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Now that everyone is done with their first food, they sat back and enjoyed the rest of the show. As the performers came on stage, Noe performed the first song he’s ever sang in front of an audience.
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The hydrophobicity of the bacterial cell envelope, important for the transport of hydrophobic substrates across the membrane and/or the release of hydrophobic products, is usually quantified as the LogP of its constituent lipid fractions (mainly phospholipids). Unfortunately, such an approach is too time-consuming to allow accurate modelling of the effects of environmental perturbations on the hydrophobicity of the cell envelope. The lipopeptide alamethicin, an amphiphilic polypeptide, acting as a channel-forming peptide that inserts into and modulates the transport properties of phospholipid membranes, was used here as a convenient and rapid alternative to lipophilic fluorophores to quantify the lipid hydrophobicity of the bacterial envelopes of Gram-negative Escherichia coli (especially of the nonpolar lipid fraction) and Gram-positive Staphylococcus aureus. The integral membrane alamethicin induces a measurable negative (hydrophilic) transmembrane potential in E. coli inner membrane vesicles that can be conveniently determined with the lipophilic potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide and, on the basis of its fluorescence intensity, serves to calculate the overall hydrophilicity of the bacterial envelopes and the membrane potential. An in situ exposure of S. aureus vesicles to gramicidin, an amphiphilic peptide inducing small pores in lipid bilayers, and of E. coli vesicles to α-toxin (which makes large pores) at steady state allows quantification of the membrane potential; the small potential induced by gramicidin is almost entirely attributed to the inner membrane of S. aureus. This approach allows a three-dimensional quantification of the hydrophilicity of the bacterial cell envelope and a precise characterization of the physical properties of individual membranes, particularly relevant to modelling the transport of hydrophobic