ge-2270-a and efrotomycin

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Gram-Negative Bacteria; Gram-Positive Bacteria; Guanosine Diphosphate; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Pyridones; Species Specificity; Thiazoles

MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

Topics: Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Poly U; Protein Synthesis Inhibitors; Pyridones; Thiazoles