Coexistence of Domains with Distinct Order and Polarity in Fluid Bacterial Membranes¶

Abstract

In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan-labeled Escherichia coli and temperature dependencies of both laurdan's GP and fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid-crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37 degrees C reflects membrane heterogeneity. Time-resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdan's emission. In the proteo-lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple-Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid-crystalline bacterial membrane: a lipid-enriched and a proteolipid domain. In bacteria with chloramphenicol (Cam)-inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature-dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam-induced domain dissipation.

Membrane-catalyzed Nucleotide Exchange on DnaA

“…However, there is an indication that clustering, rather than the concentration of acidic phospholipids, in such a membrane is essential for nucleotide dissociation (26). In the E. coli membrane, both structural and compositional heterogeneity were revealed (27,28). Domains containing cardiolipin were visualized at the cell poles (29), consistent with an enrichment of cardiolipin in minicells (30).…”

Genetic Basis of Evolutionary Adaptation by Escherichia coli to Stressful Cycles of Freezing, Thawing and Growth

“…To test whether the evolved cls À allele also changes membrane fluidity, we measured fluorescence anisotropy of the same four strain constructs after either FT treatment or stationary phase at 37°(see materials and methods for further details). Laurdan is a fluorescent probe that localizes to the cytoplasmic membrane and that is sensitive to changes in the water-lipid interface where the phospholipid head groups reside (Harris et al 2002;Vanounou et al 2002). The results of the experiments are summarized in Figure 5.…”

“…Measuring membrane fluidity: Fluorescence polarization (anisotropy) measures fluidity in the cytoplasmic membrane using various probes, each one specific to a particular region of the membrane (Harris et al 2002;Vanounou et al 2002). Fluorescence anisotropy was measured with a Spectramax M5 fluorometer (Molecular Devices, Menlo Park, CA) using two probes, 6-dodecanoyl-2-dimethylaminoaphthalene (laurdan) and 1,3-diphenyl-1,3,5-hexatriene (DPH).…”

“…Fluorescence anisotropy was measured with a Spectramax M5 fluorometer (Molecular Devices, Menlo Park, CA) using two probes, 6-dodecanoyl-2-dimethylaminoaphthalene (laurdan) and 1,3-diphenyl-1,3,5-hexatriene (DPH). Laurdan is an amphipathic molecule that localizes near the lipid polar head groups in the cytoplasmic membrane and is therefore sensitive to changes at the water-lipid interface (Harris et al 2002;Vanounou et al 2002). DPH is useful for detecting changes in saturation of fatty acyl chains in the cytoplasmic membrane (Aricha et al 2004;Beney et al 2004).…”

“…The ratio of probe to culture concentration was chosen by using the minimum concentration giving an appropriate signal-to-noise ratio (Vanounou et al 2002). Laurdan anisotropy was measured at the 355-nm excitation wavelength and 440-nm emission wavelength.…”