Natural kirromycin resistance of elongation factor Tu from the kirrothricin producer Streptomyces cinnamoneus

• Published in: Microbiology (Society for General Microbiology) Vol. 143; no. 2; pp. 617 - 624
• Main Authors: Cappellano, Carmela, Monti, Federica, Sosio, Margherita, Donadio, Stefano, Sarubbi, Edoardo
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.02.1997; Society for General Microbiology
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Bacteriology; Base Sequence; Biological and medical sciences; Dose-Response Relationship, Drug; Drug Resistance, Microbial - genetics; Escherichia coli; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetics; Microbiology; Molecular Sequence Data; Peptide Elongation Factor Tu - biosynthesis; Peptide Elongation Factor Tu - drug effects; Peptide Elongation Factor Tu - genetics; Protein Biosynthesis - drug effects; Pyridones - metabolism; Pyridones - pharmacology; Recombinant Fusion Proteins - biosynthesis; Restriction Mapping; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Species Specificity; Streptomyces - drug effects; Streptomyces - genetics; Actinomycetales; Resistance; Antibiotic; Mocimycin; Streptomycetaceae; Streptomyces; Bacteria; Actinomycetes; Genetical translation; Elongation factor EFTu
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-143-2-617

Lepetit Research Center, via R. Lepetit 34, 21040 Gerenzano, Italy 2 Author for correspondence: Stefano Donadio. Tel: +39 2 96474 243. Fax: +39 2 964T4 400. e-mail: stefanodonadio@mmd.com ABSTRACT 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 tuf 1 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 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, Thr 378, 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. Keywords: Streptomyces cinnamoneus , antibiotic, expression system, protein synthesis, tuf gene