The sensitivity of grown in batch culture at 37 °C, to ampho-tericin methyl ester (AME), judged by the concentration of AME required to induce a standard rate of leakage of K from suspensions of organisms, decreased with the time of growth. Organisms in exponential growth were sensitive to 0.1 to 0.2 μg AME/ml while organisms in the stationary phase were resistant to 4 to 60 μg AME/ml, depending on the initial concentration of glucose in the medium and the length of time for which incubation had been continued. When the initial concentration of glucose was low (0.1%, w/v), the AME resistance rose during the early stationary phase and then, after 40 h incubation at 37 °C, decreased again. Sphaeroplasts were prepared from organisms at different phases of growth and did not show these changes in AME sensitivity, but remained highly sensitive for growth up to 40 h.

Sphaeroplasts were prepared by treating suspensions of organisms with mercap-toethanol and then digesting with Streptomyces enzyme preparation. Addition of the material extracted by the digestion to suspensions of exponential-phase organisms or sphaeroplasts increased their AME resistance. Fractionation of the digest showed that the antagonistic material was contained in the neutral lipid fraction. Pure lipids fell into the following order of decreasing antagonism to AME when added together with the antibiotic to suspensions of exponential-phase organisms: sterol esters (ergosterol esters > cholesterol esters; unsaturated fatty acid esters > saturated fatty acid esters), sterols, triglycerides, unsaturated fatty acids, saturated fatty acids. The amount of antagonistic material released from stationary organisms was not markedly greater than that from exponential-phase organisms and analysis of the lipid content of wall preparations showed that the content of total lipid, neutral lipid and triglyceride of 40 h organisms was not more than 75,25 and 30%, respectively, greater than that of exponential-phase organisms.

The AME resistance of stationary-phase organisms decreased rapidly if suspensions were incubated with glucose or mercaptoethanol. The decrease in the presence of glucose was prevented by metabolic inhibitors, especially SH binding agents. Treatment of organisms with either iodoacetamide or -ethylmaleimide gave a rapid increase in AME resistance, amounting in some cases to 5- to 15-fold. The effect of iodoacetamide decreased as the organisms passed into the stationary phase and their intrinsic resistance increased. Evidence is presented which suggests that the degree of reduction of SH groups in the cell surface is an important factor in determining AME resistance.


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