitrite under anaerobic conditions to nitrogen gas. DQ acts as an electron mediator. After donation of the electrons to the respiratory system of the bacteria, produced DQ is reduced to durohydroquinone on the electrode surface electrocatalytically. P. denitrificans were exposed to low-frequency magnetic field (10 mT, 50 Hz) for 24 min. In comparison with the control samples, the reduction peak of I-E curves that represent denitrification activity of the cells decreased significantly after magnetic field exposure. The decrease of the peak current was about 20%. The CFU-colony forming units-method was used to estimate the number of surviving bacteria. After 24 min exposure of 10 mT magnetic field P. denitrificans culture on electrode indicates 21% bacterial death.
- MeSH
- draslík metabolismus MeSH
- dusičnany metabolismus MeSH
- finanční podpora výzkumu jako téma MeSH
- nigericin farmakologie MeSH
- nitrátreduktasy antagonisté a inhibitory MeSH
- Paracoccus denitrificans metabolismus účinky léků MeSH
- paraquat chemie MeSH
- protonmotorická síla fyziologie účinky léků MeSH
- sodík metabolismus MeSH
