carbocyanines and Autolysis

Quantifying fluorescent markers in cell populations using flow cytometry has been a powerful technological advance. Fluorescent properties of cyanine dyes coupled with flow cytometry allow investigators to monitor the membrane potential (MP), an important component of the proton motive force (PMF). MP (or ΔΨ) is the electrical potential across the cell membrane. The other component of the PMF is ΔpH, or the difference in interior and exterior proton concentrations. MP plays a critical role in bacterial physiology. In Staphylococcus aureus, MP is required for generation of ATP, regulating autolytic activity, maintaining ion homeostasis, and resistance to some classes of antibiotics. This protocol exploits unique spectral and physical properties of the cyanine-based molecule diethyloxacarbocyanine iodide, or DiOC, and flow cytometry technology to quantify MP in S. aureus. This assay has been used by researchers to define the electron transport chain of S. aureus as well as determine how intrinsic and extrinsic factors affect MP.

Topics: Autolysis; Bacterial Outer Membrane; Carbocyanines; Coloring Agents; Flow Cytometry; Membrane Potentials; Proton-Motive Force; Staphylococcus aureus

The effect of beta-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various beta-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus.

Topics: Anti-Bacterial Agents; Autolysis; Carbocyanines; Cefotaxime; Coloring Agents; Drug Interactions; Gramicidin; Ionophores; Membrane Potentials; Penicillin G; Streptomyces griseus