Vol. 26, No. 6ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1984, p. 857-8620066-4804/84/120857-06$02.OOIOCopyright © 1984, American Society for Microbiology

Comparison of Fortimicins with Other Aminoglycosides and Effectson Bacterial Ribosome and Protein SynthesisNICOLE MOREAU,* CHRISTINE JAXEL, AND FRANQOIS LE GOFFIC

CERCOA (Laboratoire Associe a l'ENSCP), Centre National de la Recherche Scientifique, 94320 Thiais, France

Received 7 June 1984/Accepted 19 September 1984

Fortimicins are bicyclic aminoglycoside antibiotics that contain a fortamine moiety instead of the deoxy-streptamine found in other aminoglysides. Fortimicin A had a bactericidal effect on Escherichia coli andStaphylococcus epidermnidis and was found to inhibit protein synthesis in vivo. In vitro, fortimicin A inhibitedpolyuridylic acid-directed phenylalanine polymerization and induced misreading, as shown by leucineincorporation. In contrast, fortimicin B had no effect on either polymerization or misreading. In assaysprogrammed with natural mRNA, only a weak polymerization inhibition effect was observed with fortimicinA, whereas a strong stimulation was seen in the presence of fortimicin B. Both fortimicins A and B inhibiteddissociation of 70S ribosomes into their subunits and neither was able to displace [3H]dihydrostreptomycin,[3H]tobramycin, or [31H]gentamicin from their respective binding sites on the 70S particle.

Fortimicins A and B are pseudodisaccharide antibioticsproduced by Micromonospora olivoasterospora (7 and ref-erences cited therein). The relation between structure andactivity of a few analogs has been established (26), and manyderivatives of fortimicins as well as of other compounds ofthe same family, such as sporaricins (14 and references citedtherein), istamycins (23), sannamycins (6), and dactimicins(22), have been reported in the literature during the last 3years. The interest in this new family of aminoglycosidesshown in the literature, as well as their high resistance toenzymatic inactivation, has prompted us to explore themode of action of fortimicins. Fortimicins are structurallydifferent from major aminoglycosides, since they incorpo-rate fortamine, a novel aminocyclitol moiety, instead of2-deoxystreptamine. To our knowledge, no study dealingwith the mode of action of molecules from this series hasbeen published. It was therefore interesting to carry out sucha study, and it would not be surprising to find that the modeof action of these molecules, although related, differs fromthat of the other aminoglycosides. As a matter of fact, it iswell known (24) that streptomycin and apramycin, which arestructurally different from major aminoglycosides, exertdifferent effects on protein synthesis. Moreover, it is nowwell documented (9, 18) that even the aminoglycosides of thedeoxystreptamine group do not share either a commonbinding site or a common mode of action on the bacterialribosome.

This work is divided into three parts: (i) effects on wholebacteria, (ii) study of biosyntheses in vitro; and (iii) bindingto the bacterial ribosome.

MATERIALS AND METHODSMaterials. Materials were obtained from the following

sources: fortimicins A and B, Abbott Laboratories; gentami-cin C, Unilabo; kanamycin A, Allard-Bristol; tobramycin,Eli Lilly & Co.; neomycin B, Roussel-Uclaf; streptomycin,Rhone Poulenc.[methyl-3H]thymidine (1 Ci/mmol), [5-3H]uridine (1

Ci/mmol), L-[3,4,5-3H]lysine (40 Ci/mmol), a [U-'4C]-labeledamino acid mixture (45 mCi/matom of carbon), L-[U-14C]phenylalanine (450 mCi/mmol), and L-[U-14C]leucine

* Corresponding author.

(318.6 mCi/mmol) were purchased from the Commissariat al'Energie Atomique.The amino acids were from Serva. mRNA from MS2

phage, polyuridylic acid [poly(U); K salt], tRNA fromEscherichia coli MRE600, ATP, pyruvate kinase, and coen-zyme A were from Boehringer. GTP and dithiothreitol werefrom Fluka. Phosphoenolpyruvate was from Sigma Chemi-cal Co. Spermidine and putrescine were from Aldrich Chem-ical Co. All other chemicals were from commercial sourcesand were of the highest grade. Sephacryl S200 was fromPharmacia Fine Chemicals, Inc.

Bacterial strains and media. E. coli MRE600 (RNase I-)was supplied by the Microbiological Research Establish-ment, Porton, England, as a frozen paste. E. coli K-12,Staphylococcus epidermidis BL450, and E. coli R135 (gen-tamicin resistant) were from our laboratory collection. StrainR135 synthesizes the enzyme AAC3(I) (28), able to acetylategentamicin and fortimicins. Tryptic soy broth (Merieux) wasused as a culture medium. Davis-Mingioli minimum mediumA (5) consisted of 7 g of K2HPO4, 3 g of KH2PO4, 0.5 g ofsodium citrate. 3H20, 0.1 g of MgSO4 7H20, 1 g of(NH4)2SO4, 2 g of glucose (autoclaved separately), and 1,000ml of water (final pH 7).

Preparation of ribosomes and cell-free extracts. The cell-free extracts and ribosomes were obtained from E. coliMRE600 or R135 (gentamicin resistant). The S135 extractswere prepared as previously described from cells broken bygrinding with alumina (8). The 70S ribosomes were obtainedeither by centrifugation, as previously described (21), orchromatography on Sephacryl S200, as previously described(15). The 70S ribosomes were stored as 200-,ul aliquots inliquid nitrogen or as 2-ml aliquots at -20°C in the presenceof 30% MeOH. Cell-free extracts and ribosomes from E. coliR135 (gentamicin resistant) were prepared by the sameprocedure.

Determination of MICs and effects of drugs on bacterialsurvival. The MICs were determined by the classical agardilution technique. Serial twofold dilutions of the drug weremixed with melted Mueller-Hinton agar. The plates wereinoculated with a dilute bacterial suspension, grown intryptic soy broth. The MIC (in micrograms per milliliter) wasrecorded after a 12-h incubation at 37°C. Cultures of E. coliwere grown in tryptic soy broth to about 3 x 108 cells, and

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858 MOREAU, JAXEL, AND LE GOFFIC

cultures of S. epidermidis were grown in the same mediumto about 8 x 107 cells per ml (turbidity of 50; measured witha Hach turbidimeter, model 2100A). Various concentrationsof the drugs were added to the culture tubes, which wereincubated at 37°C. After various periods of incubation,samples were withdrawn, the turbidity was measured, andthe viable counts were estimated by plating on drug-freenutrient agar. The lowest concentration which yielded nogrowth was designated the MBC.

Determination of cellular macromolecule syntheses. Mix-tures (10 ml) containing 2 x 108 E. coli cells per ml (turbidityof 20), suspended in Davis-Mingioli minimum medium, werepreincubated for 15 min at 37°C with an adequate drugconcentration. [3H]thymidine (0.25 ,uCi; 50 mCi/mmol) anddeoxyadenosine (50,ug), [3H]uridine (0.25 ,uCi; 54.3mCi/mmol), or [3H]lysine (1.42 ,Ci; 26.58 mCi/mmol) wasthen added. After 30 min of incubation, 1 ml of cold 10%trichloroacetic acid was added, and the mixtures werefiltered through Whatman GF/A glass fiber filters. The filterswere washed three times with S ml of cold 1% trichloroaceticacid, dried, and counted in 5 ml of Beckman NA scintillationfluid.

In vitro protein synthesis. In vitro protein synthesis pro-grammed by mRNA from phage MS2 was conducted aspreviously described (16), with the following minor modifi-cations: 4.7 mM Mg(OAc)2; 0.47 mM Ca2+; 7.5 mM putres-cine; 0.94 mM spermidine; 4.7 mM NH4Cl; 8.9 mM KCi;0.94 mM dithiothreitol; 0.94 mM ATP; 0.94 mM GTP; 11.25mM phosphoenolpyruvate (K salt); 1 ,ug of pyruvate kinase;4 (A260) absorbance units at 260 nm of ribosomnes; 200 ,ug oftRNA; 1 A260 of mRNA; 0.357 ,uCi of 14C-labeled amino acidmixture; 0.829 ,ug each of the 20 amino acids; and 0.6 mg ofprotein of the S150 supernatant, in a total volume of 110 ,.The pH of the mixture was adjusted to 7.5 with potassiumphosphate buffer and sodium hydroxide. A 5-,u portion ofwater or the antibiotic solution was added, the mixture wasincubated at 37°C for 30 min, and the reaction was processedas described by Jelenc and Kurland (16).For poly(U)-programmed biosynthesis, each assay was

performed as mentioned above, except for the following: 2A260 of ribosomes, 10 ,ug of poly(U); 0.055 ,uCi of[14C]phenylalanine (10.55 mCi/mmol), and 5 x 10-9 mol ofleucine. The assays for misreading were carried out in thesame way, but using [14C]leucine and 5 x 10-9 mol ofphenylalanine.

In the case of strain R135, poly(U)-directed poly-phenylalanine synthesis was carried out as mentioned above,but the ribosomes and the S150 fraction from the gentami-cin-susceptible and gentamicin-resistant strains were recip-rocally combined to reconstitute the in vitro synthesis sys-tem. Coenzyme A and Mg(OAc)2 were added to each assaymixture (final concentrations, 10-4 and 1.1 x 10-2 M,respectively) as described in reference 13.Enzyme assays. The standard phosphocellulose paper bind-

ing assay was used for enzyme assays (11). The reactionmixture contained, in 50 ,I: 0.2 mM antibiotic; 0.25 mM14C-labeled acetyl coenzyme A, 2 Ci/mol; 10 mM Mg(OAc)2;60 mM NH4CI; 6 mM ,3-mercaptoethanol; 10 mM Tris-hydrochloride, pH 7.6; and a quantity of purified enzyme(19). This led to a radioactivity incorporation of about 10,000cpm after 10 min of incubation at 37°C.

Competition between fortimicins and other aminoglycosidesfor binding on E. coli ribosomes, as evaluated by equilibriumdialysis. Equilibrium dialysis was carried out with rotatingcells, 500-,Il total volume (Dianorm instrument). Two com-partments of 250 ,ul each were formed with a semipermeable

Visking memnbrane. Aliquots of 200 ,ul of 10 mM Tri-hydro-chloride (pH 7.5-10 mM MgCl2-100 mM NH4CI buffer wereadded to one compartment and 200 RI of the ribosomes-plus-antibiotic mixture was added to the other. The compositionof the latter mixture was as follows: 0.24 puM 70S ribosomes;0.02 jiM 3H-labeled aminoglycoside (specific activity, 1,800Ci/mol for [3H]dihydrostreptomycin, 5,000 Ci/mol for[3H]tobramycin, and 90 Ci/mol for [3H]gentamicin C2); and 2nM to 2 mM fortimicin A or B. The buffer composition wasthe same as in the first compartmnent. When equilibrium wasreached (4 to 5 h at 20°C), a 100l-pl aliquot of the solution wassampled from each compartment and the radioactivity wasdetermined by liquid scintillation counting. Three assayswere carried out for each fortimicin concentration.[3H]dihydrostreptomycin was from Amersham Corp.;[3H]tobramycin (20) and [3H]gentamicin C2 were synthe-sized in this laboratory (3).

Stabilization of 70S couples. Buffer 10 consisted of 10 mMTris-hydrochloride (pH 7.5), 10 mM Mg(OAc)2, 100 mMNH4Cl, and 6 mM ,B-mercaptoethanol. Buffer 2 was the sameas buffer 10 but with 2 mM Mg(OAc)2 and 2.5% sucrose. OneA260 of 70S ribosomes was incubated at 37°C for 20 min inbuffer 10 with various concentrations of fortimicin A or B.The mixtures were diluted to obtain the concentrations ofbuffer 2, but with the same concentrations of antibiotics.They were layered on 11 ml of a 5 to 30% (wt/vol) sucrosegradient prepared in buffer 2 and containing the sameantibiotic concentration. The gradients were centrifuged for15 h at 20,000 rpm (SW41 rotor). The A260 was recorded withan Isco 185 density gradient fractionator.

RESULTS

Antibacterial activity. The MICs of fortimicin A for S.epidermidis and E. coli K-12 were 1 and 2 ,ug/ml, respec-tively. The effects of fortimicin A on bacterial survival areshown in Fig. 1. For S. epidermidis, the drug was bacteri-cidal at the MIC. For E. coli, the same effect was observedonly at drug concentrations >20 ,ig/ml.

In vivo macromolecule synthesis. No effect was observedon DNA and RNA syntheses (data not shown). For proteinsynthesis (Fig. 2), the incorporation of [3H]lysine was inhib-ited at between 8 and 40 ,iM fortimicin A (5 and 25 ,ug/ml).This concentration range corresponds to the MBC and wasobserved at the same bacterial concentration (108 bacteriaper ml).

In vitro protein synthesis. The results of the translation ofpoly(U) and MS2 phage mRNA are shown in Fig. 3 and 4,respectively. The effects of fortimicins A and B and theother aminoglycosides were measured after a 30-min incu-bation at 37°C. Our results are in agreement with thosepreviously published (2, 4, 30). Fortimicin B has practicallyno effect on poly(U)-directed elongation (not shown). At>50 ,uM, a slight inhibition (20%) of phenylalanine incorpo-ration together with an increase in misreading, evaluated asleucine incorporation, were shown. There was a strongstimulation of natural mRNA-directed synthesis when forti-micin B was present at >10 ,uM.

In the case of polyphenylalanine synthesis, fortimicin Ainduced an inhibition from 0.7 ,iM, reaching a maximum(65%) at 50 jiM, followed by stimulation up to the highestconcentration tested (200 ,uM). Misreading began at 70 ,uMand reached 40%. The natural mRNA-directed synthesiswas similarly affected. There was an initial inhibition from0.7 ,uM, corresponding to the first phase of polyphenyl-alanine synthesis. Then, instead of the stimulation noticed

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EFFECTS OF FORTIMICINS ON BACTERIAL RIBOSOME 859

with poly(U) at antibiotic concentrations of >70 p.M, astabilization phase was observed.

In vitro synthesis with resistant cell ribosomes and factors.Ribosomes and factors were prepared from a resistant E.coli strain known to synthesize an aminoglycoside-inactivat-ing enzyme which acetylates gentamicin. If a value of 100%acetylation is attributed to gentamicin, sisomicin is acetyl-ated at 100%; netilmicin, at 20%; amikacin, at 0%; andfortimicin A or B, at 100%. Four in vitro polyphenylalanine-synthesizing systems were reconstituted by reciprocal ex-changes between ribosomes and factors from susceptible(MRE600) and resistant (R135) E. coli strains. Figure 5

E.coli

0I-L-

-DEC

time (hours)

shows that, whatever the origin of the ribosomes, thesynthesis was not inhibited by fortimicin A when S150extracts from the resistant strain were used.

Competition between fortimicins and three other aminogly-cosides for binding on bacterial ribosomes. Using equilibriumdialysis, we carried out competition experiments betweenfortimicins and [3H]dihydrostreptomycin, [3H]gentamicin,and [3H]tobramycin. The fortimicins could not remove anyof the three radioactive compounds from the ribosomes,even at concentrations up to i0-' M (data not shown).Dependence of 70S ribosome dissociation on fortimicin

concentration. The 70S ribosomes dissociate into their sub-units at 2 mM Mg2>. Prevention of this dissociation byfortimicin A is shown in Fig. 6 (fortimicin B not shown).Both antibiotics stabilize 70S couples, fortimicin A beingslightly more active than fortimicin B (range, 10 to 40 ,uMand 40 to 100 ,uM, respectively).

DISCUSSIONAs observed for other aminoglycosides, fortimicin A was

bactericidal. In the case of E. coli, the MBC was 10 times theMIC. This result may be explained by an inoculum effect,well known for aminoglycosides. Our assays were run atabout 3 x 108 cells per ml, and Thornsberry et al. (27) haveobserved that, at this concentration, there is a strong inocu-lum effect for fortimicin A, the MBC rising from 2 p.g/ml at103 CFU/ml to 32 p.g/ml at 107 CFU/ml. Although numeroushypotheses have been proposed (9, 12), no conclusive ex-planation has been found for the bactericidal effect ofaminoglycosides.

Fortimicins A and B do not behave similarly towards invitro protein synthesis. The only effect observed with forti-micin B is the strong stimulation of natural mRNA-directedprotein synthesis at >10 F.M. However, this effect, whichmight be attributed to an action other than elongation, wasnot accompanied by any antibacterial activity. Fortimicin Ahas a strong effect on polyphenylalanine synthesis: theinhibition corresponds to the inhibition of one of the elon-gation steps, whereas the stimulation observed at concentra-tions of >70 ,uM may be due to the misreading observed at

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FIG. 1. Growth inhibition of E. coli K-12 (A) and S. epidermidisBL450 (B) by increasing concentrations of fortimicin A. The con-

centrations are expressed in micrograms per milliliter, and theexperimental conditions are described in the text.

0

10 100 1000

Concentration of Fortimicin A (P,M)FIG. 2. Inhibition by fortimicin A of protein synthesis in intact

cells of E. coli K-12. The experimental conditions are described inthe text. The data are expressed as percentages of the controlwithout antibiotic (100% = 7,500 cpm).

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860 MOREAU, JAXEL, AND LE GOFFIC

these concentrations. As suggested by other authors (4, 30),the latter may be due to an increase in the rate of synthesis.If we compare the effects of fortimicin A with those exertedby other aminoglycosides (Fig. 3), we observe that inhibitionbegins at least as soon as with neomycin (the most active ofthe drugs tested here) and earlier than with gentamicin.However, the inhibition percentage did not reach the valuesobserved with tobramycin or neomycin, but was higher thanthat observed with gentamicin or streptomycin. None of thefour drugs tested induced the high level of stimulationobserved at >70 ,uM fortimicin A. Misreading was observedat higher concentrations of fortimicin A (70 ,uM) than ofgentamicin, neomycin, or streptomycin. However, the levelof misreading was as high as with gentamicin at equivalentinputs. One may compare this effect with that observed byother authors with kanamycin (4, 24). Table 1 summarizesthese comparative findings.We have shown that the presence of inactivating enzyme

relieves the inhibitor effect offortimicin A, as with ribosomes,from susceptible as well as resistant strains. We may con-clude that the ribosomes from the resistant strain are actu-ally susceptible and that the S150 fraction determines theresistance through the drug-inactivating enzyme, which can

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Concentration of antibiotic (jM)

FIG. 4. MS2 phage mRNA-directed incorpQration of a mixtureof 15 '4C-labeled amino acids into polypeptides by 70S ribosomes inthe presence of fortimicins A (A\) and B (O) and gentamicin C (0)(100% = 2,500 cpm).

acetylate high quantities of fortimicin. The inability of inac-tivated fortimicin A to inhibit protein synthesis is sufficientto explain the resistance of the strain, although the presenceof the modified drug inside the cell may hamper the entry ofunmodified molecules, as suggested by Benveniste and Dav-

Concentration of antibiotic (,uM)

B

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Concentration of antibiotic (pM)

FIG. 3. Effect of fortimicins and other aminoglycosides on poly-phenylalanine synthesis and on misreading. The extent of biosynth-esis is expressed as a percentage: ItOta is the control incorporationof ['4C]phenylalanine plus [14C]leucine in the absence of antibiotic(Ytotal = 50,000 cpm). Polyphenylalanine synthesis =

(['4C]phenylalanine incorporation/Ytotal) x 100. Misreading =

(['4C]leucine incorporation/ItOtal) x 100. Influence on polyphe-nylalanine synthesis (A) and on misreading (B) of fortimicin A (A),streptomycin (A), gentamicin C (0), tobramycin (*), and neomycinB (O).

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70 700

FIG. 5. Effect of fortimicin A on polyphenylalanine synthesis,using ribosomes and S150 fractions from E. coli strains MRE600 andR135. Symbols: (A) ribosomes and S150 from strain MRE600; (O)ribosomes from strain R135 and S150 from strain MRE600; (C)ribosomes from strain MRE600 and S150 from strain R135; (0)ribosomes and S150 from strain R135.

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EFFECTS OF FORTIMICINS ON BACTERIAL RIBOSOME 861

TABLE 1. Comparative in vivo and in vitro effects of fortimicins and other aminoglycosides on E. colia

In vivo Inhibition of polyphenylalanine synthesis (,uM) MisreadingMIC inhibitionofCocCnn LMAntibiotic (pLg/mi) protein Concn ConnvCncn (M)for synhis This Reference Reference Reference (>M) 4(M) Maximum giving 50%

E. colib (ynhmi) paper 2 4 24 giving 20% giving misreading association ofE.coliU(1Lg/ml) papermZZ / gisregadiUn/go maximum (%) subparticlesmisreading

Fortimicin A 2 13 (this study) 2.4 260 >2,000 >77 25 (this study)Fortimicin B 256 >500 >2,500 15 80 (this study)Streptomycin 2 70 (10) >500 >200 100 >100 4 900 43 20% at 130

,uM (4)Gentamicin 0.5 4 (10) >500 300 1 0.2 540 78 6 (4)Kanamycin 2 55 (10) 90 1-10 2 14 (4)Tobramycin 0.5 3 (this study) 9 <70 None None None 50'Neomycin 0.5 18 (10) 1.8 0.1-1 0.27 2 27 3 (4)

a Numbers in parentheses are references.b This study.c Tangy, thesis, 1984.

ies (1). It has previously been shown (25) that acetyltobra-mycin is able to inhibit polyphenylalanine synthesis and tobind to the ribosome (17). Fortimicin is, in this respect,different from tobramycin, since our experiments show thatacetylfortimicin is unable to inhibit synthesis. It should beinteresting to compare the affinities of fortimicin and ace-tylfortimicin for the ribosome.No competition for binding to 70S ribosome was found

between fortimicin A or B and streptomycin, gentamicin,and tobramycin. The last three antibiotics belong to differentclasses of aminoglycosides and bind to the E. coli ribosomewith dissociation constants in the range of 0.1 to 1 ,uM, butare not capable of efficiently displacing each other from theirrespective binding sites (18). We can deduce from this resultthat the binding sites of the fortimicins are different fromthose of the three other molecules or overlap only slightlywith the latter.

Fortimicins are shown to stabilize 70S couples. In the caseof fortimicin A, the beginning of stabilization corresponds tomaximum biosynthesis inhibition and to the beginning of thephase attributed to misreading. As demonstrated by others(4), however, ribosomal stabilization and misreading are notnecessarily related. Furthermore, our assay for stabilizationdoes not take into account the role played by initiationfactors. The value of 10 ,uM at which fortimicin A is activecorresponds to the values found by Lando et al. (4) forgentamicin, kanamycin, and neomycin. It distinguishes for-timicin A from streptomycin (130 ,uM) and tobramycin (>50,uM) (F. Tangy, state thesis, University of Paris VI, Paris,France 1984).The purpose of this work was to compare fortimicins with

some other aminoglycosides as to their effects on the bacte-rial ribosome. We conclude that fortimicin behaves differ-

0.s

0.6

0.4

B

7 5 3 7o

3 7 5 3 7 5 3 7 5 3volum (mlt

FIG. 6. Sucrose density pattern of E. coli ribosomal subunits inthe presence of increasing fortimicin A concentrations. (A to D) 2mM Mg2"; (E) 6 mM Mg2". (A and E) No antibiotic; (B) 10.5 ,uM;(C) 40 ,uM; (D) 120 ,uM.

ently from the other molecules tested (Fig. 3 and 4, Table 1).The binding of an antibiotic to the ribosome must beaccounted for in any comprehensive description of its modeof action. We present evidence that fortimicins and therepresentatives of three different aminoglycoside families,streptomycin, gentamicin, and tobramycin, do not share acommon binding site on the ribosome. Fortimicin is notrelated to any of the other aminoglycosides tested as to itseffects on protein biosynthesis. It is unique in that it leads toa strong stimulation of biosynthesis at concentrations higherthan 70 puM. Moreover, acetylfortimicin is totally unable toinhibit polyphenylalanine synthesis, whereas tobramycindoes not lose this ability after acetylation (25). As to itseffect on ribosome dissociation, fortimicin differs from bothstreptomycin and tobramycin, but remains similar to genta-micin. Although deprived of antibiotic activity, fortimicin Bhas been included in the assays, with the view to obtainingsome information on the structure-activity relationshipsamong aminoglycosides. Some data have been obtained byother authors (29), using a mutant whose ribosomes areresistant to istamycin, a member of the same family. Tworeasons may be given to explain the lack of antibioticactivity of fortimicin B: either the drug does not enter thecell or it is deprived of an effect on the bacterial ribosome.The experiments carried out in this study favor the latterhypothesis, even if they do not exclude the former.

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12. Heller, A. H., R. Spector, and M. Aalyson. 1980. Effect ofsodium chloride on gentamicin accumulation by Escherichiacoli; correlation with bacterial growth and viability. J. Antibiot.33:604-613.

13. Hotta, K., H. Yamamoto, Y. Okami, and H. Umezawa. 1981.Resistance mechanisms of kanamycin-, neomycin-, and strep-tomycin-producing streptomycetes to aminoglycoside antibiot-ics. J. Antibiot. 34:1175-1182.

14. Iwasaki, A., T. Deushi, I. Watanabe, M. Okuchi, H. Itoh, and T.Mori. 1982. A new broad spectrum aminoglycoside antibioticcomplex, sporaricin. J. Antibiot. 35:517-519.

15. Jelenc, P. C. 1980. Rapid purification of highly active ribosomesfrom Escherichia coli. Anal. Biochem. 105:369-374.

16. Jelenc, P. C., and C. G. Kurland. 1979. Nucleoside triphosphateregeneration decreases the frequency of translation errors.Proc. Natl. Acad. Sci. U.S.A. 76:3174-3178.

17. Le Goffic, F., M. L. Capmau, F. Tangy, and M. Baillarge. 1979.Mechanism of action of aminoglycoside antibiotics. Bindingstudies of tobramycin and its 6'-N-acetyl derivative to thebacterial ribosome and its subunits. Eur. J. Biochem. 102:73-81.

18. Le Goffic, F., M. L. Capmau, F. Tangy, and E. Caminade. 1980.Have deoxystreptamine antibiotics the same binding site onbacterial ribosomes? J. Antibiot. 33:895-899.

19. Le Goffic, F., N. Moreau, and M. Chevereau. 1973. Purificationpar chromatographie d'affinitd d'une acetyltransferase et d'unephosphotransferase inactivant les antibiotiques aminosidiques.Biochimie 55:1183-1186.

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