The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system.

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts.

GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes.

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Mutant EF-Tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome.

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

Structure and expression of elongation factor Tu from Bacillus stearothermophilus.

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.

Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promoters.

The str operon of Streptomyces ramocissimus contains the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and for the polypeptide chain elongation factors G (EF-G) (fus) and Tu (EF-Tu) (tuf). This kirromycin producer contains three tuf or tuf-like genes; tuf1 encodes the regular EF-Tu and is located immediately downstream of fus. In vivo and in vitro transcription analysis revealed a transcription start site directly upstream of S. ramocissimus tuf1, in addition to the operon promoter rpsLp. Transcription from these promoters appeared to be growth phase dependent, diminishing drastically upon entry into stationary phase and at the onset of production of the EF-Tu-targeted antibiotic kirromycin. In surface-grown cultures, a second round of tuf1 transcription, coinciding with aerial mycelium formation and kirromycin production, was observed. The tuf1-specific promoter (tuf1p) was located in the intercistronic region between fus and tuf1 by high-resolution S1 mapping, in vitro transcription, and in vivo promoter probing. During logarithmic growth, the tuf1p and rpsLp transcripts are present at comparable levels. In contrast to Escherichia coli, which has two almost identical tuf genes, the gram-positive S. ramocissimus contains only tuf1 for its regular EF-Tu. High levels of EF-Tu may therefore be achieved by the compensatory activity of tuf1p.

Elongation factor Tu1 of the antibiotic GE2270A producer Planobispora rosea has an unexpected resistance profile against EF-Tu targeted antibiotics.

Sensitivity of EF-Tu1 of the GE2270A producer Planobispora rosea towards GE2270A, pulvomycin and kirromycin was determined by band-shift assays for EF-Tu1-antibiotic complex formation and by in vitro translation experiments. EF-Tu1 of P. rosea appeared to be not only totally resistant to GE2270A, but also ten times more resistant to kirromycin than EF-Tu1 of Streptomyces coelicolor. In contrast, P. rosea EF-Tu1 was found to be not resistant to pulvomycin, an antibiotic that just like GE2270A blocks EF-Tu x GTP x aminoacyl-tRNA complex formation. Previous in vivo and in vitro experiments with mixed populations of antibiotic resistant and sensitive EF-Tu species had shown that sensitivity to kirromycin and pulvomycin is dominant over resistance. In the case of GE2270A we observed, however, that sensitivity is recessive to resistance, which again points to a different action mechanism than in the case of pulvomycin. Besides the tuf1 gene encoding the regular elongation factor EF-Tu1 a gene similar to S. coelicolor tuf3 for a specialized EF-Tu was located in the P. rosea genome. The tuf1 gene was isolated and sequenced. The amino acid sequence of EF-Tul of P. rosea not only exhibits an unusual Tyr160 substitution (comparable to those described for kirromycin-resistant EF-Tus), but also shows significant changes of conserved amino acids in domain 2 that may be responsible for GE2270A resistance (the latter do not resemble those leading to pulvomycin resistance). P. rosea EF-Tu1 thus is a first example of a bacterial EF-Tu with resistance against two divergently acting antibiotics.