Leoš Doskočil

Polyhydroxyalkanoates in Bacterial Cells - More Than just Storage Materials

Since polyhydroxyalkanoates (PHAs) belong among the most widespread storage materials within bacteria genus, it can be assumed that the ability of PHAs accumulation represent significant advantage in the natural environments. Of course, probably the most obvious is the possibility to utilize PHAs when external carbon sources are depleted. Nevertheless, it is likely that PHAs play much more complex role in the stress response of bacteria. Therefore, the aim of this work was to investigate possible influence of PHA accumulation in bacteria on physical properties of the cells and their cytoplasm with respect to possible stress survival. Cells of bacteria Cupriavidus necator H16 with various poly(3-hydroxybutyrate) (PHB) content (25 – 91 % of cell dry weight) were subjected to analytical centrifugation employing LUMiSizer. We observed that slope of index instability of bacterial culture increased with rise of PHB content in cells. It indicates that accumulation of PHB granules in bacterial cells affects overall physico-mechanical properties of the cells in particular sedimentation potential and density which might influence their behavior in natural environment such as sedimentation, surface absorption and subsequent biofilm formation. Furthermore, when investigated by Cryo-SEM, the PHB containing cells showed needle-type plastic deformations while these structures were absent in the cells without polymer. This suggests that native intracellular PHB granules reveal completely different mechanical and physico-chemical properties than any other component of bacterial cytoplasm and their flexibility even in deeply-frozen state is significantly higher than that of PHB isolated from bacterial cells. Based on these observations, it can be expected that, aside from their involvement in metabolism, presence of PHB granules dramatically changes physico-mechanical properties of cytoplasm and overall properties of cells which might represent important advantage when cells are exposed to stress conditions.

A study of zwitterionic/cationic vesicle formation and the influence of hyaluronan on this formation

The aggregation behavior of the biocompatible and naturally occurring zwiterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) combined with its synthetic cationic analogue 1,2-dipalmitoyl-3-trimetylammonium-propan (DPTAP) was studied. Further, the physical properties of the formed DPPC/DPTAP liposomes were determined. The phase transition temperature of DPPC/DPTAP mixtures was studied using the steady-state fluorescence of laurdan, and the results were compared with those obtained from microcalorimetry measurements. The phase transition temperature was higher for all DPPC/DPTAP mixtures compared to that for pure DPPC and DPTAP, this increase being particularly pronounced for equimolar mixtures of DPPC and DPTAP. Membrane fluidity was determined by means of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy measurements. While the ratio of DPPC in DPPC/DPTAP liposomes was increased, the results suggest the formation of more tightly packed membranes. The interaction of DPPC/DPTAP liposomes with hyaluronan (Hya) was also studied. The formation of complexes was observed at a specific DPTAP/Hya concentration ratio independently of DPPC concentration or the molecular weight of Hya.

Fluorescence Analysis of Cu(II), Pb(II) and Hg(II) Ion Binding to Humic and Fulvic Acids

Abstract. The aim of this work was to study molecular and quantitative aspects of metal ion binding to humic substances (HS). The object of our study was characterization of two standards of humic substances (Elliott Soil standard HA 1S102H and Elliott Soil standard FA 2S102F). All samples of IHSS standards HS were characterized by elemental analysis (EA), ultraviolet-visible spectroscopy (UV/Vis), Fourier transform infrared spectroscopy (FTIR) and steady-state fluorescence spectroscopy. Chemical parameters on the complexation of Cu (II), Pb(II) and Hg(II), including the conditional stability constants and the percentage of fluorophores participating in the complexation, were estimated by the modified Stern–Volmer equation. The stability constants (log Ka) of Me(II)–ESHS complexes range from 3.70 to 5.15 in the order: Hg–ESHA>Cu–ESHA>Pb–ESHA>Cu–ESFA>Pb–ESFA. With respect to the ESHA, ESFA, which showed the smallest contents of O-containing functional groups (e.g. hydroxyl, carbonyl, ester, especially carboxyl groups on the aromatic ring) and the lowest humification degree, the ESFA was characterized by much smaller stability constants. Our findings suggest that soil HS belongs to class of important organic ligands for complexation with heavy metal ions and may significantly affect the chemical forms, mobility, bioavailability and ecotoxicity of heavy metals in the soil environment.