Aug 12, 1994 - pulvomycin and kirromycin both act by specifically disturbing ... substrate limitation. ... are sensitive (Zief et al., 1957; Akita et al., 1963; Landini.
The EMBO Journal vol.13 no.21 pp.5113-5120, 1994
Pulvomycin-resistant mutants of E.coIi elongation factor Tu
Leo A.H.Zeef, Leendert Bosch, Pieter H.Anborghl, Rengul Cetin1, Andrea Parmeggianil and Rolf Hilgenfeld2 Department of Biochemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands, 'SDI No. 61840 du CNRS, Laboratoire de Biochimie, Ecole Polytechnique, F-91128 Palaiseau Cedex, France and 2Protein Crystallography, Central Research G 865A, Hoechst AG, D-65926 Frankfurt, Germany Communicated by L.Bosch
This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring
elongation factor (EF)-Tlu Arg230 -* Cys, Arg333 -* Cys or Thr334 -X Ala which confer pulvomycin resistance. These mutations are clustered in the threedomain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu. This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface. Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu GDP. The three-domain junction changes dramatically in the switch to EFTulGDP; in EF-TuGDP this region forms an open hole. Structural analysis of the mutation positions in EF-Tu-GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF-Tu-GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin. Key words: aminoacyl-tRNA/EF-Tu/GTP binding protein/ kirromycin/ribosome
Introduction The antibiotic pulvomycin was first reported by Zief et al. (1957). Wolf et al. (1978) discovered that pulvomycin inhibited protein synthesis by acting on elongation factor Tu (EF-Tu). Among the various effects pulvomycin has on EF-Tu, a dramatic inhibition of the aminoacyl-tRNA (aa-tRNA) binding affinity of EF-Tu-GTP occurs (Wolf et al., 1978; Pingoud et al., 1982a; Anborgh and Parmeggiani, 1991). Since EF-Tu has the essential cellular function of mediating the interaction of aa-tRNA with the ribosome during translation, this observation led Wolf © Oxford University Press
et al. (1978) to the generally accepted hypothesis that pulvomycin inhibits protein biosynthesis by causing a substrate limitation. This was further supported by the inhibition of enzymatic binding of aa-tRNA to the ribosome by pulvomycin. Initially pulvomycin was assigned an incomplete chemical structure (Akita et al., 1964) which was later shown by Smith et al. (1985) to be missing an unusual
22-membered lactone ring (Figure 1). Apart from leading to faulty calculations of pulvomycin concentrations, the early incomplete structure stimulated speculation on the similarity between pulvomycin and the well-known kirromycin. Subsequently, Pingoud et al. (1982a) showed that the two antibiotics bind to different sites on EF-Tu. Although pulvomycin affects EF-Tu in a unique way, both antibiotics influence the interaction of the protein with all its ligands simultaneously, indicating that their action is to disrupt the allosteric control mechanisms of EF-Tu (Chinali et al., 1977; Wolf et al., 1978). Another property that the two antibiotics share is their limited range of activity, since only Gram-positive bacteria are sensitive (Zief et al., 1957; Akita et al., 1963; Landini et al., 1993). However, EF-Tu species from Gram-negative bacteria are even more sensitive to inhibition in vitro than EF-Tu from Gram-positive bacteria (Landini et al., 1993). The apparent resistance of Gram-negative bacteria is the result of membrane impermeability to pulvomycin. In this paper we report a method to reduce the membrane impermeability of Escherichia coli so as to allow the selection of mutants resistant to pulvomycin by virtue of mutations in EF-Tu. Three EF-Tu mutants were isolated by targeted mutagenesis of the tufA gene. All three mutants did not confer resistance to pulvomycin when wild-type EF-Tu (wtEF-Tu) was present, indicating that the action of pulvomycin, when bound to wtEF-Tu, is to actively obstruct translation rather than starve the ribosome of aa-tRNA, as currently believed.
Results Isolation of pulvomycin-resistant mutants Although EF-Tu from E.coli is very sensitive to pulvomycin (Wolf et al., 1978; Landini et al., 1993), the bacterium is resistant by virtue of membrane impermeability. Consequently, the generation of pulvomycinresistant mutants required increasing the uptake of antibiotic through the membrane. A similar problem exists for kirromycin, for which it has been found that low concentrations of ethylene diamine tetraacetic acid (EDTA) in agar plates enhance sensitivity without significantly inhibiting the growth rate (van de Klundert et al., 1978). The greater sensitivity to kirromycin induced by EDTA can be explained in terms of increasing the permeability of the outer membrane, since EDTA treatment of Ecoli 5113
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-1.5. Table I. Minimum inhibitory concentrations (MIC) of pulvomycin or kirromycin for strains containing pulvomycin-resistant EF-Tu mutants EF-Tu mutation
R230C R333C T334A Wild-type
Doubling time (min)
MICp (ig/ml) ApB::Mu
MICp (jg/ml)
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Ap encodes a pulvomycin-resistant EF-Tu mutant; B::Mu, EF-TuB gene inactivated by Mu phage insertion; Bs encodes wtEF-TuB; MICP, MIC pulvomycin; MICK, MIC kirromycin. aLZ34L (ApB::Mu). bLZ32L (ApB::Mu). CLZ33L (ApB::Mu). dEV4L F'cam (AsB::Mu). eLZ37L (ApBS).
ILZ35L (ApBs).
8LZ36L (ApBS). hMG1655L F'cam (ASBS).
cells is known to lead to a loss of lipopolysaccharides, a key element in the protective function of this membrane (Nikaido and Vaara, 1987). In the presence of 1.5 mM EDTA, the minimum inhibitory concentrations (MIC) measured on agar plates of kirromycin and pulvomycin for MG1655 were 40 and 100 gg/ml, respectively. When performing M 13-based targeted mutagenesis according to Zeef and Bosch (1993), we discovered that the M13 lysogens that were formed by this technique had a still further increased antibiotic sensitivity in the presence of 1.5 mM EDTA, achieving a pulvomycin MIC of 20 gg/ml. Studies on a different filamentous coliphage, fl, indicated that the protein product of gene III is responsible for the changes in membrane properties brought about by filamentous phage infection (Boecke et al., 1982). The advantage of using this EDTA-M13 lysogen technique over methods using mutants hypersensitive to hydrophobic antibiotics (Nikaido and Vaara, 1987; Vuorio and Vaara, 1992) is that the mutated target gene and hypersensitivity are introduced simultaneously with the M13 construct, thus reducing the risk of isolating spontaneous reversion mutations in the gene causing hypersensitivity to antibiotics. Following mutagenesis and a 3 day incubation at 37°C on plates containing 100 jg/ml pulvomycin, 50 ,ug/ml tetracycline (tet) and 1.5 mM EDTA, three colonies were detected by microscopy (under identical conditions, 15 kirromycin-resistant mutants were scored). When streaked on fresh selection plates (containing 50 ,ug/ml pulvomycin), these strains (LZ32L, LZ33L and LZ34L; Table
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Time (min) Fig. 2. Protection against Phe-tRNAPhe deacylation by EF-Tu mutants. 200 pmol of EF-Tu-GDP were converted into EF-Tu-GTP by treatment with 2 mM phosphoenolpyruvate (PEP), 1 mM GTP and pyruvate kinase (PK) (40 ig/ml) in 97 il of standard buffer for 15 min at 30°C and then cooled on ice. 3 ,l (75 pmol) [14C]Phe-tRNAPhe (224 c.p.m./ pmol) were added on ice. After 15 min, incubation at 24°C was started and 15 gl samples were taken at the times indicated. Samples were spotted onto Whatman 3MM filters, which were dipped into cold 10% (w/v) TCA, washed twice in cold 5% (w/v) TCA and dried in ether. Symbols: EF-TuR230C (A); EF-TuR333C (X); wtEF-Tu (A); no EF-Tu added (0).
I) required 24 h for normal colony formation, indicating that the inability to form colonies detectable with the naked eye after 3 days growth on the initial selection plates was due to competition from a background layer of sensitive cells (see also Table I). Mutations were retrieved from the chromosome using the genetic retrieval technique described in Zeef and Bosch (1993). For each mutant, 10 M13mp9Zam2O phage clones retrieved in this way were screened for the ability to confer pulvomycin resistance. The whole tufA gene of a resistant phage was sequenced to identify the mutations. Each gene contained a single mutation which gave rise to the EF-Tu mutants Arg230 -* Cys, Arg333 -> Cys or Thr334 -4 Ala.
Genetic characterization reveals a dominance of pulvomycin sensitivity The mutations conferring pulvomycin resistance in strains LZ32L, LZ33L and LZ34L were localized in the tufA locus by P1 phage transduction mapping (see Materials and methods). Table I shows the MIC for strains harbouring a pulvomycin-resistant tufA gene and a tufB gene inactivated by Mu phage insertion (ApB::Mu). The pulvomycin resistances of ApB::Mu strains (5.0-7.5X wild-type level) were severely reduced by the presence of a wild-type tufB gene (ApBs), indicating that sensitivity was dominant for all three mutants. A similar result was found when ApB::Mu strains were transformed with the plasmid pVE 1.15 encoding wtEF-TuA.
Biochemical characterization of EF-TuR230C and R333C The two mutant species had slightly lower GDP dissociation rates than wtEF-Tu. The intrinsic GTPase (tested in the presence of increasing concentrations of KCI) and the thermostability of EF-TuR230C were both similar to those of wtEF-Tu (results not shown).
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Tll> Fig. 3. PAGE under non-denaturating conditions of EF-Tu in the presence of pulvomycin. 40 pmol of EF-Tu in complex with GDP or GTP were incubated for 15 min at 0°C in standard buffer (50 mM Tris-HCI, pH 7.5, 60 mM NH4CI, 10 mM MgCl2, 1 mM DTT) in the presence of pulvomycin at the concentrations (jiM) indicated below the lane number. Electrophoretic conditions were as described by Anborgh and Parmeggiani (1991). Migration direction from top to bottom (+). In (A), lanes 1-6 contain EF-TuR230C GDP; lanes 7-11 contain wtEF-Tu-GDP. In (B), lanes 1-6 contain EFTuR333C-GTP; lanes 7-12 contain wtEF-Tu-GTP. In (B), the residual protein at the origin consists mainly of pyruvate kinase added during preincubation, together with phosphoenolpyruvate to convert EFTu-GDP into EF-Tu-GTP (Anborgh and Parmeggiani, 1991).
As shown in Figure 2, EF-Tu(R230C) GTP was significantly weaker in aminoacyl ester bond protection of aa-tRNA than wtEF-Tu and EF-TuR333C. This indicates a reduced affinity for aa-tRNA as a whole, or an anomalous binding of the CCA end of aa-tRNA by EF-TuR230C. Despite this, LZ34L (ApB::Mu) has a high growth rate, suggesting that aa-tRNA binding affinities well below wtEF-Tu levels can be tolerated. Figure 3 shows that -50% of wtEF-Tu-GDP is bound at 15 ,uM pulvomycin (Figure 3A, lane 8) and a little more than 50% of wtEF-TuGTP is bound at 15 jM (Figure 3B, lane 8). EF-Tu-GTP therefore has a slightly higher affinity for pulvomycin than EF-Tu-GDP. The pulvomycin-bound EF-Tu GDP complex of mutant R230C (lower band in Figure 3A, lanes 2-5) is smeared, even at very high pulvomycin concentrations. This may indicate that pulvomycin is released during the electrophoresis period. In addition to this, pulvomycin-bound EFTuR230C does not migrate as far as wtEF-Tu-GDP-pulvomycin or EF-TuR333C pulvomycin. The same result was found for GTP-bound EF-TuR230C. A similar trend is found for EF-TuR333C (GTP-bound complex shown in Figure 3B). A pulvomycin-bound band is formed at 15
loglpulvomycin] Fig. 4. Inhibition of poly(Phe) synthesis as a function of pulvomycin concentration (A) and dominance of wtEF-Tu over pulvomycinresistant EF-TuR333C (B). (A) Reaction mixtures (70 gl) contained 20 mM Tris-HCI, pH 7.5, 90 mM NH4Cl, 7 mM 2-mercaptoethanol, I mM ATP, 0.5 mM GTP, I mM PEP, PK (40 jug/ml), 0.3 jiM EF-Ts, 0.1 ,uM EF-G, 0.4 jM ribosomes, poly(U) (80 mg/ml), a saturation amount of Phe-tRNA synthetase, 120 jig bulk tRNA, 5 jM [14C]phenylalanine (200 c.p.m./pmol) and I p1 of a solution of pulvomycin in dimethylformamide at 70-fold the indicated concentration, while I pl of dimethylformamide was added to those reaction mixtures that did not contain pulvomycin. In addition, the reaction mixtures contained 0.4 jM of either wtEF-Tu (0) or EFTuR230C (A). The polymerization reaction took place for 10 min at 37°C. Thereafter, samples (60 pu) were withdrawn and assayed for incorporation of [14C]phenylalanine into hot trichloroacetic acid insoluble material (Anborgh et al., 1992). The absolute values of poly(Phe) synthesis at 0 ,ug/ml pulvomycin (taken as 100%) were: wtEF-Tu, 87.5 mmol/mol EF-Tu-min; EF-TuR230C 91, mmol/mol EFTu-min. (B) Reaction mixtures and incubations were as described in (A), except that reaction mixtures contained 0.4 ,iM wtEF-Tu (0), 0.4 jiM EF-TuR333C (U) or 0.2 ,uM wtEF-Tu + 0.2 iM EFTuR333C (*). The absolute values of poly(Phe) synthesis were: wtEFTu, 123 mmol/mol EF-Tu-min; EF-TuR333C, 112 mmol/mol EFTu-min; EF-TuR333C + wtEF-Tu, 157 mmol/mol EF-Tu-min.
iM pulvomycin, but even at 80 ,uM some unbound EFTu-GTP exists. These results suggest that pulvomycin resistance for the mutants is the result of efficient release of pulvomycin, and therefore indicate a lower binding affinity for the antibiotic.
Dominant sensitivity is also found in poly(Phe) synthesis Figure 4 shows that EF-TuR230C and EF-TuR333C are three to four times more resistant than wtEF-Tu in the poly(Phe) synthesis assay (pulvomycin concentrations giving 50% inhibition were -8 ,uM for mutant and 3 ,uM for wtEF-Tu). This is low compared with the 6- to 7.5fold increase in the MIC of the E.coli mutants (Table I). 5115
L.A.H.Zeef et al.
Fig. 6. Close up view of the three-domain junction interface of EFTu.GppNHp. The orientation has been rotated 1800 horizontally with respect to Figure 5. The peptide backbone is shown with ribbons (domain colouring as for Figure 5). Only residues that form salt bridges in the network of electrostatic interactions have been shown (from left to right: D369, R333, R233, E232, R230, R204 and D99), together with T334. Blue spheres have been placed on atoms of the positively charged guanidinium groups of Arg residues, whereas red spheres have been placed on atoms of the negatively charged carboxylate groups of acidic residues. Ecoli EF-Tu numbering has been given. All residues are identical to Tthermophilus EF-Tu, except for D369 (TT E381) which has been modelled to the Ecoli residue D.
Pulvomycin resistance mutations destabilize the EF-Tu-GTP conformation Figure 5 shows that all three mutation positions are located in the three-domain junction interface of EF-Tu-GTP (Berchtold et al., 1993). This interface opens in the switch to EF-Tu.GDP, so that R230 is exposed to solution whereas R333 and T334 remain part of the domain 2-3 interface (Kjeldgaard and Nyborg, 1992; the domain 2-3 interface remains basically unchanged in the switch from GTP- to GDP-bound conformations of EF-Tu; Kjeldgaard and Nyborg, 1992; Berchtold et al., 1993; Kjeldgaard et al., 1993). Mutations R333C and R230C. As shown in Figure 6, the three-domain junction interface of EF-Tu-GTP contains a network of ion pair interactions, in addition to many hydrogen bonds (H-bonds) which are not shown here for clarity. R230 and R333 make up a part of this electrostatic network, so that mutations to Cys are likely to weaken Fig. 5. Structures of EF-Tu-GppNHp (above), pulvomycin (middle) and EF-Tu-GDP (below) showing positions of mutations causing pulvomycin resistance (green spheres). For EF-Tu.GppNHp and EFTu-GDP, from top to bottom, R333, T334 and R230. Domain I is red, domain 2 is yellow and domain 3 is blue. The pulvomycin coordinates were created and energy minimized (AMBER force field) in vacuo using the program Macromodel version 3.0 (Mohamdi et al., 1990) and structural information from Smith et al. (1985).
Despite the modest increase in resistance observed during the poly(Phe) synthesis assay, it was still possible to measure the dominance of sensitivity with this assay when using a mixed population of resistant and wtEF-Tu (Figure 4B). EF-TuR230C and EF-TuR333C were as sensitive as wtEF-Tu during poly(Phe) synthesis in similar experiments using instead the antibiotic GE 2270 A (results not shown).
5116
this network and the interface. These two mutants are also the most resistant EF-Tu species (Table I), suggesting that weakening of this electrostatic interaction is linked to pulvomycin resistance. The seven residues (D99, R230, R204, E232, R233, R333 and D369) making up the electrostatic network are highly conserved in EF-Tu species (apart from D369 which is often an E in other species). With the exception of R233 and D369, they are also conserved in EF1a species (R204 and R333 have undergone relatively conservative changes to Lys and His, respectively, in EF- 1 a). R333 (TI R345; IT denotes the homologous residues of the EF-Tu from Thermus thermophilus) can form ion pairs with D369 (7T E38 1) and E232 (IT E243) of domain 2 in the GTP-bound EF-Tu conformation (Figure 6). R230 (TT R241) forms salt bridges with E232, as well as with
Pulvomycin-resistant mutants of EF-Tu
D99 (7T DIOO) of domain 1. D99 is just beyond the Cterminus of the 'switching B helix' that transmits the conformational signal from the G domain (domain 1) to the other two domains, depending on the nucleotide bound (Berchtold et al., 1993; Kjeldgaard et al., 1993). Based on structural considerations, R230 involvement in these regions crucial to EF-Tu function may be expected (in line with the high conservation of R230 and D99 in both EF-Tu and EF- 1 a classes); however, our results contradict this. The mutation R230C is a radical change, eliminating all possibilities of ion pair formation with E232 (TT E243) and D99 (TT DI 00). Despite this, EF-TuR230C gives rise to a viable EF-Tu species which maintains a high growth rate in E.coli (Table I). This seems to refute the idea that R230 plays an essential role in the switch mechanism. Instead, it is possible that the residue is important in aa-tRNA binding since we found a poor protection against non-enzymatic hydrolysis of the aminoacyl ester bond by this mutant EF-Tu (see Figure 2 and Discussion). Mutation T334A. Mutation T334A has a poor resistance in vivo which was reflected by our inability to measure resistance in vitro (results not shown). Together with R333, T334 forms a 3-hairpin loop between 3-strands 2 and 3 of domain 3. T334 is located in the domain 2-3 interface of the GTP- and GDP-bound EF-Tu conformations. This is a closely packed interface stabilized by van der Waals interactions and H-bonds. T334 (7T T346) can form a number of H-bonds in the domain 2-3 interface in both the EF-Tu-GDP and EF-Tu-GTP structures. The mutation T334A would have a destabilizing effect on the domain 2-3 interface due to a loss of van der Waals interactions. This would destabilize the three-domain junction interface of EF-Tu-GTP as well, suggesting this destabilization as a common structural principle underlying the pulvomycin resistance of all three EF-Tu mutations. It is significant that although the least resistant, mutation T334A has the most deleterious effect on growth rate of the three mutants (Table I). So it is likely that T334A leads to the most significant structural defect of the three mutations.
Discussion All three mutations that confer pulvomycin resistance (R230C, R333C and T334A) are located in the threedomain junction interface of the EF-Tu GTP conformation (Figure 5). This reveals a remarkable parallel with mutations that induce resistance to kirromycin, which are located in the domain 1-3 interface region of the GTPbound form of EF-Tu (Mesters et al., 1994). These two interface regions are crucial for the switch from the GTP to the GDP form of EF-Tu, since their rearrangement is the means of amplifying the conformational signal given by the nucleotide bound (Berchtold et al., 1993; Kjeldgaard et al., 1993). This suggests that these antibiotics interfere with the switch mechanism and indicates that kirromycin and pulvomycin act by derailing the elegant allosteric regulation of the protein. Our structural analysis of the mutations suggests that the mutations destabilize the three-domain junction interface of EF-Tu-GTP. The disturbance of a network of
electrostatic interactions in this three-domain junction appears to induce the highest resistance to pulvomycin. Berchtold et al. (1993), when describing the EF-Tu-GTP domain 1-2 interface, point out the significance of the polar nature of this interface. The interface is designed to open and close, since in the open EF-Tu-GDP conformation the residues interact with solvent. These authors also note the directional nature of polar interactions which leads to a higher specificity. Mutations in this region would therefore be expected to have deleterious effects on EF-Tu functions. However, the growth rates of strains harbouring the two most resistant species (R230C and R333C), although lower than wild-type, are still high (Table I; better than kirromycin-resistant mutant strains, for example; Zeef and Bosch, 1993). Also, the in vitro properties of the EFTu mutants (GDP dissociation and GTPase activities) are similar to wtEF-Tu. With respect to the structural and functional basis for resistance, native gel electrophoresis indicated that EFTuR333C, and particularly EF-TuR230C, have a reduced binding affinity for pulvomycin (Figure 3). This suggests that the pulvomycin binding site is disturbed. In this regard it may be significant that EF-TuR230C showed a reduced aa-tRNA binding affinity, as measured by the deacylation protection assay (Figure 2). Since pulvomycin inhibits aa-tRNA binding, the binding sites of these two ligands may be identical or overlap. Below we consider the evidence available concerning the location of the pulvomycin binding site on EF-Tu. Our results suggest a location near the EF-Tu-GTP threedomain junction interface, where the mutation positions are found. A disturbance of the pulvomycin binding affinity for the mutants is in line with this, as well as the observation of Anborgh (1993) that the deletion of domain 3 abolishes pulvomycin binding. A pulvomycin binding site near the three-domain junction can explain the wide range of effects pulvomycin has on EF-Tu as this region undergoes a profound change in the switch from EFTu-GTP to EF-Tu-GDP, as mentioned above. Binding of pulvomycin would prevent the tight association of domains 1 and 2, which form the proposed aa-tRNA binding cleft of EF-Tu-GTP (Berchtold et al., 1993). This would explain how aa-tRNA binding is inhibited by pulvomycin, and is in line with the IH NMR measurements of Romer et al. (1981) who reported that pulvomycin 'freezes' EF-Tu-GTP in the EF-Tu-GDP conformation. The possibility that pulvomycin prevents the tight association of domains 1 and 2 in EF-Tu-GTP implies that the three-domain junction interface cannot be formed when pulvomycin is bound. If pulvomycin were to bind directly to this region, the binding site could therefore resemble the open domain 1-2 contact of EF-TuGDP (Kjeldgaard and Nyborg, 1992), or a hybrid EF-Tu conformation. However, a pulvomycin binding site in the threedomain junction interface of EF-Tu-GTP is not readily accommodated on a structural basis. Firstly, this region is polar in nature, whereas pulvomycin has a largely hydrophobic character, with only the sugar moiety being hydrophilic (Figure 1). It is also difficult to explain why weakening of the EF-Tu-GTP structure would lead to reduced pulvomycin binding affinity. These considerations suggest instead that mutations at the three-domain junction alter pulvomycin binding at a different site on EF-Tu by
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L.A.H.Zeef et aL Table II. Bacteria, phages and plasmids Strain, plasmid or phage Escherichia coli EV4 LBE2040 LZ32La LZ33La LZ34La LZ35La LZ36La LZ37La MG 1655 Phage and plasmids M 13mp9Zam2O pTS32 pVEI.15
Relevant traits or insertion
Reference or source
tufB::Mu A(pro-lac) rpoB fus tufAb tufB::Mu A(pro-lac) rpoB F'Camc tufAd tuJB::Mu A(pro-lac) rpoB F'Camc tufAe tuJB::Mu A(pro-lac) rpoB F'Camc tufAb F'Camc tufAd F'Camc tufAe F'Camc wild-type
Vijgenboom et al. (1985) van de Klundert et al. (1978) this work this work this work this work this work this work Berget et al. (1988)
Tetr tufA Camr Tetr tsf Ampr tufA
Zeef and Bosch (1993) Hwang et al. (1989) Zeef and Bosch (1993)
Superscript 'r' denotes resistant. aStrains which end with the letter L are M13mp9Zam2O lysogens. bThe tufA mutation is R333C. CA chloramphenicol-resistant F factor, F1 lac-pro TnlOdCam from strain PB759 (Berget et al., 1988). dThe tufA mutation is T334A. eThe tufA mutation is R230C.
long-range effects. This would be in contrast to mutations conferring kirromycin resistance, which are believed to alter the kirromycin binding site directly (Mesters et al., 1994). A study by Pingoud et al. (1982a) concluded that the pulvomycin binding site is different to the kirromycin binding site. This is in line with our localization of the mutations that cause pulvomycin resistance in a different region to that of kirromycin-resistant mutations. Also, we have found previously (Anborgh and Parmeggiani, 1991) that these antibiotics induce different mobility shifts of EFTu*GTP on non-denaturing PAGE. Recently, we concluded that three kirromycin resistance mutations, G316D, A375T and A375V, stabilize the domain 1-3 interface in EFTu-GTP (Mesters et al., 1994). With the pulvomycin resistance mutations a destabilization should occur for the three-domain junction interface of EF-Tu-GTP. Kirromycin and pulvomycin resistance mutations thus seem to have opposite effects on EF-Tu-GTP in terms of stabilization. In line with this result, some ApB::Mu strains were more sensitive to kirromycin than AsB::Mu strains (Table I). Conversely, enhanced pulvomycin sensitivity was found for a kirromycin-resistant strain LZ12L (Zeef and Bosch, 1993; AQ124KB::Mu, MIC 4 ,ug/ml) with respect to a strain with wtEF-Tu, EV4L F'cam (ASB::Mu, MIC 20 ,ug/ml). Wolf et al. (1978) reported that the kirromycin-resistant EF-TuA375V was also more sensitive to pulvomycin in poly(Phe) synthesis, but we could not detect a greater sensitivity for the kirromycin-resistant strain harbouring EF-Tu mutant A375V, or strains harbouring kirromycinresistant EF-Tu mutations G316D or A375T (results not shown). In general, these results support our conclusions based on crystal structure, i.e. that mutations causing kirromycin and pulvomycin resistance have opposite effects on EF-Tu. A consideration of the kirromycin and pulvomycin resistances of diverse bacteria also shows that resistance or sensitivity to these antibiotics are independent from one another (Landini et al., 1993). Like kirromycin, pulvomycin does not inactivate eukaryote protein synthesis
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(Londei et al., 1986). Unlike kirromycin, pulvomycin is active against some archaebacterial EF- I x (Londei et al., 1986), in addition to chloroplast and eubacterial EF-Tu species (Worner et al., 1983; Glockner and Wolf, 1984; Landini et al., 1993). The cross-kingdom activity of pulvomycin suggests that the pulvomycin binding site might be a primeval trait of an ancestral elongation factor (Londei et al., 1986). EF-Tu from Gram-negative eubacteria are more sensitive than Gram-positive bacteria (Landini et al., 1993). No sequences are available from an EF-Tu species that is known to be resistant to pulvomycin. All three sites R230, R333 and T334 are highly conserved in 64 available EFTu and EF- 1 cx sequences (R333 has undergone a relatively conservative change to His in EF-loc). Concerning the mechanism of protein synthesis inhibition, it has been assumed previously that pulvomycin inhibits protein synthesis by preventing the EF-Tumediated delivery of aa-tRNA to the ribosome only, since pulvomycin destroys the aa-tRNA binding ability of EFTu GTP (Wolf et al., 1978). Our observation that pulvomycin sensitivity is dominant over resistance, both in vivo (Table I) and in vitro in poly(Phe) synthesis (Figure 4B), is in conflict with this assumption; if simple starvation of the ribosome for aa-tRNA was the case, then a pulvomycinresistant EF-Tu should be able to alleviate this regardless of the presence of pulvomycin-sensitive EF-Tu. It follows that wtEF-Tu-GTP-pulvomycin must inhibit protein synthesis by active disturbance of synthesis sustained by a resistant EF-Tu species, rather than passive substrate limitation. Obviously, this is a much more effective mechanism of action for an antibiotic. Much lower concentrations are needed to knock out translation than the ribosome substrate starvation model, which would require systematic inactivation of most of the cellular EF-Tu. Added to this, EF-Tu is the most abundant protein in the bacterial cell (Pedersen et al., 1978) and cellular growth rate is not very sensitive to a drop in EF-Tu concentration (growth rate drops by 17-20% when the tufB gene is
Pulvomycin-resistant mutants of EF-Tu
interrupted and the EF-Tu concentration drops by 35-40%; van der Meide et al., 1982; Tubulekas and Hughes, 1993). An explanation for dominant sensitivity must account for the active disturbance of EF-TuAp functions by wtEFTu. Since dominance is observed in vitro in poly(Phe) synthesis, one can discount inhibition due to excessive intrinsic GTPase activity or to disturbance of alternative regulatory functions that have been proposed for EF-Tu (Young and Bernlohr, 1991). A mechanism of dominant inhibition through sequestering EF-Ts, as reported by Hwang et al. (1989) for EF-Tu mutants at position 136, can be discounted since the pulvomycin sensitivity of ApBs strains remained unchanged when transformed with the EF-T's overproducing plasmid pTS32 (results not
shown). At present the mechanism of dominant sensitivity is
unknown, although we favour two possible mechanisms involving the ribosome. The first entails a blockage of the ribosome by the EF-Tu-pulvomycin-ribosome interaction, as for kirromycin, although evidence for this is not strong. For kirromycin, translation is inhibited by an anomalously strong EF-Tu GDP-aa-tRNA-ribosome complex that does not leave the ribosome following GTP hydrolysis, thereby inactivating the ribosome (for a review see Parmeggiani and Swart, 1985). The formation of this anomalous complex requires both aa-tRNA and mRNA (Wolf et al., 1974, 1977), which indicates that a different mechanism occurs for pulvomycin since pulvomycin prevents aa-tRNA binding by EF-Tu-GTP. However, kirromycin also lowers the affinity of EF-Tu-GTP for aa-tRNA (Abrahams et al., 1991), although this drop in affinity by three orders of magnitude does not prevent ternary complex formation since the cellular concentration of aa-tRNA is extremely high (100 ,uM; Pingoud et al., 1982b). The two antibiotics also affect the ribosome-stimulated EF-Tu GTPase differ-
ently; pulvomycin lowers, whereas kirromycin strongly stimulates this activity (Wolf et al., 1978; Anborgh and Parmeggiani, 1991). This may reflect that pulvomycin weakens the EF-Tu-ribosome interaction (repudiating an EF-Tu/ribosome model for the action of pulvomycin) or it may reflect that EF-Tu-GTP-pulvomycin binds stably to the ribosome in a way that does not result in GTP
hydrolysis. An alternative model to explain dominance centres on evidence of a cooperative interaction between more than one EF-Tu in the elongation cycle (Vijgenboom et al.,
1985; Ehrenberg et al., 1990; Anborgh et al., 1991; Weijland and Parmeggiani, 1993, 1994). Pulvomycin may disturb this as yet unclear cooperativity so that wtEFTu inactivates EF-TuAp Further experiments to explain sensitivity dominance are required and are being performed at present.
Materials and methods Genetic techniques and growth media The genotypes, traits and sources of bacterial strains, phages and plasmids are presented in Table II. Standard molecular genetic methods were employed (Berget et al., 1988). A T7 DNA polymerase sequencing kit (Pharmacia) was used, with single-stranded M 13 phage DNA as template. The rich growth medium used (LC medium), antibiotic resistance selections and M 13 genetic techniques were as described in Zeef and Bosch (1993). Pulvomycin, 50% pure, was isolated according to Smith et al. (1985) and used as a 10 mg/ml stock solution, dissolved in
methanol and kept at -20°C. Kirromycin was a generous gift from GistBrocades NV (Delft, The Netherlands). MI 3-mediated targeted mutagenesis of the chromosomal tufA gene was performed as described in Zeef and Bosch (1993) with Ml3mp9Zam20 [similar to MI 3mp9Zam2, except that the tet resistance (tetr) genes were cloned as a HindIlI fragment in M 13mp9Zas2 in the opposite orientation to that shown for M13mp9Zam2]. The following alterations of the standard protocol were made: 10 ,l M13mp9Zam2O mutagenized with EMS was used to infect I ml EV4 F'cam (ASB::Mu) cells in logarithmic growth phase. After 1 h incubation at 37°C, cells were pelleted by centrifugation and unabsorbed phage was removed as supernatant. Cells were resuspended in 1 ml LC medium and then incubated for a further hour at 37°C. Cells were then pelleted by centrifugation, resuspended in 50 gl supernatant and plated onto agar plates (35 mm diameter Petri dishes; Greiner Labotechnik) containing 100 gg/ml pulvomycin, 50 ,ug/ ml tet and 1.5 mM EDTA. Due to increased lysis in liquid cultures in the presence of EDTA, MIC assays for pulvomycin and kirromycin were performed by streaking growing bacteria to single cells on agar plates containing 25 .g/ml tet, 1.5 mM EDTA and varying concentrations of antibiotics (0-150 ,ug/ml for pulvomycin and 0-100 ,ug/ml for kirromycin). The MIC was determined as the concentration preventing any colony formation after a 24 h incubation period at 37'C. Growth rate determinations were performed in liquid cultures (LC medium) by measuring the time required for a doubling of the optical density at 560 nm in the logarithmic growth phase. P1 phage transduction mapping with phage lysates of LZ32L, LZ33L or LZ34L (ApB::Mu) was performed by infecting strains EV4 (ASB::Mu) and LBE2040 F'Cam (AsBsfus) and then scoring for gain of tetr (Ml3mp9Zam2O contains a tetr gene). Due to our limited supply of pulvomycin, only 10 colonies of each mutant were tested for cotransduction of pulvomycin and fusidic acid resistance. This was performed by patching colonies on agar plates and then picking and patching further onto pulvomycin plates. In this way, cell concentrations were low enough to allow for the clear determination of phenotype. For EV4 (ASB::Mu) transductions, all 30 tetr transductants (ApB::Mu) tested were also found to be pulvomycin-resistant, showing that the pulvomycin resistance mutations were closely linked to the tetr marker (Ml3mp9Zam2O lysogen). For LBE2040 F'Cam (AsBsfus), all 30 tetr transductants [LZ35L, LZ36L and LZ37L (ApBS)] were found to be sensitive to fusidic acid (500 gg/ml). This showed that the tetr markers (and so also the pulvomycin resistance mutations) were linked to the fus gene, the third gene of the str operon of which tufA is the fourth. All 30 LBE2040 F'Cam (ASBS) tetr transductants (ApBS) were pulvomycin-sensitive (Table I).
Isolation of biological components and in vitro methods wtEF-Tu was purified from strain MG 1655, and EF-TuR230C, EFTuR333C and EF-TuT334A were isolated from LZ34L, LZ32L and LZ33L, respectively. Cells were grown in LC medium containing 25 tg/ ml tet and were harvested in the late logarithmic growth phase. The EFTu mutants were isolated to 90-95% purity by DEAE-Sepharose chromatography followed by Aca44 chromatography, essentially as reported (Anborgh et al., 1992). EF-Ts, EF-G, ribosomes and PhetRNAPhe were prepared as reported (Parmeggiani and Sander, 1981). For in vitro studies, working stocks of 2.5 mM pulvomycin in N,Ndimethylformamide were prepared. Isoelectric focusing (according to O'Farrell, 1975) of EF-TuR230C and EF-TuR333C confirmed the expected acidic shift with respect to wtEF-Tu (data not shown). Poly(U)-directed poly(Phe) polymerization and dissociation rate constants of EF-Tu-GDP complexes were measured as described by Anborgh et al. (1992). The protection of Phe-tRNA against non-enzymatic hydrolysis was determined according to Pingoud et al. (1977). The GTPase activity of EF-Tu was measured by sequestering the 32Pi that was cleaved from [y-32P]GTP according to Ivell et al. (1981).
Methods of structural analysis Comparison of 64 EF-Tu and EF- I a sequences retrieved from the EMBL and GenBank data banks was performed by means of the 'pileup' function of the GCG computer program (Genetics Computer Group, 1991). We found, as did other authors (e.g. Cousineau et al., 1992), that this alignment produced three classes of proteins that correspond to the three kingdoms of evolutionary descent: 28 eukaryote EF- 1 a, six archaebacterial EF- 1 a and 31 chloroplast, mitochondrial and eubacterial EF-Tu (Zeef, 1994). These data were considered, together with the crystal structures of
5119
L.A.H.Zeef et aL Ecoli EF-Tu-GDP (Kjeldgaard and Nyborg, 1992) and Tthermophilus EF-TuGppNHp (Berchtold et al., 1993). Since the EF-Tu sequences from these two species have high homology (70% identity; Yokota et al., 1980; Kushiro et al., 1987), we have made the assumption that the structure of Ecoli EF-Tu GTP (which is at present unknown) is similar to that of Tthermophilus EF-Tu-GTP. The high resolution (better than 1.7 A) of the Tthermophilus EFTu.GppNHp structure (Berchtold et al., 1993) allows the prediction of side-chain interactions in the wild-type and mutant proteins. However, in the EF-Tu-GDP structure this is not possible with the same confidence because of the lower resolution and higher crystallographic thermal factors (Kjeldgaard and Nyborg, 1992; J.Nyborg, personal communication).
Acknowledgements We are grateful to R.van Mill for help in creating coordinates for pulvomycin, to R.Cool for supplying sequence oligomers, to D.Hughes for supplying plasmid pTS32 and to B.Kraal for help in completing this manuscript.
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