Increased binding of a hydrophobic, photolabile probe to Escherichia coli inversely correlates to membrane potential but not adenosine 5'-triphosphate levels.

Abstract

We describe conditions for a quantitative determination of azidopyrene binding to Escherichia coli cells. In addition, we define conditions whereby irradiation of azidopyrene in the presence of cells leads to irreversible association of probe with celLs. This is presumably due to the light-dependent generation of reactive nitrenes and subsequent incorporation of nitrenopyrene moieties into cellular components. These methods allowed us to determine that the amount of azidopyrene bound to cells was inversely correlated with the magnitude of the cellular membrane potential, but was not correlated with high or low adenosine 5'triphosphate levels per se. Cells bound more azidopyrene if the AA' was low. Cellbound azidopyrene was found to be entirely associated with the inner and outer membrane. We suggest that the decreased association of hydrophobic probes upon energization of whole cells reflects a rapid transition in structural properties of the cell envelope.

Photochemistry of phenyl azide: chemical properties of the transient intermediates

“…(2) (3) advantage of the unusually high reactivity of short-lived intermediates formed when these azides are irradiated with ultraviolet light. Our objective is to identify the structure of these intermediates and to characterize their chemical and physical properties.…”

Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin R1: change in permeability of the cell envelope

“…It shows that pyocin Ri causes an increased binding of these probe molecules to the cells. It has been recently reported that membrane deenergization by energy poison and colicin K results in the increased binding of parinaric acid and azidopyrene to the cell envelope of E coli (25,27). In addition to these recent findings, results of a transition in fluorescence polarization of DPH and a deflection in fluorescence intensity of ANS around 20°C (26) indicate that hydrophobic probes are distributed in the cell envelope where the phase transition of lipid is observed (20) in spite of pyocin RI treatment.…”

Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein

“…A 50ml culture of strain CSR603(pCA6) was grown in M9 medium, irradiated in 10-ml portions, and labeled with [3S]methionine as described above. Cells were converted to spheroplasts and were separated into soluble and membrane fractions by using a modification of previously described procedures (49,50).…”

“…Vesicles were prepared as described in the text. (A) Vesicles(50 Il) from strains JK381(pBR322) (2.38 mg/ml) (0 and U) and JK381(pKP37) (2.45 mg/ml) (O and O) were assayed for [H]TPMP+ uptake after a single freeze-thaw cycle in the presence ofdifferent amounts of colicin Ia (O and 0) or colicin Ib (U and 0). [3H]-TPMP+ uptake determined in the absence of colicin (100% level) was 8,430 cpm for vesicles from strain JK381(pBR322) and 7,180 cpm for vesicles from strain JK381(pKP37).…”