2I~ BIOCHIMICA ET BIOPHYSICA ACTA

8BA 95973

ALTEI~ED RIBOSOMES IN SPIRAMYCIN-RESISTANT MUTANTS OF

B A C I L L U S S U B T I L I S

ASAD AHMED* Bioclmnistry Section, Food and Drug Research Laboratories, Departmont of National Healtk and Welfare, Ottawa (Canada) (Received April ~gth. x068)

SUMMARY

Tire mechanism of action of the antibiotic spitamy¢in has been studied by comparing the properties of spiramycin-sensltive and resistant strains of Bac-il~us sub~i~/s. Ceil-free extracts from a resistant mutant exhibited significantly greater synthesis of protein in the presence of spiramysin than those derived from the sen- sitive strain. In addition, the mutant extracts also acquired simultaneous resistance to other macrolide antibiotics v/z., erythromycin, magnamycin, and ole-¢ndomycin. Protein synthesis by reconstituted systems containing ribosome and supernatant fractions originating from the two strains indicated that resistance is lhked to the ribosomes. The 5o-S subunit was identified as the resistant component by using ribo- somes hybridized from sensitive and resistant subunits. Mutations to spiramycin resistance did not affect the binding of [~4C]spiramycin 'to the ribosomes. Similarly. the sensitive and resistant ribosomes did not exhibit any significant differences in their susceptibility towards spiramycin-induced inhibition of the binding of [liC]- phenylalauine tR'qA over a wide range of antibiotic, aminoacyl-tRN'A, and magnesium concentrations. It is proposed that spiramycin acts primarily by binding the 5o-S subunit of the ribosome and blocking growth of the poiypeptide chain, by interferene~ with the peptidyl transfer step. At higher concentrations, the antibiotic exhibits secondary effect and causes inhibition of the binding of aminoacyl-tRNA to the ribo- somes and the 3o-S subunlts. Genetic resistance is acquired against the former mechanism by alterations of a component of the 5o-S subuuit.

INTRODUGTION

Stndies with cell-free systems ~:em Es~l~i*hia cell have indicated that the macrelide antibiotic spiramycinL2 is a specific inhibitor of protein synthesis s,4. Inhibition occurs after the formation of aminoacyl-tRNA at some step during the polymerization of amino acids on ribosomes. The only effect which could be clearly discerned was the inhibition of the binding of aminoaeyl-tRNA to the 7o-S ribosomes

Abbrevi~.tions: tRNA. transfer l~lA; Italy U, ]~olyurldyBc acid. • Present address: Department of Genetics, University of Albcrt~, Edmonton, Canada.

BiocMm. Bte#hy*..~¢m, x66 (I968) 2x8--228

SPIRA,~YCII~'-RESISTAIqT MUTANTS OF B, s ~ ZI~

and their 3o-S subunlts. However. no definite corre]at ian was found between the in,, hibitinn of polypeptide synthesis and the hindiug of ~ c y l - t l ~ A . Moreover. in preliminary experiments [aC]spiramycin appeared to b~d preferentially the 5o-S~ rather than the 3o-S suhanits. These obserwtions suggested that inldbitlon of e~nlr~ acyl-tRNA binding to the 3o-S subunits may not represent the prhnary n ~ u l s m of action of spiramycin. This problem can be elucidated through i~ ~ ' ~ studies. How,. ever, the inability of the antibiotic to enter intact cells of E. co/i rendered these studies difficult. I have, therefore, taken advantage of the greater permeability o[ Bac~l&~ sub,ills to spiramycin and have attempted to investigate its primary mode of actio~ by comparing the properties of sensitive and resistant strains. I t has been found that resistance to spiramycin is caused by specific mut~tianul ulterations of a c~mponen~: of the 5o-S subunit cf the ribosome. Such ~ltsratious enable the nbos~mes to carry on amino acid polymerization in the presence of splrantycin, although their affinity ~cr the antibiotic remains unaffected. Moreover, it has been found that inhibition ~ amincacyl-tRNA binding to the ribosomes by spiram~ht is a secondary effect which seen~s to be operational only at high antibiotic concentrations.

MATERIALS AND METHODS

Bacterial s~aius and isolation oJ s~n'ramy¢in-fesistant mutants B. subtilis strain W 23 was kindly supplied by Dr, V, N. IYEn. Cafleton Uni-

versity. Ottawa. Growth of this strain was completely inhibited by 4,3,1o -e M spiramycin. Resistant mutants of spontaneous origin were obtained by spreading approx. 8- xo e cells on brain heart infusion plates (37 g Difco brain heart infusion, 3 g yeast extract, I g tryptone, and zz g agar, per l) contalning4.3, xo-e M spir~anycin. Several spiramycin-resistant colonies appeared after 2o h incubatinn at 37% In- dividual colonies were picked, purified, and maintained on nutrient broth slants. The spiramycin-sensitive strain (W 23) and the resistant mutant (sp/-x) used through- out tbese studies grew with a generation time of 36 rain in nutrient broth at 37 °.

Gro~h o~ bacteria and pr~aration of extracts The bacterial strains were grown in nutrient broth (8 g Difco nutrient broth and

5 g glucose, per l) with vigorous aeration at 37 °. When the cultures reached an ab- sorbaneo of 0.60 at 6o0 ;'~, the cells were harvested by centtifugatio~ in a refrigerated Lourdes centrifuge. The yield was about x g fresh packed cells per l of the growth medium. The coils were washed and stored frozen at - - z 8 ° u n t i l needed.

S-3o and S-zoo extracts, and the ribosomes were prepoxed following the pro- cec~ure described by NII~NBERn ~ with the modification that the pH of the e.xtraction and assay buffers was maintained at 7,4 (tel 6 ). Protein was estimated by the methed of Low~y e~ al., as described by LAVN~ 7.

Assay [or ~oly U-direc2ed call*free pradn sy~#he~s The assays were carried out by the procedure described in the accompanying

communication*, with the exception that the pH of the reaction mixture was main-

aio~im. Biophys. Acta, x65 (19~S) ~erS-'2z3

~ 2 0 A. AHMED

tained at 7.4. In some experiments, the S-3o fraction was replaced by dialysed S-Ion fraction (x5o?sg protein) and the indicated Am m~ units of ribosomes or ribosomal subunits. Radioactivity was determined in toluene-2,5-diphenyloxazole-x,4-bis-~-(4- methyl-5-phenyloxaselyl)-benzene sdntillation mixture s in a Packard Tri-Carb Spectrometer at 79 % efficiency for x~C.

Prepatolio~ o/ribosomal subu~i#s The ribosomal subnnits were prepared by zone centrifugation in low mag-

nesium buffer as described earlier ~.

Ddsaion e/ [14C]spimmydn-~ibosom¢ complex The [t4C]spiramycin-ribosome complexes were separated by gel filtration on

Sephadex G-5o columns. Reaction mixtures contained in a final volume of o.~2 rift: o.oz M Tris-HCl (pH 7.4), o.o14 M magnesium acetate, 0.o6 1~ KCI, o.oo6 M 2- mercaptoethanol, 44.o Am m~ units of ribosomes, and i31 ooa counts/rnin of [t~C]- spiramyein I. Where indicated, non-radioactive erythromycin (9" zo-a M) was added to the reaction mixture before the addition of [laC]spiramycin. The reaction mixtures were incubated for 30 min at 37 °. After incubation, 200# samples were placed on pre.equilibrated IO.SCm×I.zcm Sephadex G-5o columns, and eluted with the 'standard buffer '5 at pH 7.4 and 25 °. Seven-drop fractions (o.35 ml) were collected. Radioactivity was determined on ~5o t~l aUquots in BRAY'S solution 9 at 7I % effi- ciency, and absorbance measured on so pl samples after suitable dilution.

Assay/or [~4C]phvnylalanin¢ tRNA binding to ~I~ ~'ibesom~s B. subtilis and E. cell tRNA were charged with [t*C]phenylalanine by the

method of YAMANE ANY SvEoga ~°. The final preparations contained zo/q~moles [~*C]phenylaladine per rag of B. subtilis tRNA and 363 ppmoles [t4C]phenylalanine per mg of E. coli tRNA.

Binding assays were carried out by the procedure described by NIR~BgRG AND LEDER 11,12. The reaction mixtm'es contained, in a final volume of 75 ph o.o2 M Tris-aeetate (pH 7.4), o.o2 i~ magnesium acetate, o.n 5 M potassium acetate; 2.97 A~ora~ units of B. sub¢ilis sensitive ribosomes or 3.oo 4~oms units of resistant ribo- somes, sSpg of poly U, 6.66-zo-aM spiramycin, and 3oopg (3.xpFmoles) of B. s~tilis /a~C]pbenylalanine tRNA or Ioopg (36.31*#moles) of E. coli [14C]phenyl- alanlue tRNA. Individual variations are given in legends to the figures. After in- cubation for 3 ° rain at 37 °, the reaction mixtures were washed on Millipore filters (pore size o.45 p)n and radioactivity determined as described previously.

Cl~mic~s ATP, GTP, phospboeuolpyruvate and phosphoeno[pyruvate kinase were ob-

talued from Sigma Chemical Company, St, Louis, Mo, Sepbadex G-5o was purchased from Pharmaeia Ltd., Montreal. [t~C]Pbenylalanine (specific activity 393 mC/mmole) was obtained from ;flew England Nuclear Corp., Boston, Mass. Stripped tRNA from

Bioch~m. Biophys. Atta, 166 0968) 2IS--zz8

SpI~GIN-RESISTANT MUTANTS OF B. subtit~TS

B. subtilis strain 6o09 and E. cell Kxz were purchased from General Biochemieals, Chagrin Falls, Ohio, Poly U was obtained from Miles Laboratories, Elkhart, Indiana. Spiramyein base and [MC]spiramyein I (o.24/*C/mg) were generously provided by 1~. GuY MAnmn of the Poulene Ltd., .Montreal. Erythromyein lactobionate was donated by the Abbott Laboratories, Montreal, and oleandomycin phosphate and magnamycin base were rite gift of l~r. R. B. TAYLOR, Pfizer Co. Ltd., bfontreal.

Growth of B. subtilis W 23 was completely inhibited in the presence of 4.3" to ~ M spiramyein, t{oweves, when a sufficient number of cells were spread on plates con- taining spiramyein, a few resistant colonies appeared after overnight incubation. These colonies were purified and, on further analysis, were found to have also ac- quired resistance to other maereilde antibiotics m'z., erythromyein (r.8-xo ~ NIL magnamyein (z.3- xo'4 ~), and oleandomyein {2.5' xo -~ M) in addition to spiramyein. Since it has been shown that spiramyein is a specific inhibitor of protein synthesis *.t, it was expected that these mutants might be altered in their protein synthesizing system in such a manner that these were no longer eermitive to the inhibitory action of these antibiotics. Several experiments were carried out to identify the nature of this altered component utilizing the cell-ires protein synthesizing systems described by NIRENBERG 6 and HIRASHIMA, ASANO AND TSUGITA 6. The results are described in the following sections.

Inhibition o/cell-Ires protein synthesis by spiramyein and other macrolide anlibioties in exlraas of sensitive and rss~tant strains

The effect of sp'tramyein concentration Olt the relative incorporation of [t4C]- phenylalanine by sensitive and resistant extracts is shown in Fig. L The sensitive extracts showed 60 % inhibition of protein synthesis at 4" to-61~ spiramycin and inhibition increased up to 80 % at 4' xo-n 1K. On the other hand, the resistant extracts showed, only zo % inhibition at 4" to"* M which increased to about 40 % at 4" ro~ ~'~ spiramycin, Thus, the resistant extracts exhibited a uniform gain of nearly 4 ° % activity over the sensitive strains in the presence of a wide range of spiramyein con- centrations.

Single-step mutations to spiramyein-resistanee were found to confer in vivo resistance to other macrolide antibiotics simultaneously. This was also demonstrated by in vitro experiments. The relative incorporation of [xIC]pbenylalaulne by sensitive and resistant extracts in the presence of 4.1o-* M magnarayein, exythromycin, and uleandomyein is shown in Table f, I t can be seen that, in every case, the resistant extracts exhibited relatively less inhibition of protein synthesis in comparison to the sensitive extracts.

Inhibition o/systems reconstltute.4 /tom ribosomes and supernatant /factions o] sensitive and resistant strains

In order to identify the component altered by mutation, ribosomes and su-

Bio¢kim. Biophys. Aaa, 166 (i96S) 218-~28

!.°

4O

~0

o , , , i 4 - 1 0 "6 a . l o -~ . i = 1 o . , a . l o -a Splrmm¥cio Concentratio~ fa l l

Fig. x. Inhibition of cell-free protein synthesis by spiramycin as a function of 5piramycin concen- tration in extracts of sensitive and resistant strains of B. subtills. In the ab~nce of added splta- mycin, the sens~tlve and resistant extracts incotlmrated 26.o/*/~moles and 2g-5/*/*moles of iuC] phenylalanlne respectively {m xoo).

TABLE I

1NHIBXT|ON OF CELL'FREE PROTEIN ,~YNTH~]S BY MACIgOLIDE ANTIBIOTICS nq EETRA~5 DERIVED 1~tOM 51PIRAMYCIN-SENSITIV~ AND RESIS~'ANI~ MUTANTS OF B. Sgf~ti~$

The antibiotics were used at 4 " to'* M finM concentration

Antibiotic lUC]P~nylalanlne incorporaled

Sensitive ~traa Resiaar~ ez;lract I~tanoles % t.~maes %'"

Control 2z,2 too 63.2 I0o Splramycin 5.8 ~6 36,x S7 Erythromycin Io.o 45 4 6.t 73 Magn~mycin 4.9 :~2 44.2 7 ° Oleandomytln 8,z 37 3I-3 49

pernatant fractions from sensitive and resistant strains were mixed in various com- binations and their sensitivity to spiramyein was determined. Table I I shows the results of art experiment on the inhibition of such reconstituted systems. Proteinsyn- thesis was strongly inhibited when sensitive ribosomes were combined with superna- tan ts derived from either the sensitive or resistant strain. On the other hand, inhibition was considerably reduced when resistant ribosomes were employed, and this was in- dependent of the sanree of the supernatant fraction used. This result clearly indicated

Biodnm, Biophys. Aaa, t66 (x~) 218-~28

SPIRAMYCIN-RESISTANT MUTANTS OF B. S U ~ S Z2 3

TABLE II

llffHIB|TION OP PROTEDI SYHTIiESIS BY ePIRAMYCIN IN SYeT~I~5 RBCO~S~flTUTKD FROM I~JBOSOMK AND BUFERNATANY FRACTIONS DEKIVKD FROM SpIRA~yCIN-~NS[T[~ At~D RESISTANT STRAI~S OF B, #ubHlts Each reaction mixture contait led 3.o A~60 m~ t 1 ai'L~ of lqbo~omes a~d I5O fig of g-loO protein. The concentration of spiramycin used was 4 " Io4 M.

£,ourc* o//raaie~ [tac] p/Je~ty/a/ani~ incorpevaled

Ribosomes Superna tan l --5plramyrin + Sp~ramycln

##moles % tq~moles %

Sensitive Sensitive 7.6 tco 3.7 49 Sensitive Resistant 9.z zoo 4.4 49 Resistant Sensit ive I~.8 leo 9,7 76 Resistant Resistant 18. 4 1oo *5.3 83

tha t ribosomes from the resistant strain were responsible for the observed resistance to spiramyein.

Inhibition with ribosomes r¢conailuVd /tom 5o-S and 3o*S subuni¢s derived from sensitive and r~sis~ant slmins

Attempts to locate spiramyein resistance on the ribosome subunits p~oved to be difficult owing to the low act ivi ty of reconstituted ribosomes. This is an agreement with the findings by TAK~PA AND LII, MAN~ la tha t 3 0 6 subunits of B. snbtiHs are extremely fragile and show low amino acid polymeriaing activity. Nevertheless, results of an experiment using reconstituted ribosomes containing 5o-5 and 3o-S subunits derived from the sensitive and resistant strains, are presented in Table HI . I t appears

T A B L E I I I

[~4HIBITION OF PROTEIN SYHTHESIS BY Sp[RAMYCIN ON HYBRID RIBOSOI,[]~S CONTAIHING 5o-S AND $O-S SUBUIq~s DERIVJED FROM SPIRAMYCIN..SENSITIVE AND RESISTANT STRAINS OF B . S t i f l e r s

Each reaction mixture contained *5o/*g of 5-1oo protein from sen,~tive strain and the following quantities of ribosome subunits in ,4 f~o m# units: Sensitive main, 5o-S, 0.75. ~ d 3o-S, o.55; and resistant strain, 5o-S, 0,80, and 3e-S, o.55. The concentration oJ spiramy~in used was 4 ' zo-~ M.

Source of ribosomal ~uhunits [uC]Ptwnylalanlne incorporated

5o-S 3o.S --Spiramy~in + SjMramy61n

/q, moles % lqano~ts %

Sensitive Sensitive 0.39 too o.z 9 74 Sensitive Resistant o,4~ too o.z8 67 Ee~ietant Sensitive 0.42 zoo 0.37 88 11edstaut Resistant o.45 *co 0.39 87

tha t reassociated ribosomes containing resistant 5o-S subunits were relatively more resistant to inhibition, and this seemed to be independent of the source of the 3o-S subunits u~ed.

Biachim, Bi~hy~. Aaa, ,66 (i968) ~,8-z28

zz 4 A, AI~ED

Ribosome hybrids in which 3o-S subunits of E. coil were associated with 5o-S subunits from sensitive or resistant strains of B. subIilis t~ did not show appreciable differences in their sensitivity to spisamycin.

~iC]Spiramycin bi~ding to sensitive and resiatant ribosomes The formation of s specific complex between [~C]spiramyein and E. coli

ribosomes has been reported elsewhere s,4. The same procedure was used to determine the relative binding of ~t~]spiramyein to the sensitive and resistant ribosomes of B. subiilis. Fig. za and b show the elution patterns of ~4C]splramycin-ribc*ome

. . . . . . . . . . . . . . . t . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . ' . . . . i

! 1 - r " l [ -

Fig. ~t. Binding of [~C]spiramycln to B. Nlil~s ribosomes derived from (a) sensitive and (b) re- sistant strains, and (e) its displacement by erythromy¢in from sensitive ribosomes. Experimental conditions are described in tIAT~m^LS AND ~E~HODS.

complexes for the two strains, It is evident that there were no detectable differences in the binding oi the antibiotic to the sensitive and resistant ribosomes. The presence of erythromycin in the reaction mixture (Fig. 2c) displaced some of the uC counts from the ribosomes.

Inhibition of aminoacfl-tRNA binding to sensitive and msis~an~ r/bosomes Previous experiments with the E. coZ/systemS, a have indicated that high con-

centrations of spiramycin cause a marked inhibition of the binding of aminoacyl- tRI~A to the ribosomes. However, it appeared doubtful if this was a true reflection of the primary mechanism of action of the antibiotic. In order to answer this question specifically, the effect of spiramycin on the binding of aminoacyl-tRNA to sensitive and resistant ribosomes from B. sub~ilis was investigated. Binding assays were car- tied otit by the metho~l of NZm~N1;ERG and co-workers tLas. The effect of spiramycin concentration on inhibition of the binding of [UC]pbenylalanine tRNA is shown in

BiecAim. Biophys. Acta, z66 (t968) 2t8--2~8

SPIRA~YCIN-RE~ISTAIqT MUTA~q'['S OF .B. subtilis

IgO

ig

zz5

Biochim. Biopkys. Act~, 166 ( [ 968 ) 2x8-228

oe.lo ,~, ~a=:o'se6=l#'l b.e.lo "= 5piramycin Concentroeion (M/

Fig. 3. Inhibition o{ the binding of B. subtilis [t4C~phenylalanine tI~NTA to sensitive and resistant rlbosomea by spLramycin a.s a function of splramyein eoneen~'ation. In the absence of sp'tramyein. the sensitive ~ d resistant ribosomes bound o.8E and t.ot/q~moles of [t*C]phenylalanine tRNA, respectively (= zoo).

Fig. 3, I t can be seen tha t both the sensitive and resistant ribosomes underwent equal inhibition a t all antibiotic concentrations tested, maximal inhibition (nearly 40%)

G

~ ee

ta~

~o~

}o, [ ~ Ph#n¥/llenine IRNA added (~plaoles]

Fig, 4, Inhibition of the binding of//. sub~ilts [a~C]phenylalaaine tRNA t~ sewitive slad resistant ribosomes by spiramycin ~6.66 - [o -s M) as a function of [14C]phonylala~e tRNA coaceataation.

ZZ6 A. AHMED

was exerted a t 6.6 • zo -a M spiramyein, whereas practically no inhibition (less than ~%) was observed a t 6 ,6 . Io ~ M. Similarly, changes in [UC]phenylalahine tRNA concentration (Fig. 4) or magnesium concentration (Fig, 5) had no effect or~ inhibi-

~ s

~ 6

. d •

/

o ~ $ oolo o~15 o~2o o~os oolo oo15 0020 Mog~*s/vm Conce~tratlon {M] Mugne*/.m Co~cuntrolion [M)

Fig. 5. Inhibition of the binding of F~ c~?:! [uC lphenylalanine tR~qA to sensitive and resistant ribo- somes by spiramycin {e.66 - xo-J M) as ~ function of magnesium concentration. The reaction mix- tures contained 3.4 ° AHo ~ units of sel~sltive ribosomes and S.44 A .o mp units of resistant ribo- somes.

tion ot binding to the s~nsitive or resistant ribosomes. The small variations observed in total binding seem to be caused by slight differences in growth conditions and the age of ribosomal preparations used. Taken together, these results indicate tha t within limits of sensitivity of the assay system used, the sensitive and resistant ribosomes do not show any significant differences in their relative susceptibilities towards spira- mycin-induced inhibition of the binding of aminoacyl-tR.~IA.

I t may be pointed out that the inhibition of arninencyl-tRNA binding to 3o-S subunits, as shown earlier in E. c~lP,~, could not be demonstrated for B. subtilis because of a rapid deterioration of their binding activities in the isolated state.

DISCUSSION

In a separate communication 4, i t has been shown that the m~crolide antibiotic spiramycin is a specific inhibitor of cell-free protein synthesis in E. coli. I t was found that inhibition occurs after the formation of aminoaeyl-tRNA, during the transfer of the growing peptide chain from peptidyl- tRNA to tile incoming a~noacy l - tRNA on the ribosome. This inhibition of polymerization is not aPImrently caused by tufa- coding, release of incomplete polypeptide chains, b r~kdown of polyribosome$, or inhibition of the binding of messenger RNA to the ribosomes. The only effect of spir~-

Bio~him. Bfop~s; At4#, z66 (1968) 2zg-z2g

SPIRAMYCIN-RESISTANT MUTAI~TS OF e. b~b~illS 227

mycin that could be detected was the inhibition of binding of ~C]pheny]alanme tRNA to the 7o-S ribosomes', specifically the 3o-S subunits. However, no definite correlation was found between the inhibition of polypoptide synthesis and that of aminoaeyl-tRNA binding.

In order to determine the mechanism of action of spirsmycin more precisely, advantage has been taken of tbo properties of spiramycin-sensitive and resistant strains of B. subtilis. These mutants acquired simultaneous resistance to other related macrolides, indicating a common site of action. I t is expected that these mutants will also map in the same region of the B. subfflls chromosome lB. The acquisition of i~: t~vo resistance was also demonstrated by ~n vitro studies. Extracts from the resistant mu- tant were considerably more resistant to inhibition of ~eC]phenylalanine hlcorpora- finn by spiramycin Ol other macrolides than the sensitive c~acts . Expe~me~,ts ~ t ~ reconstituted systems led to the identification of the ribosome as the site of action and the component harboring genetic resistance. This was supported by the isolation of a specific complex between [x~]sp, ir~.~ycin and the ribosome. Ho.,sever, an un- expected observation was that there did rot seem to be any significant difference in the relative affinity of sensitive and resistant ribosomes for [t4C]spiramyein. LEON A};D BROCK ¢° have also reported no difference in the binding of ~4C]streptomycin to streptomycin-sensitive and resistant ribosomes of E. cot*;. However, TAUBMA~ a a~, t*, have found that erythromycin-resistant mutants of B. subtilis bind less [*H]erythro. myein to the 5o-S subunits of their ribosomes.

Several lines of evidence indicate that the primary site of action of spiramycin is located on the 5o-S subunit of the ribosome. Incorporation experiments with hybrid ribosomes suggest that resistance is linked to the resistant 5o-S subunits. Moreover, (a) the preferential binding of ~C~spiramycin to 5o.S sobuults of E. eMi ribosomes mentioned easlie#, Co) the i~ ~ivo and in vitro eross-rest*tance of spiramycin with erythromyein, known to affect the 5o-S snbtmit an'is in B. subtlZis, and (c) the partial displacement of [14C]spiramycin frolri ribosomes by erythromycin, strengthen the notion that the site of action of spiramycin must be located on the 5o-S subunit. VAZ- ~LrE#D and VAZQUEZ AND MO~EO x4 have also arrived at a similar conclusion recently.

If the primary site of action of spiramycin is located on the 5o-S subuult, then the observed inhibition of aminoacyl-tRNA binding to the 3o.S subunit should be the reflection of a secondary mechanism. This question has been answered by a com- parison of the relative inhibition of aminoacyl-tRlqA binding to the sensitive and resistant ribosomes. Such a comparison carried out under different conditions re- vealed that both sensitive and resistant ribosomes axe equally susceptible to iuldbi- tion by spiramycin, Thus, mutations to spiramycin resistance apparently do not alter this effect on the ribosome and, therefore, inhibition of aminoacyl-tRNA binding to the ribosomes can not be the physiologically significant mechanism of action, of spira- mycin.

On the basis of these results s~t it is proposed that spiramycin inhibits protein synthesis by two mechanisms which operate at different antibiotic concentrations. At low concentrations (approx. 4" xo~ NI), spiramycin binds to a site (or sites) on the 5O.S subnhit of the ribosome and inhibits amino acid polymerization by interfering

• In ~, recent publication ~, V̂ Z~LtlZZ ~ o Mol~ao hove also relmrted im!xibition of [u~;]phenyl- alatflBe.tRNA binding to the 7o-S rilxmomes of E. ~oli by spiramycitt IIL However, they did not find a.~y effect on the 3o*S plume of binding.

B~ochim. Biophy*. Aeta, x66 (x968) aiS-z28

z28 A. AHMED

wi th the trsnsfe~ of the pept idyl g roup from t R N A to smincacyl- tRNA. This brings about an immediate cessation of the growth of polypeptide chain, wi thout causing breakdown of polydbosomes or release of pept ide fragments. The spiramycin-resis- tan* mutan t s are probably altered in this component (peptidyl transferase) o~ the 5o-S subuni t which carries ou t the transfer function. At relatively h igh concentrations, the antibiotic acts .secondarily b y binding both subonits , thereby inhibi t ing the bind- ing of amincacyl-tRN'A to the 7o-S ribosomes and the 3o-S subunits , This proposal satisfactorily accounts for all the observations tha t Lave been made concerning the mode of action of spiramyein.

I t hank Dr. W. J. JOllNSO~ of the Food and D ru g Laboratory, Ot tawa, for his enthusiast ic support, and Mlle. LtETTE CHASS~ for technical assistance dur ing the course of these investigations. This work was par t ly supported b y NRC gran t A-44.x¢.

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