Abstract
Using a cell-free protein-synthesis system, we have established that the elongation factor (EF) Tu (EF-Tu) of the actinomycete Planobispora rosea, the producer of the thiazolyl peptide GE2270, a specific EF-Tu inhibitor, is highly resistant to its own antibiotic, while it is completely inhibited by kirromycin, which is another inhibitor of this factor. P. rosea was found to possess a single tuf gene, located between fus and rpsJ, encoding other components of the protein-synthesis machinery. The P. rosea tuf gene was expressed as a translational fusion to malE in Escherichia coli, and the resulting EF-Tu with an N-terminal Gly-Met extension was able to promote poly(U)-directed poly(Phe) synthesis in cell-free systems. This activity was not affected by GE2270, and the recombinant protein was incapable of binding the antibiotic, indicating that the P. rosea EF-Tu is intrinsically resistant to this inhibitor. Inspection of the translated tuf sequence revealed a number of amino acid substitutions in highly conserved positions. These residues, which are likely to be involved in conferring GE2270 resistance, map in EF-Tu domain II, as do the only two known mutations conferring resistance to this class of thiazolyl peptides in Bacillus subtilis.
MeSH terms
-
ATP-Binding Cassette Transporters*
-
Actinomycetales / physiology*
-
Amino Acid Sequence
-
Anti-Bacterial Agents / pharmacology*
-
Carrier Proteins / biosynthesis
-
Carrier Proteins / genetics
-
Cloning, Molecular
-
Conserved Sequence
-
Dose-Response Relationship, Drug
-
Drug Resistance, Microbial / genetics
-
Escherichia coli / genetics
-
Escherichia coli Proteins*
-
Genes, Bacterial
-
Maltose-Binding Proteins
-
Models, Molecular
-
Molecular Sequence Data
-
Monosaccharide Transport Proteins*
-
Mutation
-
Peptide Elongation Factor Tu / antagonists & inhibitors*
-
Peptide Elongation Factor Tu / biosynthesis
-
Peptide Elongation Factor Tu / genetics
-
Peptides, Cyclic / pharmacology*
-
Periplasmic Binding Proteins*
-
Pyridones / pharmacology
-
Recombinant Fusion Proteins / biosynthesis
-
Recombinant Proteins / biosynthesis
-
Sequence Analysis, DNA
-
Sequence Homology, Amino Acid
-
Thiazoles / pharmacology
Substances
-
ATP-Binding Cassette Transporters
-
Anti-Bacterial Agents
-
Carrier Proteins
-
Escherichia coli Proteins
-
MalE protein, E coli
-
Maltose-Binding Proteins
-
Monosaccharide Transport Proteins
-
Peptides, Cyclic
-
Periplasmic Binding Proteins
-
Pyridones
-
Recombinant Fusion Proteins
-
Recombinant Proteins
-
Thiazoles
-
maltose transport system, E coli
-
Peptide Elongation Factor Tu
-
GE 2270 A
-
mocimycin