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.

FIG. 1.

Restriction map of the S. ramocissimus tuf3 region. Only the relevant SacII restriction site is shown (#). The probe used for S1 nuclease mapping of tuf3 transcripts (the asterisk indicates the ³²P-labeled 5′ end) is shown above the restriction map. Dashed line, nonhomologous pUC18-derived extension.

FIG. 2.

(a) Growth curve of S. ramocissimus B7. Arrowheads indicate the time points at which samples were taken for RNA isolation and kirromycin quantitation. The shaded box labeled kirromycin denotes the presence of the antibiotic in the filtrate. The doubling time during exponential growth was 2.5 h. (b) S1 nuclease protection analysis of the tuf3 transcripts in RNA isolated at the time points indicated in panel a (30 μg of RNA per sample). EXP and STAT indicate exponential and stationary growth phases, respectively, and the shaded box indicates the transition phase. tuf3p, a transcript initiated at tuf3p. Lane T3 shows the location of the 950-nt full-length tuf3 probe. Lane M contains end-labeled HpaII-digested pBR322 size markers (in nucleotides).

FIG. 3.

S1 nuclease protection analysis of the tuf3 transcripts in RNA isolated from surface-grown cultures at the time points (h) indicated, using 40 μg of RNA per sample. VEG, AER, and SP indicate the appearance of vegetative mycelium, aerial mycelium, and spores, respectively, and the shaded area corresponds to the transition phase. tuf3p indicates a transcript initiated at tuf3p; lanes T3 and M are as in Fig. 2b.

FIG. 4.

Translational activity of S. ramocissimus EF-Tu3. (a) Translational activity of cell extracts of S. coelicolor LT2 harboring expression vector pISRT3-1 with (⋄) and after removal of (♦) endogenous EF-Tu1His. A Ni²⁺-NTA-treated cell extract of S. coelicolor LT2 harboring pIJ487, the parental vector without tuf3, was used as a control (○). (b) In vitro translation of an EF-Tu-depleted S. coelicolor LT2 cell extract supplemented with S. ramocissimus EF-Tu3 (•) and S. ramocissimus EF-Tu1 (○). The translation of the poly(U) messenger was studied by measuring the incorporation of [¹⁴C]Phe at 30°C as a function of time (a) and of the concentrations of the EF-Tu species during a 10-min incubation (b).

FIG. 5.

Translational activities of S. ramocissimus EF-Tu1 and EF-Tu3 in the presence of antibiotics. In vitro translation of an EF-Tu-depleted S. coelicolor cell S30 extract supplemented with 0.5 μM S. ramocissimus EF-Tu1 (○) or S. ramocissimus EF-Tu3 (•). The translation of the poly(U) messenger was studied by measuring the incorporation of [¹⁴C]Phe at 30°C as a function of the concentrations of kirromycin (a), pulvomycin (b), and GE2270A (c). For both EF-Tu1 and EF-Tu3, activity in the absence of the antibiotics was normalized to 100% (2,700 dpm for EF-Tu1 and 1,800 dpm for EF-Tu3).

FIG. 6.

Translational activity of a mixture of S. ramocissimus EF-Tu1 and EF-Tu3 in the presence of the antibiotics kirromycin and GE2270A. In vitro translation of an EF-Tu-depleted S. coelicolor LT2 cell S30 extract supplemented with 0.5 μM EF-Tu3 (black bars), with 0.5 μM EF-Tu1 (white bars), or with a mixture of 0.5 μM EF-Tu3 and 0.5 μM EF-Tu1 (hatched bars). Poly(U) translation was studied by measuring the incorporation of [¹⁴C]Phe in the presence of 10 μM kirromycin (K) or 2 μM GE2270A (G). Activity of the EF-Tu mixture in the absence of antibiotics was taken as 100%.

FIG. 7.

Translational activity of cell extracts of S. coelicolor LT2 harboring the S. coelicolor tuf3 expression vector pISCT3-1 with (hatched bars) and after removal of (black bars) endogenous EF-Tu1His by Ni²⁺-NTA treatment (NTA). A cell extract of S. coelicolor LT2 containing pIJ487, the parental vector without tuf3, was used as a control (white bars). Poly(U) translation was studied by measuring the incorporation of [¹⁴C]Phe in the presence of 10 μM kirromycin (K) or 2 μM GE2270A (G). Activity of the cell extract in the absence of antibiotics was taken as 100%.

FIG. 8.

Location of conspicuous residues on the crystal structures of Thermus thermophilus EF-Tu-GDP complexed with aurodox (N-methyl derivative of kirromycin) (a), E. coli EF-Tu-GDPNP complexed with enacyloxin IIa (b), T. thermophilus EF-Tu-GDPNP complexed with pulvomycin (c), and T. thermophilus EF-Tu-GDPNP complexed with GE2270A (d). The antibiotics are shown as stick figures. Hydrogen bonds are shown as black dotted lines, and the salt bridge in panel b is shown as a cyan dotted line. Selected side chains and stretches of the EF-Tu backbone that are likely involved in conferring antibiotic resistance are shown in green on the schematic representation. Carbon atoms are either in beige (antibiotics) or in green (side chains changed in EF-Tu3 that are very likely involved in conferring antibiotic resistance). Oxygen atoms are red, nitrogens are blue, the chlorines in enacyloxin IIa are purple, and the sulfurs in GE2270A are yellow. Numbering of the residues is according to E. coli EF-Tu. The figures were prepared using Pymol (8).