Membrane Deenergization by Colicin K Affects Fluorescence of Exogenously Added but Not Biosynthetically Esterified Parinaric Acid Probes in Escherichia coli

Abstract

Fluorescence of the conjugated polyene fatty acid, parinaric acid (PnA), was studied in membranes of Escherichia coli during deenergization by colicin K. The free fatty acid and biosynthetically esterified forms of cis -PnA (9,11,13,15- cis,trans,trans,cis -octadecatetraenoic acid), both of which are sensitive to E. coli lipid-phase transitions, were compared. When free cis -PnA was added exogenously to respiring bacteria, dissipation of the energized state of the membrane resulted in a dramatic increase in cis -PnA fluorescence; all-trans -PnA was much less sensitive. Neither spectral shifts nor a change in cis -PnA fluorescence polarization were observed. Analysis of the PnA content of extracellular fractions of deenergized and control cells revealed a difference in probe distribution: the membranes of energy-poisoned E. coli bound about 77% of exogenously added cis -PnA, whereas membranes of actively respiring controls bound only about 44%. No fluorescence enhancement was observed in cells centrifuged to remove unbound cis -PnA before colicin treatment. When cis -PnA was biosynthetically esterified to phospholipids of an unsaturated fatty acid auxotroph of E. coli , the fluorescence did not change during membrane deenergization. In double-probe experiments, membrane deenergization resulted in fluorescence enhancement of exogenously added N -phenyl-1-naphthylamine, without change in esterified PnA fluorescence. We conclude that deenergization of E. coli membranes leads to increased binding and fluorescence of exogenously added PnA and cannot be detected from within the inner and outer membranes by PnA esterified in vivo.