Natural kirromycin resistance of elongation factor Tu from the kirrothricin producer Streptomyces cinnamoneus.
The antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocardia lactamdurans, producers of antibiotics of the Kr class, are known to possess an EF-Tu resistant to Kr. Both micro-organisms appear to possess a single tuf gene and we have characterized the one from S. cinnamoneus, which belongs to the tuf1 family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to malE, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-Tu was able to catalyse polyU-directed polyPhe synthesis in two heterologous cell-free systems, even as an uncleaved fusion. When tested for antibiotic sensitivity it behaved like the natural S. cinnamoneus protein, showing equivalent resistance to Kr but sensitivity to the antibiotic GE2270, indicating that all the determinants for Kr resistance are intrinsic to the EF-Tu sequence. Multiple sequence analysis of EF-Tu proteins, together with knowledge of mutations conferring Kr resistance, allowed the identification of key residues as likely candidates for the natural Kr resistance of the S. cinnamoneus EF-Tu. One of these, Thr378, was mutated to the consensus Ala and the resulting mutant protein was sensitive to Kr. Interestingly, it retained some activity (30% of the control) even at high Kr concentrations.
Cappellano
Carmela
C
Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy.
Monti
Federica
F
Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy.
Sosio
Margherita
M
Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy.
Donadio
Stefano
S
Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy.
Sarubbi
Edoardo
E
Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy.
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D011728:Pyridones / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013302:Streptomyces / Q000187:drug effects* / Q000235:genetics
D000900:Anti-Bacterial Agents; D011728:Pyridones; D011993:Recombinant Fusion Proteins; C035937:kirrothricin; D010444:Peptide Elongation Factor Tu; C008536:mocimycin