Proteolytic activity of Staphylococcus aureus strains isolated from the colonized skin of patients with acute-phase atopic dermatitis.

Staphylococcus aureus strains isolated from the colonized skin lesions of 26 patients with acute-phase atopic dermatitis were reported to produce various extracellular proteolytic enzymes. Using the skim-milk-agar culture plating method, it was shown that 97% of the strains (65 of 67 examined) produced proteolytic activity, with 61% (42 strains) producing activity comparable to that of the proteolytically hyperactive reference strain Staphylococcus aureus V8. This observation was confirmed by azocasein degradation with culture supernatants, which indicated that 91% of the strains produced extracellular proteinases and 43% exceeded the 2% activity threshold of the reference strain. Control strains were isolated from the nose vestibules of 18 healthy carriers; the proteolytic activity of these strains never exceeded 2.5% of the activity of the reference strain. In 54% of the patients examined ( n=14), the activity of the strains was higher than that determined for the isolates from the control group. The combined use of assays incorporating azocasein and a synthetic chromogenic substrate, N-CBZ-Phe-Leu-Glu- pNA, showed that two staphylococcal enzymes, Staphylococcus aureus metalloproteinase (SAMP) and Staphylococcus aureus serine proteinase (SASP), contributed to the total proteolytic activity released by the strains examined. The contribution of each of the two enzymes varied greatly between different isolates. The undamaged skin of the patients was not colonized with Staphylococcus aureus. The presence of several strains with atypical proteinase characteristics was also reported, suggesting the possible involvement of enzymes other than serine- and metallo-proteinases in the proteolytic activity of Staphylococcus aureus. Taken together, the results of the study imply that staphylococcal proteinases may contribute to the pathogenicity of atopic dermatitis.

Hexavalent chromium stimulation of riboflavin synthesis in flavinogenic yeast.

Flavinogenic yeast overproduce riboflavin (RF) in iron-deprived media. In optimal growth media supplemented with Fe, hexavalent chromium 'Cr (VI)' treatment led to elevated RF synthesis in all cases of 37 flavinogenic strains studied. The level of RF production exceeded the rate observed at iron-deficient conditions. At sublethal Cr concentrations the RF oversynthesis over time correlated well with the growth-inhibitory adaptational period as manifested by the prolonged lag phase. The consecutive logarithmic biomass growth was accompanied by a drop in RF biosynthesis. Cr (VI)-induced RF overproduction was not a result of cellular iron level decrease. The treatment of yeast with Cr (VI) led to the stimulation of GTP-cyclohydrolase and RF-synthase activities, the key enzymes of the RF biosynthesis pathway.

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha. An application to real wastewater treatment.

The application of methylotrophic yeast Hansenula polymorpha to the treatment of methanol and formaldehydecontaining wastewater was experimentally verified. A variety of real wastewater samples originating from chemical industry effluent were examined. The yeast cell culture could grow in the wastewater environment, revealing low trophic requirements and a very high adaptation potential to poor cultivation conditions. The proliferation of cells was accompanied by a concomitant xenobiotic biodegradation. Grown, preadapted cellular suspension at a density of about 1 x 10(7) cells/ml proved to be able to utilize formaldehyde present in wastewater at concentrations up to 1750 mg/l, levels toxic to most microorganisms. The biological waste treatment method presented shows the enhanced potential by means of specific enzymatic activities of monocarbonic compound oxidations through methylotrophic pathway reactions. The need to obtain mutants highly resistant to formaldehyde has also been rationalized.

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha in a model wastewater system.

In search of the optimal way to reduce the hazards of environmental contamination by formaldehyde (FD) and methanol the use of unconventional yeasts is proposed as exemplified by the methylotrophic yeast Hansenula polymorpha. In a very simplified environment of a model wastewater solution, H. polymorpha cells were able to grow on, and metabolize formaldehyde and methanol, applied as sole carbon sources, at concentrations typical for wastewaters of the chemical industry. Several experimental conditions were tested for cell growth and biodegradation kinetics. It was found that the yeast culture inoculated at low cell density was able to grow on initial FD levels up to 400mg/l and the biomass yield was dependent on both, the amount of total carbon added and the physiological state of the cells. When high density of pre-adapted cell culture was used, the methylotrophs were fully viable and able to degrade formaldehyde present at initial concentrations up to 700 mg/l. The maximum limiting FD consumption rate was determined as approx. 400 mg/1 per hour. Methanol, at concentrations up to 2%, was easily utilized and did not have a negative effect on cell growth and respiration. It is suggested that in real wastewaters the eukaryotic microorganisms--in contrast to bacteria--might reveal greater adaptation potential to toxic levels of formaldehyde as well as to other wastewater constituents.

Effect of Staphylococcus aureus serine proteinase on the respiratory burst in phagocytic cells in vitro.

The in vitro effects of the Staphylococcus aureus serine proteinase (SASP) on the respiratory burst of human blood mononuclear phagocytes and rat lung macrophages were investigated. The generation of reactive oxygen species (ROS), determined by means of luminol-based chemiluminescence, was stimulated by treatment with SASP in both types of the defense cells. Cell activation depended on the concentration of the enzyme and the response of monocytes was an order of magnitude stronger relative to macrophages. The chemiluminescence emission kinetics were different for both cell types and the maximum signal was achieved in approximately 3 and 17 min, respectively. In experiments involving further cell activation by latex particles, macrophages pretreated with various SASP concentrations reacted with enhanced ROS generation whereas for monocytes, the latex-induced chemiluminescence was weakened by the enzyme. The results concerning the modification of the phagocytic host cell activity by SASP suggest that this enzyme might play an important role in pathomechanisms of staphylococcal infections in vivo.

Fluorescence and phosphorescence study of Tet repressor-operator interaction.

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O1 and tet O2 operators. Binding of Tet R to tet O1 operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O2 operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O1 and that with the O2 operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 x 10(9) M(-1) sec(-1). In contrast to the Tet R-tet O2 complex, the Stern-Volmer acrylamide quenching constant Ksv of the complex with tet O1 operator changes from 7.5 M(-1) at 5 mM NaCl to 22 M(-1) at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0-0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0-0 vibronic bands of Trp 43 to 413 and 412 nm for tet O1 and tet O2 operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0-5.5 to 3.5-3.3 sec. The thermal phosphorescence quenching profile ranged from -200 degrees C to -20 degrees C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O1 and tet O2 operators.

Tet repressor-tetracycline interaction.

Previous studies [Wasylewski et al. (1996), J. Protein Chem. 15, 45-58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7 x 10(9) M-1 sec-1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%). Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg.tc]+ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.

A fluorescence quenching study on protoporphyrin IX in a model membrane system.

The interaction of protoporphyrin IX (3,7,12,15-tetramethyl-8, 13-divinyl-2,18-porphyrine-dipropionic acid) (PPIX) with unilamellar dimyristoyl-L-alpha-phosphatidylcholine (DMPC) phospholipid vesicles has been studied by means of steady-state fluorescence quenching spectroscopy. The method of fluorescence-quenching-resolved spectroscopy has been applied in order to resolve the complex emission spectrum of a membrane-bound PPIX into two component spectra, attributed to distinct fluorophore species with different accessibilities to the iodide quencher. It is shown that PPIX associated with liposomes exists in two different microenvironments. One part of the fluorophore is embedded inside the lipid bilayer and is inaccessible to iodide. Its fluorescence spectrum exhibits the maximum characteristic of protoporphyrin found in the apolar medium. The other fraction of PPIX is located near the membrane surface, close to the polar phospholipid heads. Its emission is blue-shifted, resembling that of PPIX in a polar environment. It is quenched by iodide, although it reveals significant shielding from the quencher as compared to a buffer PPIX solution. Fluorescence quenching using 1-oxyl-4-oxo-2,2,6,6-tetramethyl-piperidine (TEMPONE) does not discriminate between the two protoporphyrin species. However, the accessibility of protoporphyrin IX to this quencher is much lower in a liposome system than in water.

Fluorescence-detected polymerization kinetics of human alpha 1-antitrypsin.

The time dependence of the human alpha 1-antitrypsin polymerization process was studied by means of the intrinsic fluorescence stopped-flow technique as well as the fluorescence-quenching-resolved spectra (FQRS) method and native PAGE. The polymerization was induced by mild denaturing conditions (1 M GuHCl) and temperature. The data show that the dimer formation reaction under mild conditions was followed by an increase of fluorescence intensity. This phenomenon is highly temperature sensitive. The structure of alpha 1-antitrypsin dimer resembles the conformation of antithrombin III dimer. In the presence of the denaturant the polymerization process is mainly limited to the dimer state. The alpha 1-antitrypsin activity measurements confirm monomer-to-dimer transition under these conditions. These results are in contrast to the polymerization process induced by temperature, where the dimer state is an intermediate step leading to long-chain polymers. On the basis of stopped-flow and electrophoretic data it is suggested that both C-sheet as well as A-sheet mechanisms contribute to the polymerization process under mild conditions.

A fluorescence study of Tn10-encoded tet repressor.

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor from Escherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to the tet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant, kq equal to 2.6 x 10(9), 2.8 x 10(9), and 2.0 x 10(9) M-1 sec-1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85 x 10(9) and 0.28 x 10(9) M-1 sec-1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.

Fluorescence-quenching-resolved spectra of melittin in lipid bilayers.

The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.

Fluorescence-quenching-resolved spectra of fluorophores in mixtures and micellar solutions.

The application of a new fluorescence-quenching-resolved spectroscopic method [Wasylewski, Z., Koloczek, H. and Wasniowska, A. (1988) Eur. J. Biochem. 172, 719-724] for resolving fluorescence emission spectra of a mixture of fluorophores into components is described. Contrary to fluorescence lifetime measurements, in this method the overlapping spectra can be decomposed even if the components have similar or the same fluorescence lifetimes, but differ in bimolecular-rate-quenching constants. Using this technique, we have resolved the emission spectra of a two-component mixture of fluorescein and riboflavin, which have very similar fluorescence lifetimes. To illustrate the utility of this approach in the study of fluorophores in compartmentalized biological systems such as lipid bilayers, we have also used the method to resolve the emission spectra of a two-component mixture of fluorophores commonly used in biological studies which undergo partition between water and a micellar phase.