Fe/Mn superoxide dismutase-encoding gene in Paracoccus denitrificans is induced by azide and expressed independently of the FNR-type regulators.

Paracoccus denitrificans cells undergo changes in protein composition upon exposure to azide, a known activator of the fumarate-nitrate reduction (FNR)-type transcription factor NarR. One of the most prominent protein species inducible by azide is a Fe/Mn-family superoxide dismutase (SOD). Azide induces SOD at protein, mRNA transcript, and enzyme activity levels in the aerobically growing cells. Since SOD expression remains unaffected in the fnrP-, nnr-, and narR-mutant strains, we postulate a mechanism independent of the known FNR-type regulators but involving a redox signal arising from the respiratory chain.

Nitrate reductase whole-cell assay: side effects associated with the use of benzyl viologen.

A comparative examination of reduced methyl [MV·](+) and benzyl [BV·](+) viologens (as artificial electron donors for quantitative estimation of the respiratory periplasmic (Nap) and membrane-embedded (Nar) nitrate reductases) using a newly constructed nap mutant strain of Paracocccus denitrificans was done. The activity with [MV·](+) was high in whole-cell assays, confirming that this compound donates electrons to Nar. Initial rates of the more lipophilic [BV·](+) were considerably lower, which was interpreted to be caused by an inhibition of the active transport of nitrate into the cells. Anionophoric activity of [BV·](+) was detectable but too low to effectively circumvent the inhibition of nitrate transporter.

Control of gene expression by FNR-like proteins in facultatively anaerobic bacteria.

Facultatively anaerobic bacteria are able to adapt to many different growth conditions. Their capability to change their metabolism optimally is often ensured by FNR-like proteins. The FNR protein of Escherichia coli functions as the main regulator during the aerobic-to-anaerobic switch. Low oxygen tensions activate this protein which is expressed constitutively and is inactive under aerobic conditions. The active form is dimeric and contains a [4Fe-4S]2+ cluster. The direct dissociation of the cluster to the [2Fe-2S]2+ cluster by the effect of oxygen leads to destabilization of the FNR dimer and to loss of its activity. The active FNR induces the expression of many anaerobic genes; the set comprises over 100 of controlled genes. Many other bacteria contain one or more FNR analogues. All these proteins form the FNR family of regulatory proteins. Properties of these proteins are very distinct, sometimes even among representatives of different strains of the same bacterial species. FNR-like proteins together with other regulators (e.g., two-component system ArcBA, nitrate-sensing system NarXL, etc.) control a complicated network of modulons that is characteristic for every species or even strain and enables fine tuning of gene expression.

[Analysis of the results of mycological examination of skin samples from 1997 to 1998]. / Analiza rezultata mikoloskih pregleda uzoraka koze u periodu od 1997. do 1998. godine.

Mycological findings of 2,447 skin specimens were analyzed. The aim of the study was to estimate the methodology for mycological examination, frequency of particular fungal species isolation and localization on the skin. Fungi were found in 591 specimens: 53.64% microscopically and isolated in cultures, 31.47% only microscopically and 14.89% only isolated in cultures. Total number of isolated strains was 405 (356 strains grew on Sabouraud agar with antibiotics and 272 on mycobiotic agar). The most frequently isolated species were: Trichophyton mentagrophytes (27.9%), Trichophyton rubrum (20%) and Candida albicans (9.14%). The most frequent localizations on skin where fungi had been found were foot (41.47%), toe-nail (22.06%) and finger-nail (10.88%). Sabouraud agar is more reliable for isolation of dermatophytes and mycobiotic agar for Candida species. Usage of both media for primoisolation provides better and faster identification of fungi causing dermatomycoses. Comparing our with other studies, the patterns of fungal appearance in our population had changed and the number of nail specimens, where fungi were found had increased.

The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase.

The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site-directed mutagenesis, and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat. C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino-acid residues of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.

Fungus-like hyphochytrids associated with human disease.

We report two cases, with liver and brain abscess, respectively, where fungus-like organisms belonging to the Hyphochytriomycota were found at the site of inflammation together with Peptococcus in the first and Cysticercus cellulosae in the second case. This is the first time these groups of organisms have been reported in human material. The role of hyphochytrids in human pathology remained uncertain as they were found together with already known human pathogens.

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.

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 microM pseudoazurin; the reaction was retarded by increasing ionic strength.

Evaluation of relative contributions of two enzymes supposed to metabolise hydrogen peroxide in Paracoccus denitrificans.

A biosensor exploiting an electrochemically mediated enzyme-catalysed reaction was used to quantify relative contributions of cytoplasmic catalase and periplasmic cytochrome c peroxidase to the overall rate of hydrogen peroxide breakdown in cells of Paracoccus denitrificans. The effects of antimycin (an inhibitor of electron flow to cytochrome c peroxidase), the reaction rate versus substrate concentration profiles for the whole cells and subcellular fractions, and the time courses of oxygen concentration demonstrated a profound decrease in the capacity of cytochrome c peroxidase to reduce H2O2 under in vivo conditions. The reason is suggested to be a competition for available electrons between the enzyme and terminal oxidases metabolising oxygen produced by catalase.

Iron as a possible mediator of the oxic-to-anoxic transition in Paracoccus denitrificans.

Expression of the lacZ gene from the Fnr-regulated FF-melR promoter on a plasmid in iron-deprived Paracoccus denitrificans cells required not only a decreased oxygen tension but also supplementation with iron. The levels of beta-galactosidase and 5-aminolevulinate synthase showed comparable responses to changes in iron availability. The presence of soluble and particulate enzymes catalyzing the reduction of Fe(III) by NADH suggests a hypothesis in which the redox state of the cytoplasmic NAD-couple determines the concentration of free Fe(II) and thereby modulates the activity of a common regulator of the Fnr type.

Oxygen increases the steady-state level of nitrate in denitrifying cells of Paracoccus denitrificans.

The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [14C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.

Determination of coenzyme Q in human plasma.

Coenzyme Q10 (CoQ10) levels in human plasma were determined by high-performance liquid chromatography (HPLC) with UV detection. CoQ10 was dissociated from lipoproteins by methanol and subsequently cleaned-up on silica gel and octadecyl silica solid-phase extraction cartridges. HPLC separation was performed on a C18 reversed-phase column. The methanol-hexane mobile phase provided a greater possibility of separation procedure adjustment allowing the shortest possible elution time without loss of resolution than a two-alcohol mobile phase. Quantitation was based on the peak heights using a standard addition method. The lower limit of detection was 8 ng on-column, corresponding to 90 micrograms ubiquinone per litre of plasma in an actual sample. Thirty-one randomly selected plasma samples from apparently healthy, 18 to 56-year-old individuals (males and females) were analyzed for total CoQ10. The average level in these subjects was 0.47 +/- 0.18 mg/l with the range of 0.26-1.03 mg/l. The method was also applied to the determination of ubiquinone plasma level changes in one healthy volunteer over a period of one month and after oral intake of CoQ10.

Respiratory inhibitors activate an Fnr-like regulatory protein in Paracoccus denitrificans: implications for the regulation of the denitrification pathway.

Unlike the uncoupler carbonyl cyanide 3-chlorophenyl-hydrazone, the respiratory inhibitors CN-, N3-, NO2- and rotenone enhanced the formation of nitrate and nitrite reductases in highly aerated cultures of the Paracoccus denitrificans ex-conjugant PD1222 (pRW2A/FF). A maximal effect was observed at concentrations partly blocking electron transport to O2. The level of beta-galactosidase reporting the activity of an Fnr-like regulatory protein showed a similar concentration dependency. It is concluded that oxygen is sensed by Fnr in an indirect way, possibly via the redox state of a cellular component.

Coordinate expression of the enzymes of nitric oxide metabolism in Paracoccus denitrificans.

Cellular activities of nitric oxide producing nitrite reductase (NiR) and nitric oxide reductase (NOR) in Paracoccus denitrificans increased in parallel upon anaerobic adaptation of aerobically grown cells. In addition, both activities exhibited comparable dependences on oxygen supply into the growing culture. The results suggest that the maintenance of a sufficiently high NOR/NiR activity ratio plays an important role in preventing nitric oxide accumulation and its toxic effects on the cells during denitrification.

Relationship between cardiolipin content and cytochrome c oxidase activity of cytochrome aa3 in Paracoccus denitrificans.

Using cultivation at different oxygen tensions, the molar ratio of cardiolipin to haem a in cells of Paracoccus denitrificans was varied systematically from 30 to 300. The molecular activity of cytochrome aa3 (with N, N, N', N'-tetramethyl-p-phenylenediamine as substrate) remained unchanged in this interval, this ruling out any regulatory effect of physiological cardiolipin levels on the terminal oxidase. Titration of anaerobically grown cells with cyanide indicated the presence of cytochrome aa3 which accounted for about 1/4 of the total electron flow from TMPD to oxygen.

Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans.

The metabolism of nitric oxide in Paracoccus denitrificans has been studied using a Clark-type electrode. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the SH reagent N-ethylmaleimide, both of which released nitric oxide from cells respiring nitrite, were found to be efficient inhibitors of nitric oxide reductase activity. Control experiments with another uncoupler, pentachlorophenol, showed that the inhibitory effect of CCCP was not the result of a decrease in membrane potential. The denitrification pathway in cells with partly inhibited nitric oxide reductase, or in a reconstituted system containing purified nitric reductase and membrane vesicles, exhibited marked sustained oscillations of nitric oxide concentration. The occurrence of the oscillations was strictly dependent on the initial concentration of nitrite. The observed oscillatory kinetics is considered to reflect two regulatory signals destabilizing the denitrification pathway, namely the inhibition of nitric oxide reductase by nitric oxide and/or by nitrite.

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.

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.

Determination of nitrate by conversion to nitrite using Paracoccus denitrificans.

A new method of determination of nitrate was developed, utilizing the nitrate reductase activity of Paracoccus denitrificans in which a further reduction of nitrate is blocked either by a mutation affecting formation of cytochromes c or by inhibition of the electron flow to nitrite reductase by mucidin. After deproteinization of the sample with zinc acetate the nitrite produced is determined colorimetrically.