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

Abstract

The antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocadia 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 5. cinnamoneus, which belongs to the tuff family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to ma/€, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-T u 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, Tht.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.

Avoidance of suicide in antibiotic-producing microbes

“…And in either event, tuf genes have not been encountered in the respective antibiotic-biosynthetic gene clusters. The kirrothricin producer Streptomyces cinnamoneus has a single tuf gene [25] and EF-Tu from this organism is resistant to kirrothricin, kirromycin and efrotomycin [63] but is sensitive to GE2270A [25]. Single tuf genes also confer resistance in the producers of efrotomycin and GE2270A (Table 4).…”

The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu

“…Exponentially growing S. ramocissimus B7, cultured in S medium, was used as a source for 4 Cl, 1 mM dithiothreitol, 10 M GDP, 10 M phenylmethylsulfonyl fluoride, 10% [vol/vol] glycerol) and kept frozen at Ϫ80°C. For the purification of S. ramocissimus EF-Tu1 and EF-Tu2, the method described by Olsthoorn-Tieleman et al (24) for S. coelicolor EF-Tu1 was used with the following modifications.…”

“…S30 cell extracts of S. coelicolor LT2 (if necessary harboring pISRT2-1 or pIJ487), from which His 6 -tagged EF-Tu1 was removed by treatment with Ni 2ϩ -NTA-agarose beads, were obtained as described by Olsthoorn-Tieleman et al (24). The extracts, supplemented with purified EF-Tu species and antibiotics at various concentrations, were incubated in translation buffer (final concentrations: 50 mM Tris-HCl [pH 7.6], 9 mM MgCl 2 , 60 mM NH 4 Cl, 1 mM dithiothreitol, 1 mM ATP, 1 mM GTP, 6 mM phosphoenolpyruvate, 50 g of pyruvate kinase/ml, 0.1 mg of poly[U]/ml, 0.2 mg of E. coli tRNA/ml, and 13.2 M […”

“…Various mechanisms are exploited by antibiotic-producing microorganisms to protect themselves from the toxic action of their own products (5); these include the use of an efficient drug efflux system, intracellular storage of the antibiotic in an inactive form, modification of an otherwise sensitive target, and (temporary) expression of a resistant target. The mechanism used by producers of kirromycin-type antibiotics to protect themselves against their own products is only partially known; some producers contain an intrinsically kirromycin-resistant EF-Tu (4,9).…”

Inhibitory Mechanisms of Antibiotics Targeting Elongation Factor Tu

“…Some store the drug in an inactive form while a sensitive target is present, some prevent the interaction of the drug with the target through active efflux of the drug combined with a permeability barrier, while others evolve a resistant form of the target that is otherwise functionally sound. The latter strategy has been shown to be the case in the GE2270A producer Planobispora rosea [32,37] and for the kirromycin producers Streptomyces cinnamoneus and Nocardia lactamdurans [38]. On the other hand, some kirromycin producers such as Streptomyces ramocissimus or Streptomyces collinus express kirromycinsensitive EF-Tu [39][40] and therefore must use an alternative guarding mechanism.…”