JOURNAL OF BACTERIOLOGY, JUlY 1986, p. 265-271 Vol. 167, No. 10021-9193/86/070265-07$02.00/0Copyright © 1986, American Society for Microbiology

Unique Antibiotic Sensitivity of Archaebacterial PolypeptideElongation Factors

PAOLA LONDEI,l JOSE LUIS SANZ,2 SERGIO ALTAMURA,' HEIDI HUMMEL,3 PIERO CAMMARANO,l*RICARDO AMILS,2 AUGUST BOCK,3 AND HEINZ WOLF4

Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Policlinico Umberto I, 00161, Rome, Italy'; Centro deBiologia Molecular, Consejo Superior de Investigaciones Cientificas and Universidad Autonoma Madrid, Madrid, Spain2;and Lehrstuhl fur Mikrobiologie der Universitat Munchen, D-8000 Munich 19,3 and Institut fur Biologie II, Lehrstuhl fur

Mikrobiologie I der Universitat, D-7400 Tubingen,4 Federal Republic of Germany

Received 5 August 1985/Accepted 24 February 1986

The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA toribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and thetranslocation of peptidyl-tRNA (elongation factor G [EF-GI for eubacteria and elongation factor 2 [EF2] foreucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycm andpulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitorswith the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey ofmethanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organismsbelonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibitdifferent antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EFl)-equivalentprotein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer,whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineageheterogeneity of elongation factors not encountered within each of the two reference (eubacterial andeucaryotic) kingdoms.

The two classically recognized lines of cellular descent,eubacteria and eucaryotes, are each endowed with a specificset of polypeptide chain elongation factors whose compo-nent parts usually can only cooperate with ribosomes of theirown lineage (14, 18). Within each lineage, however, factorsand ribosomes are functionally exchangeable for in vitropolypeptide synthesis (5).

Elongation factors and ribosomes of the recently discov-ered archaebacteria (7) appear to define a third class offunctional specificity encompassing organisms as taxonomi-cally diverse as methane-producing (Methanococcus vanni-elii) and sulfur-dependent (Thermoplasma acidophilum)archaebacteria (13); that is, reciprocal combinations of fac-tors and ribosomes from distant archaebacterial sources areactive in polypeptide synthesis, but archaebacterial factorsare functionally restricted to ribosomes of their own lineage.The aim of the present study was to establish whether

intralineage distinctions among archaebacterial elongationfactors could be detected on structural grounds. For thispurpose, factors from a variety of archaebacteria coveringmost known divisions of the "third kingdom" were probedfor the presence or absence of specific sites which, ineubacteria and eucaryotes, interact with factor-targeted in-hibitors of protein synthesis. This approach also providesinsight into the degree of structural relatedness amongfunctionally homologous factors of the three kingdoms.Indeed, a search for common traits among factors of thethree kingdoms has revealed that a specific domain which isADP ribosylatable by diphtheria toxin is shared by the

* Corresponding author.

peptidyl-tRNA translocating proteins of both eucaryotes andarchaebacteria (10-12).Three factor-targeted drugs, pulvomycin, kirromycin, and

fusidic acid, have been used to reveal eubacterial andeucaryotic traits on archaebacterial factors. Kirromycin (25)and pulvomycin (26) both act upon the aminoacyl-tRNA-binding factor of most eubacteria (elongation factor Tu[EF-Tu]) but not upon its eucaryotic counterpart (elongationfactor 1 [EF1]); fusidic acid interacts with the peptidyl-tRNA-translocating factor of both eubacteria (elongationfactor G [EF-G]) and eucaryotes (elongation factor 2 [EF2])(20, 21).The principal result presented here is that archaebacteria

are endowed with distinct sets of factors differing in theirsensitivities to certain factor-targeted drugs.

MATERIALS AND METHODS

Organisms. The following organisms were used:Halobacterium mediterranei, "Halobacterium maris-mortui," Methanosarcina barkeri (DSM 1232), Metha-nospirillum hungatii (DSM 864), Methanobacteriumthermoautotrophicum (DSM 1053), Methanobacteriumformicicum (DSM 1312), Methanococcus vannielii (DSM1224), Sulfolobus solfataricus (DSM 1616), Thermoplasmaacidophilum (DSM 1278), Thermoproteus tenax (DSM2078), Desulfurococcus mobilis (DSM 2161), Thermococcusceler (DSM 2476), Escherichia coli D10 (8), " Thermusthermophilus" HB8, and Saccharomyces cerevisiae Y166.The sulfur-dependent archaebacteria were generously sup-pied by W. Zillig as frozen cell pastes.

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Poly(U)-programmed polyphenylalanine synthesis.Halobacterial ribosomes and ribosome-free supernatantfractions were prepared by a modification of the method ofBayley and Griffith (1). Cells from the exponential phase ofgrowth (A420, 0.7 to 1.0) were broken with alumina powder(16) while 2.0 volumes (per gramn of cells [wet weight]) ofribosome extraction buffer containing 30 mM Tris hydro-chloride (pH 7.6), 3.4 M KCl, 60 mM magnesium acetate, 7.0mM mercaptoethanol, and 2 ,ug of RNase-free DNase(Worthington Diagnostics) per ml were gradually added. Thecell extracts were centrifuged twice at 20,000 x g and theupper two-thirds of the resultant supernatant was centri-fuged in a Beckman 50 Ti Spinco rotor at 50,000 x g and 7°Cfor 3 h. The ribosome pellets were dissolved in extractionbuffer, and the ribosome-free supernatant (termed S150fraction) was divided into portions and stored at -70°C.Incubation mixtures for the assay of polyphenylalaninesynthesis by halobacterial ribosomes contained (per millili-ter) 30 Rxmol of Tris hydrochloride (pH 9.0), 400 ,umol ofNH4Cl, 1.5 mmol of (NH4)2SO4, 1.0 mmol of KCI, 30 ,molof magnesium acetate, 2.1 ,mol of ATP, 5.0 ,umol ofphosphoenolpyruvate, 7.0 ,umol of mercaptoethanol, 2.5 ,ugof phosphoenolpyruvate kinase, 1.0 mg of yeast tRNA(Boehringer Mannheim Biochemicals), 800 ,ug of poly(U), 36nmol of [3H]phenylalanine (specific activity, 140 mCi/mmol),10 A260 units of ribosomes, and an optimal amount (ca. 2.0A260 units) of the S150 fraction.

Poly(U)-directed polyphenylalanine synthesis by Methano-bacterium formicicum, Methanobacterium thermoautotro-phicum, and Methanococcus vannielii ribosomes was car-ried out by protocols optimized for each organism (A. DiGiambattista, H. Hummel, A. Bock, and C. Cocito, Mol.Gen. Genet., in press). Poly(U)-directed cell-free systemsfrom Methanospirillum hungatii and Methanosarcinabarkeri were prepared and polyphenylalanine was assayedby the methods of Hummel et al. (9).

Poly(U)-directed cell-free systems from Sulfolobussolfataricus were prepared by the method of Cammarano etal. (3, 4). The reaction mixtures (65 RI) contained (permilliliter) 15 ,umol of Tris hydrochloride (pH 7.3), 6.0 p.molof NH4Cl, 18 ,umol of magnesium acetate, 1.0 Rmol ofdithiothreitol (DTT), 3.0 ,umol of spermine, 2.4 pmol ofATP, 1.6 p.mol of GTP, 20 nmol of [3H]phenylalanine(specific activity, 100 mCi/mmol), 160 ,ug of poly(U), 10 A260units of ribosomes (240 pmol), and an optimal amount of theribosome-free, 100,000 x g supernatant (termed S100 frac-tion) (3, 4) (ca. 2.0 mg of protein). Poly(U)-directed cell-freesystems from D. mobilis and Thermococcus celer wereprepared and polyphenylalanine synthesis was assayed asdescribed for Sulfolobus solfataricus with several modifica-tions (see Fig. 3).

Thermoproteus tenax could only be used as unfractionatedcell extracts because of the loss of activity consistentlyobserved upon ribosome isolation. The cells were brokenwith alumina powder while 2.0 volumes (per gram of cell[wet weight]) of extraction buffer containing 20 mM Trishydrochloride (pH 7.3), 10 mM magnesium acetate, 10 mMNH4Cl, 0.5 mM DTT, and 2 ,ug of RNase-free DNase per mlwere gradually added. The homogenates were centrifugedtwice at 30,000 x g, and the supe-natant fractions (termedS30 extracts) were divided into portions, shock frozen, andstored in small aliquots at -70°C. The incubation mixturesfor the assay of polyphenylalanine synthesis by T. tenaxribosomes contained (per milliliter) 230 ,ul of S30 extract(equivalent to 75 mg of cells [wet weight]), 15 ,umol of Trishydrochloride (pH 7.3), 10 ,umol of NH4Cl, 15 p.mol of

magnesium acetate, 1.0 ,Lmol of spermine, 3.0 ,umol of DTT,2.0 ,umol of ATP, 1.0 ,umol of GTP, 10 nmol of [3H]phenyl-alanine (specific activity, 100 mCi/mmol), and 200 ,ug ofpoly(U).Poly(U)-directed polyphenylalanine synthesis by

Thermoplasma acidophilum ribosomes was assayed by themethod of Klink et al. (13) with the following modifications:N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid(HEPES) (pH 7.0) was 20 mM, KCl was substituted for byNH4Cl (100 mM), and spermine was 1.0 mM.

Cell-free systems from the reference eubacterial (E. coliand "Thermus thermophilus") and eucaryotic (Saccharomy-ces cerevisiae) species were prepared and polyphenylalaninesynthesis was assayed as detailed by Cammarano et al. (4).Temperature conditions and radioactivity measurements.

Reaction mixtures (65 to 125 ,ul) were incubated at thefollowing optimal temperatures: E. coli and methanogenicbacteria, 37°C; halobacteria, 40°C; Thermoplasmaacidophilum, 58°C; Sulfolobus solfataricus, Thermoproteustenax, D. mobilis, Thermococcus celer, and "Thermusthermophilus," 70°C, unless otherwise specified; and Sac-charomyces cerevisiae, 30°C. At the end of the incubationperiod (35 to 75 min), the hot trichloroacetic acid (TCA)-insoluble radioactivity was assayed by the filter papermethod of Mans and Novelli (15) or by filtration throughglass fiber disks as described previously (6).

Preparation of [3H]phenylalanyl-tRNA. Bulk tRNA wasisolated from Sulfolobus solfataricus as described previ-ously (3); charging with [3H]phenylalanine (specific activity,9 Ci/mol) was performed by the method of Tome et al. (22)with Sulfolobus solfataricus ribosome-free supernatant(S100 fraction) as the source of aminoacyl-tRNA syn-thetases. The specific activity of [3H]phenylalanyl-tRNAwas 150,000 cpm/A260 unit.

RESULTS

Interactions of drugs with elongation factors. The interac-tions of pulvomycin, kirromycin, and fusidic acid witharchaebacterial elongation factors were assayed by usingpoly(U)-programmed cell-free systems under optimal ionicand temperature conditions for polyphenylalanine synthesis.The three factor-targeted drugs act by different mechanisms.Pulvomycin inhibits the formation of a ternary complexbetween EF-Tu, aminoacyl-tRNA, and GTP (26), whereaskirromycin prevents EF-Tu-GDP from leaving the ribosome(25); fusidic acid acts by stabilizing the ribosome-GDP-EF-G and EF2 complexes (23). Whereas the former two com-pounds interact with sensitive factors in the absence ofribosomes, fusidic acid only binds to a ribosome-boundfactor-GDP complex.Methanogenic archaebacteria. The effects of pulvomycin,

kirromycin, and fusidic acid on the polyphenylalanine-synthesizing capacities of cell-free systems derived fromrepresentative members of the family Methanobacteriaceae,the family Methanomicrobiaceae, and the family Methano-coccaceae, and from the reference eubacterial (E. coli) andeucaryotic (Saccharomyces cerevisiae) species (dashedlines) are shown in Fig. 1.The significant result in Fig. 1 is the evidence that a

uniform pattern of antibiotic sensitivity encompasses thephylogenetically diverse groupings investigated. As Fig. 1shows, the methanobacterial EF-Tu and EF1 [EF-Tu(EF1)]-equivalent factor was blocked by pulvomycin concentrationsequal to or lower than those required to block eubacterialEF-Tu activity; unlike the eubacterial factor, however, the

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ANTIBIOTIC (MOL/L)

FIG. 1. Effects of pulvomycin, kirromycin, and fusidic acid on the polyphenylalanine-synthesizing capacities of methanogen ribosomes.Symbols: A, E. coli; A, Saccharomyces cerevisiae; 0, Methanosarcina barkeri; 0, Methanospirillum hungatii; O, Methanobacteriumthermoautotrophicum; v, Methanococcus vannielii; *, Methanobacteriumformicicum. Synthetic capacities (phenylalanines polymerized perribosome in 45 min) corresponding to 100% values on the ordinate were as follows: Methanosarcina barkeri and Methanococcus vannielii,10; Methanobacterium thermoautotrophicum and Methanospirillum hungatii, 5; Methanobacterium formicicum, 2; E. coli, 20; andSaccharomyces cerevisiae, 8.

methanobacterial factor appeared to be either insensitive tokirromycin up to lo-3 M or only inhibited at very high drugconcentrations (Methanobacterium formicicum and Meth-anobacterium thermoautotrophicum). The EF-G(EF2)-equivalent factor of all the methane-producing archae-bacteria surveyed was systematically inhibited by fusidicacid within the same range of effective concentrations as thataffecting the functionally homologous factors of E. coli andSaccharomyces cerevisiae.

Halophilic archaebacteria. The effects of the three factor-targeted drugs on the activities of poly(U)-directed systemsfrom Halobacterium mediterranei and "Halobacteriummaris-mortui" are shown in Fig. 2. It is evident that theantibiotic sensitivity spectrum of the halobacteria, having akirromycin-insensitive but pulvomycin-sensitive EF-Tu(EF1)-equivalent protein and a fusidic acid-sensitive EF-G(EF2)-equivalent protein, was essentially superimposableon that of the methanogenic bacteria. This result reinforcesthe notion (7, 27) that a close phylogenetic relationship existsbetween methane-producing and halophilic archaebacteria.The fusidic acid sensitivity of the halobacterial peptidyl-tRNA translocases was less pronounced than that of themethanobacterial factor. It is open to debate whether this iscaused by nonspecific effects arising from the extremely highsalt concentration of the halobacterial assay system orwhether this reflects a weaker fusidic acid-binding site thanexists in methanobacteria.

Sulfur-dependent (thermophilic) archaebacteria. It hasbeen shown previously that pulvomycin, kirromycin, andfusidic acid all fail to affect polyphenylalanine synthesis incell-free systems derived from the thermoacidophilic, sulfur-dependent archaebacterium Sulfolobus solfataricus (3, 4).

Here, assays of factor-targeted drugs were extended to othersulfur-dependent thermophiles representative of the orderThermoproteales (Thermoproteus tenax, D. mobilis, andThermococcus celer) and to the seemingly isolated orderhaving Thermoplasma acidophilum as its sole known repre-sentative (27). The effects of pulvomycin, kirromycin, andfusidic acid on the polyphenylalanine-synthesizing capaci-ties of cell-free systems derived from representatives of theorder Thermoproteales and Thermoplasma acidophilum areshown in Fig. 3, together with the results of control assayswith "Thermus thermophilus" and E. coli as the referencespecies.The control experiments (Fig. 3, dashed lines) demon-

strated that kirromycin and fusidic acid were both capable ofefficiently interacting with sensitive target factors of"Thermus thermophilus" at 70°C (the optimal temperaturefor the archaebacterial assay systems), whereas pulvomycin-treated samples that had been preheated at 65°C for 30 minretained their full inhibitory activity when assayed in the E.coli system at 37°C.As the plots show, the sensitivity of sulfur-dependent

archaebacteria to factor-targeted drugs was quite distinct,albeit less uniform, than that of the methanogenic bacteria.The thermophilic EF-Tu(EF1)-equivalent factors sharedwith their methanogenic and halophilic counterparts a lackof sensitivity to kirromycin; unlike the methanogens andhalophiles, however, the thermophiles were also unaffectedby pulvomycin, with the only exception being Thermo-coccus celer, whose EF-Tu(EF1)-equivalent factor superfi-cially resembled those of the methanogens and halophiles inbeing kirromycin insensitive but pulvomycin sensitive.The thermophilic EF-G(EF2)-equivalent factor also dif-

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KIRROMYCIN

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FIG. 2. Effects of pulvomycin, kirromycin, and fusidic acid on the polyphenylalanine-synthesizing capacities of halobacterial ribosomes.Symbols: A, E. coli; A, Saccharomyces cerevisiae; 0, Halobacterium mediterranei; 0, "Halobacterium maris-mortui". Halobacterialribosomes statistically polymerized 10 to 12 phenylalanine residues per ribosome in 45 min.

fered from the functionally homologous proteins of themethanogens and halophiles in being sensitive to fusidicacid, with the exception of Thermoproteus tenax, whosepeptide chain translocating factor responded to fusidic acid

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concentrations that were considerably lower (about twoorders of magnitude) than those required to produce acomparable inhibition of the two reference assay systems.The possibility that the fusidic acid sensitivity of

ANTIBIOTIC ( MOL/L )FIG. 3. Effects of pulvomycin, kirromycin, and fusidic acid on the polyphenylalanine-synthesizing capacities of ribosomes of sulfur-

dependent archaebacteria. Symbols: A, E. coli; A, E. coli assayed with preheated antibiotics; ', "Thermus thermophilus"; 0, Thermoplasmaacidophilum; 0, Thermoproteus tenax; M, D. mobilis; 0, Thermococcus celer. Synthetic capacities (phenylalanines polymerized perribosome in 45 min) corresponding to 100% values on the ordinate were as follows: Thermoplasma acidophilum and Thermococcus celer, 20;D. mobilis, 10; Thermoproteus tenax, 5; "Thermus thermophilus", 3; E. coli, 15. D. mobilis and Thermococcus celer poly(U) systems werecomposed as described for Sulfolobus solfataricus (see Materials and Methods), with the following modifications: the D. mobilis systemcontained 1.0 A260 unit per ml of Sulfolobus solfataricus unfractionated tRNA; and the Thermococcus celer system contained 1.0 A260 unitper ml of Sulfolobus solfataricus tRNA, 100 mM NH4Cl, and 0.2 mM spermine.

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ARCHAEBACTERIAL ELONGATION FACTORS AND ANTIBIOTICS 269

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bols: 0, clproteins attem contaThermoplaing Thermvannielii Scoccus va,proteins. Isystems w

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-AXistraight-forward because fusidic acid will only interact witha ribosome-bound factor-GDP complex (23). However, (i)bacterial mutants that are resistant to fusidic acid all have analtered EF-G protein (21), and (ii) Fig. 4 shows that thespecificity of the in vitro response to fusidic acid resides onlyin the factor. Most likely, therefore, fusidic acid interactsprimarily, if not exclusively, with the factor rather than theribosome.Perhaps the most unexpected result in the present survey

is that, much unlike the case for eubacterial and eucaryoticelongation factors, archaebacterial elongation factors dis-play a remarkable heterogeneity in their response to factor-targeted drugs.

Antibiotic sensitivity spectra of archaebacterial factorsfrom this and previous reports (3, 10) are summarized inTable 1. It is evident that factors belonging to methanogenic-

\ ^ halophilic and sulfur-dependent archaebacteria possess bothsome common and some distinguishing features. The fea-tures in common are the insensitivity of the EF-Tu(EF1)-

I'-6 -5 -4 3 equivalent protein to kirromycin and the sensitivity of the0 106 10- 104 10- EF-G(EF2)-equivalent protein to ADP ribosylation by the

ANTIBIOTIC (MOL/L) diphtheria toxin reaction. The distinguishing features are theEffect of fusidic acid on hybrid cell-free systems. Sym- sensitivities to pulvomycin and fusidic acid. Here, a sharp

ell-free system containing Methanococcus vannielii S100 distinction exists between methanogens and halophiles onnd Methanococcus vannielii ribosomes; 0, cell-free sys- the one hand and sulfur-dependent thermophiles on thelining Thermoplasma acidophilum S100 proteins and other. Organisms in the former group all contain aisma acidophilum ribosomes; A, cell-free system contain- pulvomycin-sensitive EF-Tu(EF1)-equivalent factor and azoplasma acidophilum ribosomes and Methanococcus fusidic acid-sensitive EF-G(EF2)-equivalent factor. By con-100 proteins; A, cell-free system containing Methano- trast, those in the latter group are unaffected by bothnnielii ribosomes and Thermoplasma acidophilum S100 antibiotics. There are, however, two exceptions: Thermo-rhe composition of the homologous and hybrid cell-free coccus celer, whose EF-Tu(EFl)-equivalent protein is sen-,ere as for Thermoplasma acidophilum (see Materials and sitive to pulvomycin, and Thermoproteus tenax, whoseexcept that the temperature was 47°C (instead of 58°C) EF-G(EF2)-equivalent protein is blocked by fusidic acid. Inienylalanyl-tRNA (10,000 cpm per assay; specific activity, Fequres, ins is tockedrbycin and Inzm/A260 unit) was used instead of free [3H]phenylalanine general, two features, insensitivity to kirromycin and sensi-rials and Methods). tivity to diphtheria toxin, are systematic. Two others, sen-

sitivity to pulvomycin and sensitivity to fusidic acid, areunevenly distributed among the archaebacteria. The distri-

roteus tenax might be due to the use of S30 extracts bution of the antibiotic sensitivity spectra in Table 1 shows aerials and Methods) was discounted because S30 correlation with the division of the archaebacteria based onfrom Sulfolobus solfataricus were as insensitive to similarities in 16S rRNA sequences (7) and subunit compo-Lcid as were the reconstituted cell-free systems sitions of the DNA-dependent RNA polymerases (27). TheselOt shown). split the third kingdom into two main branches, one embrac-)ssibility that the specificity of the response to ing methanogens and halophiles, and the other encompass-,id resides in the ribosomes, rather than the factors, ing sulfur-dependent thermophiles. It is still unclear, how-ounted by the results presented in Fig. 4. These ever, whether Thermococcus celer is to be placed in thehowed that a hybrid cell-free system containing sulfur-dependent or methanogenic-halophilic branch (C. R.omponents of fusidic acid-sensitive Methanococcus Woese, personal communication); its unique sensitivity toand ribosomes of fusidic acid-insensitive Thermo- pulvomycin may suggest a closer relationship with the lattericidophilum was as sensitive to fusidic acid as was group.logous system containing Methanococcus vannielii One interesting aspect of the results is that all the factor--s and factors; conversely, a reciprocal combination targeted antibiotics, except kirromycin, are effective in moreng Methanococcus vannielii ribosomes and than one kingdom. The possibility that identical antibiotic-,lasma acidophilum soluble components was unaf- binding sites arose independently in more than one line offusidic acid up to i0-3 M. cellular descent seems unlikely. A simpler explanation of the

data would be that the recurrent (or shared) features (sensi-DISCUSSION tivity of peptidyl-tRNA translocases to fusidic acid and to

iree antibiotics used in this study are sensitive diphtheria toxin; sensitivity of aminoacyl-tRNA-binding fac-or the detection of specific traits on elongation tors to pulvomycin) correspond to primeval traits of thePulvomycin and kirromycin bind, in the absence of ancestral factors differentially retained by the three primary-s, to different sites on the EF-Tu protein (17). The lineages after their divergence from the progenitor stage (7).on EF-Tu of two sites, one for kirromycin and one Accordingly, the distribution of pulvomycin and fusidic acid

imycin, is further supported by the evidence in Fig. sensitivities among the archaebacteria may reflect a differ-shows that the EF-Tu(EF1)-equivalent proteins of ential loss of these two features during archaebacterialgenic and halophilic archaebacteria are sensitive to evolution. The kirromycin-binding site, which appears onlycin but insensitive to kirromycin. The binding of in eubacteria would, in this case, represent a more recentLcid to the EF-G(EF2)-equivalent protein is less trait which arose within the eubacterial lineage.

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TABLE 1. Antibiotic sensitivity of archaebacterial polypeptide elongation factors

Sensitivitya of indicated elongation factor to indicated drug

Organism EF-Tu(EFl) EF-G(EF2)

Kirromycin Pulvomycin Fusidic acid Diphtheria toxin

Methanobacterium formicicum 0 + + +Methanobacterium thermoautotrophicum 0 + + +Methanosarcina barkeri 0 + + +Methanococcus vannielii 0 + + +Halobacterium mediterranei 0 + + +"Halobacterium maris-mortui" 0 + + +

Thermococcus celer 0 + 0 +Thermoproteus tenax 0 0 + +Thermoplasma acidophilum 0 0 0 +Sulfolobus solfataricus 0 0 0 +Desulfurococcus mobilis 0 0 0 +

Escherichia coli + + + 0Saccharomyces cerevisiae 0 0 + +

a 0, Insensitive; +, sensitive.

We should emphasize that the use of protein synthesisinhibitors as probes for tracing the evolution of certaintertiary structure domains rests upon the assumption that therelevant binding sites on factors or on ribosomes possess ahigh degree of evolutionary stability; that is, antibioticsensitivity spectra are conserved within each of the tworeference (eubacterial or eucaryotic or both) kingdoms inde-pendently of physiological specialization or taxonomic kin-ship of the organisms and regardless of niches colonized bythe species along their evolutionary course. The three factor-targeted drugs used in this study meet the requirements for adeep chemical probe suited for the detection of specific traitswhich, in eubacteria or eucaryotes or both, have beenmaintained throughout long periods of the evolutionary past.For instance, "extremophiles," such as Bacillus stearother-mophilus (4), Bacillus acidocaldarius (3), and "Thermusthermophilus" (3, 24; this report), are just as sensitive tokirromycin and pulvomycin as are enteric bacteria or plantchloroplasts (24). Insensitivity to both drugs within theeubacteria appears to be limited to Lactobacillus strains(24). Sensitivity to fusidic acid appears to be generalized ineucaryotes and in eubacteria, including chloroplasts ofhigher plants (U. Tiboni, personal communication). Further-more, where primary structure data exist, these support thenotion that antibiotic-binding sites on elongation factorshave been stringently conserved. For instance, in EF2, theamino acid sequence in the vicinity of the diphthamideresidue that is ADP ribosylated by diphtheria toxin is main-tained in divergent eucaryotic species (2).

This raises the question as to the extent of primarystructural differences among the antibiotic-insensitivearchaebacterial factors and their antibiotic-sensitive coun-terparts in eubacteria or eucaryotes or both. The evidenceavailable shows that mutant factors resistant to factor-targeted drugs may differ from susceptible factors merely byone amino acid. In fact, substitutions of alanine at position375 within the allosterically important kirromycin-bindingdomain of E. coli EF-Tu result in kirromycin resistance (19).We note, however, that the basic phylogenetic stability ofthe drug-sensitive phenotype within the eubacterial kingdomindicates that the site involved in kirromycin binding issubject to severe selective constraints. That is, occasionalresistant mutants do not prevail on a phylogenetic level. Thesystematic insensitivity of archaebacteria to kirromycin

strongly suggests that primary structural differences be-tween the kirromycin-binding domain of the eubacterialfactor and the corresponding region of the archaebacterialfactor are far more extensive than those occasionally causingresistance.The lack of a uniform sensitivity of archaebacterial elon-

gation factors to factor-targeted drugs is to be added to thelist of diversities in fundamental molecular traits within thethird kingdom. These include the different subunit composi-tions of the DNA-dependent RNA polymerases in methano-genic-halophilic and sulfur-dependent archaebacteria (27),the different sensitivities of methanogenic, halophilic, andsulfur-dependent bacteria to a variety of ribosome-targetedinhibitors of protein synthesis (4, 6, 9), and the existence inarchaebacteria of two physicochemically distinct ribosomeclasses (P. Cammarano, A. Teichner, and P. Londei, Syst.Appl. Microbiol., in press). This amount of heterogeneity isin sharp contrast with the high degree of intralineage con-stancy of translational and other basic molecular processeswithin each of the two classically recognized lines of cellulardescent.

ACKNOWLEDGMENTS

We thank W. Zillig for generously supplying cells of sulfur-dependent thermophiles and Ute Bar, Gabriele Heller, and DionisioUreina for skillful technical assistance.This work was supported by grants from the Deutsche

Forschungsgemeinschaft, the Italian Ministry of Public Education,and the Comision Asesora of Spanish Research and by EuropeanMolecular Biology Organization short-term fellowships to J. L. Sanzand S. Altamura.

LITERATURE CITED1. Bayley, S. T., and E. Griffith. 1968. A cell free amino acid

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