Jürgen Baier

The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria

New antibacterial strategies are required in view of the increasing resistance of bacteria to antibiotics. One promising technique involves the photodynamic inactivation of bacteria. Upon exposure to light, a photosensitizer in bacteria can generate singlet oxygen, which oxidizes proteins or lipids, leading to bacteria death. To elucidate the oxidative processes that occur during killing of bacteria, Staphylococcus aureus was incubated with a standard photosensitizer, and the generation and decay of singlet oxygen was detected directly by its luminescence at 1,270 nm. At low bacterial concentrations, the time-resolved luminescence of singlet oxygen showed a decay time of 6 ± 2 μs, which is an intermediate time for singlet oxygen decay in phospholipids of membranes (14 ± 2 μs) and in the surrounding water (3.5 ± 0.5 μs). Obviously, at low bacterial concentrations, singlet oxygen had sufficient access to water outside of S. aureus by diffusion. Thus, singlet oxygen seems to be generated in the outer cell wall areas or in adjacent cytoplasmic membranes of S. aureus. In addition, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. When singlet oxygen luminescence was measured at higher bacterial concentrations, the decay time increased significantly, up to ≈40 μs, because of oxygen depletion at these concentrations. This observation is an important indicator that oxygen supply is a crucial factor in the efficacy of photodynamic inactivation of bacteria, and will be of particular significance should this approach be used against multiresistant bacteria.

Refinement of the x-ray structure of the RC-LH1 core complex from Rhodopseudomonas palustris by single-molecule spectroscopy

A unique combination of single-molecule spectroscopy with numerical simulations has allowed us to achieve a refined structural model for the bacteriochlorophyll a (BChl a) pigment arrangement in reaction center–light-harvesting 1 core complexes of Rhodopseudomonas palustris. Details in the optical spectra, such as spectral separation and mutual polarizations of spectral bands, are compared with results from numerical simulations for various models of the BChl a arrangement that were all well within the 4.8-Å limit of the accuracy of the available x-ray structure. The experimental data are consistent with a geometry where 15 BChl a dimers, each taken homologous to those from light-harvesting 2 complex from Rhodopseudomonas acidophila, are arranged in an overall elliptical structure featuring a gap on the long side of the ellipse.

Epifluorescence, confocal and total internal reflection microscopy for single‐molecule experiments: a quantitative comparison

Epifluorescence, confocal and total internal reflection microscopy are the most widely used techniques for optical single‐molecule experiments. Employing these methods, we recorded the emission intensity of the same single molecule as a function of the excitation rate under otherwise identical experimental conditions. Evaluation of these data provides a quantitative comparison of the signal‐to‐background ratios that can be achieved for the three microscopic techniques.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center–light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX− strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

Spectral Diffusion and Electron-Phonon Coupling of the B800 BChl a Molecules in LH2 Complexes from Three Different Species of Purple Bacteria

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

Spectral diffusion of the lowest exciton component in the core complex from Rhodopseudomonas palustris studied by single-molecule spectroscopy

We have recorded fluorescence-excitation spectra from individual RC–LH1 complexes from Rhodopseudomonas palustris. The spectra feature a few broad bands accompanied by a sharp line at the low-energy side of the spectrum which is ascribed to the lowest exciton state of the BChl a assembly. Recording several fluorescence-excitation spectra from the same individual complex in rapid succession reveals that the linewidth of the lowest exciton transition is determined by spectral diffusion which increases for higher excitation energies.

The Influence of Symmetry on the Electronic Structure of the Photosynthetic Pigment-Protein Complexes from Purple Bacteria

The primary reactions of purple bacterial photosynthesis take place in two pigment-protein complexes, the peripheral LH2 complex and the core RC-LH1 complex. In order to understand any type of excitation-energy transfer in the LH system detailed knowledge about the correlation between the geometrical structure and the nature of the electronically excited states is crucial. The interplay between the geometrical arrangement of the pigments and the transition probabilities of the various exciton states leads to key spectral features, such as narrow lines, that are clearly visible with single-molecule spectroscopy but are averaged out in conventional ensemble experiments. Combining low-temperature single-molecule spectroscopy with numerical simulations has allowed us to achieve a refined structural model for the bacteriochlorophyll a (BChl a) pigment arrangement in RC-LH1 core complexes of Rps. palustris. The experimental data are consistent with an equidistant arrangement of 15 BChl a dimers on an ellipse, where each dimer has been taken homologeous to those from the B850 pigment pool of LH2 from Rps. acidophila.