Elongation Factor Tu3 (EF-Tu3) from the Kirromycin Producer Streptomyces ramocissimus Is Resistant to Three Classes of EF-Tu-Specific Inhibitors

Abstract

The antibiotic kirromycin inhibits prokaryotic protein synthesis by immobilizing elongation factor Tu (EF-Tu) on the elongating ribosome. Streptomyces ramocissimus, the producer of kirromycin, contains three tuf genes. While tuf1 and tuf2 encode kirromycin-sensitive EF-Tu species, the function of tuf3 is unknown. Here we demonstrate that EF-Tu3, in contrast to EF-Tu1 and EF-Tu2, is resistant to three classes of EF-Tu-targeted antibiotics: kirromycin, pulvomycin, and GE2270A. A mixture of EF-Tu1 and EF-Tu3 was sensitive to kirromycin and resistant to GE2270A, in agreement with the described modes of action of these antibiotics. Transcription of tuf3 was observed during exponential growth and ceased upon entry into stationary phase and therefore did not correlate with the appearance of kirromycin in stationary phase; thus, it is unlikely that EF-Tu3 functions as a resistant alternative for EF-Tu1. EF-Tu3 from Streptomyces coelicolor A3(2) was also resistant to kirromycin and GE2270A, suggesting that multiple antibiotic resistance is an intrinsic feature of EF-Tu3 species. The GE2270A-resistant character of EF-Tu3 demonstrated that this divergent elongation factor is capable of substituting for EF-Tu1 in vivo.

Avoidance of suicide in antibiotic-producing microbes

“…A similar conclusion applies to the kirromycin producer Streptomyces ramocissimus. Remarkably, this organism has three tuf genes [245], one of which (tuf3) encodes a product (EF-Tu3) that is highly resistant to kirromycin, GE2270A and pulvomycin [167]. However, expression of tuf3 is marginal at the best of times and was not detectable during, or immediately before, kirromycin production.…”

“…By far the bulk of EF-Tu in this organism is encoded by tuf1 and is drug-sensitive. That, and the dominance of kirromycin-sensitivity over resistance, means that EF-Tu3 cannot possibly play any significant role in resistance, so S. ramocissimus must ensure its own well-being in some other, uncharacterized way [167].…”

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365

“…These strains still are able to produce kirromycin levels comparable to the wild type, indicating additional resistance mechanisms. A different kirromycin producer, S. ramocissimus, codes for 3 EF-Tu genes (tuf1, tuf2 and tuf3) [52], of which the gene product of tuf3 (EF-Tu3) displays a kirromycin-resistant phenotype [38]. The S. collinus Tü 365 genome codes for two elongation factors, EF-Tu1 (WP_020941664.1), and EF-Tu3 (WP_020938678.1) including a homolog to S. ramocissimus EF-Tu3.…”

Elfamycins: inhibitors of elongation factor‐Tu

“…This standard version is constitutively expressed and is sensitive to kirromycin. In 2007, it was shown that S. ramocissimus produces a minor quantity of EF-Tu from tuf3 in exponential phase, and this version of EF-Tu (65% amino acid homology to tuf1) is resistant to kirromycin (as well as pulvomycin and GE2270 A) (Olsthoorn- Tieleman et al, 2007). While this seemed like a plausible resistance mechanism for circumventing the toxicity associated with producing kirromycin, antibiotic synthesis actually occurs in stationary phase.…”

“…The strategy of inactivating one copy of tuf allowed a rush of studies identifying EF-Tu amino acid substitutions that conferred resistance to this understudied group of compounds. These studies, done in the late 1990s, are cited in modern reviews as tables of resistance mutations in EF-Tu; an example being Olsthoorn- Tieleman's (Olsthoorn-Tieleman et al, 2007) reporting of Abdulkarim's (Abdulkarim et al, 1994) findings of mutations causing resistance to kirromycin. However, this citation and others fail to mention the caveat that the original studies identifying these mutations were done in strains that contained one inactivated copy of EF-Tu (Abdulkarim et al, 1994;Mesters et al, 1994).…”