Erythromycin was recovered in high yield after incucation with gram-negative bacteria. The cell-free protein-synthesizing preparation from gram-negative bacteria is equally as susceptible to the antibiotic as is that from gram-positive bacteria. Thus, neither destruction of erythromycin nor the absence of the step susceptible to the antibiotic plays an important role in the resistance mechanism of gram-negative bacteria. A 100-fold d fference in accumulation of erythromycin between gram-positive and gram-negative bacteria was observed. This alone explains the resistance of gram-negative bacteria to erythromycin. Furthe-more, data showed that the inhibition of growth is closely related to the accumulation of erythromycin. The concentration of intracellular erythromycin in gram-positive bacteria was found to be 44to 90-fold greater than that of the extracellular medium. However, the antibiotic did not accumulate on the cell walls, nor was the accumulation energy-dependent. It is proposed that it takes place by the binding of erythromycin to the bacterial ribosomes, forming a very stable complex. The dissociation constants of erythromycin-Staphylococcus aureus complex and erythromycin-Bacillus subtilis complex were determined to be 1.1
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