An elongation factor Tu (EF‐Tu) resistant to the EF‐Tu inhibitor GE2270 in the producing organism Planobispora rosea

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.

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.…”

A Derivative of the Thiopeptide GE2270A Highly Selective against Propionibacterium acnes

“…5C) well correlates with the MIC values for the cognate microorganism (Table 1). It should be noted that GE2270A is able to effectively inhibit all EF-Tus tested so far, with natural resistance documented only in the producer strain Planobispora rosea (25) and in the Corynebacterium species tested here (Table 2). This is consistent with the fact that EF-Tu is a highly conserved protein with limited variation among divergent bacterial taxa.…”

“…With one exception, all resistant colonies harbored one mutation in the tuf gene for a total of 8 independent changes at 6 different positions (Table 4). Some of the observed mutations (G260R and G260C) occur at a residue (G257 in E. coli and Bacillus subtilis EF-Tu) previously reported to confer GE2270A resistance through the G257S substitution (11,25,26). Interestingly, in the three-dimensional (3D) structure of the complex between Thermus thermophilus EF-Tu, GTP, and GE2270A, a Gln residue (equivalent to Q99 in the P. acnes protein) makes direct contact with the antibiotic.…”

Preparation of Some Thiazolyl Hydrazone Derivatives and Evaluation of Their Antibacterial Activities

“…While all thiazolylpeptides exert their action by inhibiting bacterial protein synthesis, GE2270, together with the closely related molecules, amithiamycin, 4 is a potent and selective inhibitor of bacterial elongation factor Tu. 5,6 Thus it differs in its molecular target from thiostrepton and related compounds, which bind to the L11 protein in the 23S ribosomal complex. 7 The biosynthesis of thiazolylpeptide antibiotics has been investigated in several instances.…”