Affinity of Intact Escherichia Coli for Hydrophobic Membrane Probes is a Function of the Physiological State of the Cells

The fluorescence parameters of several common membrane probes in the presence of whole E. coli have been examined. The probes included electrically neutral lipophilic molecules N-phenyl-1-naphthylamine, pyrene, and 1,6-diphenyl-1,3,5-hexatriene as well as the negatively charged molecule 8-anilino-1-naphthalene sulfonate. It is demonstrated in each case that certain fluorescence parameters are a function of the state of energization of the cells. All the probes appear to monitor structural changes in the E. coli envelope which accompany the energization and de-energization of the cells. tthe phenomenon is completely reversible as demonstrated by re-energizing anoxic cells by the addition of oxygen, or starved cells by the addition of substrate. All the results are qualitatively consistent with an increased binding of probe by de-energized cells and a subsequent expulsion of probe when the cells are re-energized. A pyrene substituted with a photosensitive group, 1-azidopyrene, has been synthesized. Photolysis in the presence of a suspension of energized E. coli reveals a relatively small amount of probe irreversibly bound to the cells. However, in the presence of cells that have been de-energized the amount of irreversibly bound probe is dramatically increased. This molecule should be useful for localizing the regions of the bacterial envelope that are involved in the structural changes being monitored in these experiments.