JOURNAL OF BACrERioLOOY, Mar. 1969, p. 1262-1271Copyright 0 1969 American Society for Microbiology

Vol. 97, No. 3Printed In U.S.A.

Resistance Factor-mediated Streptomycin ResistanceJ. H. HARWOOD AND DAVID H. SMITH

Departments of Bacteriology & Immunology and Pediatrics, Harvard Medical School,Children's Hospital Medical Center, Boston, Massachusetts 02115

Received for publication 21 December 1968

Resistance (R) factor-mediated streptomycin (Sm) resistance differs from clas-

sical, high-level, chromosome-borne Sm resistance in its dominance over sensitivityand in the level of its effectiveness (in Escherichia coil -25 pg/ml versus >2,000 jg/ml). In addition, an R factor-containing strain, unlike high-level Sm-resistantbacteria, showed an inoculum effect with respect to its level of Sm resistance. Crudeextracts of this strain destroyed the inhibitory activity of Sm and bluensomycin(Blue) on in vitro protein synthesis. The ribosomes from this strain proved to besensitive to Sm in vitro. The requirements for in vitro inactivation of Sm (and Blue)were determined to be: extract, adenosine triphosphate or deoxyadenosinetri-phosphate, and Mg+. Chromatographic techniques with radioisotopes revealedthe formation of an inactivated form of Sm containing adenosine (or deoxyadeno-sine), phosphate, and Sm in equimolar amounts. The adenylate moiety is coupledto the streptobiosamine residue, rather than to the streptidine ring, of the Sm mole-cule. The adenylating enzyme, which is not induced by Sm, is located in the peri-plasmic space of the R factor-containing strain.

Resistance (R) factors are extrachromosomalgenetic elements, or episomes, that mediate resist-ance to various antibacterial drugs and were de-fined originally in species of Shigelia in Japan(26). They have subsequently been found to havea world-wide distribution in all species of entericbacteria and are now the most common basis fordrug resistance among such organisms isolated inthe United States (D. H. Smith, Proc. Intern.Symp. Infect. Multiple Drug Resistance, in press).The mechanism by which R factors confer drugresistance has, therefore, medical as well as bio-logical importance.Drug resistance associated with R factors was

originally thought to result from decreased per-meability to the drugs (12, 15); evidence has beenpresented recently, however, which suggests thatthese episomes bear genes for individual enzymeswhich inactivate chloramphenicol (10, 17, 18),kanamycin (Kana; 10, 23), dihydrostreptomycin(10, 24), neomycin (Neo) and paromomycin (22),and ampicillin (1, 4).

In this communication, we present evidencethat cells bearing R factors contain an enzymelocated in the periplasmic space which inactivatesstreptomycin by adenylation. Some of these re-sults were reported previously (D. H. Smith,Intern. Symp. Infect. Multiple Drug Resistance,in press). While this work was in progress, Ume-zawa et al. (24) and Yamada, Tipper, and Davies(27) presented similar findings.

MATERIALS AND METHODSReagents and drugs. Nucleotides and their deriva-

tives were obtained from Sigma Chemical Co. (St.Louis), synthetic polynucleotides from Miles Chemi-cal Co. (Elkhart, Indiana), MN cellulose powder 300from Brinkmann Instruments, Inc. (Westbury, N.Y.),and polymerized ethylene imine (PEI) from ChemiradCorp. (East Brunswick, N.J.).

Radioactive amino acids and IH-dihydrostrepto-mycin (labeled at R3; see Fig. 6) were obtained fromNew England Nuclear Corp. (Boston, Mass.), andradioactive nucleoside triphosphates were obtainedfrom Schwarz Bio Research, Inc. (Mount Vernon,N.Y.). l4C-streptomycin (uniformly labeled) was thegift of Merck Sharpe and Dohme Research Labora-tories (Rahway, N.J.). Bluensomycin (Blue), specti-nomycin (Spec), streptidine, and streptobiosaminewere the gifts of George Whitfield, Upjohn Co.(Kalamazoo, Mich.). Kana and Neo were obtainedfrom Bristol Laboratories (Syracuse, N.Y.).

Bacterial strains and techniques. The techniquesemployed for bacterial culture and conjugation havebeen described previously (19, 25). The R factorstudied, RE130, originated from a natural isolate ofEscherichia coli and transferred resistance to strepto-mycin (Sm), Blue, Spec, tetracycline, chlorampheni-col, and sulfonamide.

E. coli W was obtained from B. D. Davis. E. coliAB1932 (AzRi) is a spontaneous, azide-resistant mu-tant of AB1932 (K12, F- arg- met- xyl- gal lac--T6R) obtained from E. A. Adelberg.

Bioassays. The biological activity ofSm was assayedby a tube dilution method (20) and by an agar platediffusion technique with E. coli W as the indicator

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strain. For this assay, 0.1 ml of the solution to beassayed was placed into wells (5 mm in diameter)made in 30-ml tryptic digest agar (agar 1.5%) platescontaining the indicator strain dispersed throughoutthe agar. The plates were incubated at room tempera-ture overnight, then at 37 C for 12 hr. Growth in-hibition was measured as the radius drawn betweenthe outer edge of the well and the outermost edge ofthe zone of no growth.

Decrease of active Sm in culture medium. E. coliB/RE130 was grown overnight to stationary phase intryptic digest broth containing 15 ;sg of Sm per ml.The cells were pelleted by centrifugation, and thesupernatant fluid was concentrated by heating at about60 C under vacuum. The amount of biologically activeSm in this concentrated medium was evaluated by bio-assay (see above). The following controls were used.(i) E. coli B was substituted for B/RE130; becausestrain B is sensitive to 1 MAg of Sm per ml, the initialconcentration of Sm in the broth was 0.5 ,ug/ml. Aftergrowth of E. coli B to stationary phase, the culturesupernatant fluid was concentrated under vacuumsufficiently so that its estimated final Sm concentrationwas equivalent to that of the concentrated B/RE130supernatant fluid, assuming no inactivation of Sm byeither strain. (ii) A volume of broth, uninoculatedwith any bacterium and containing 15 or 0.5 Mg ofSm per ml initially, was concentrated under vacuumto an estimated final Sm concentration which wascomparable, as in the first control, to that of the ex-perimental.The osmotic shock procedure has already been

described (8).In vitro protein synthesis. Cells were grown, washed,

extracted, and fractionated into ribosomes and ISin0[supernatant fluid from 100,000 X g centrifugation ofcrude cell extract which had been incubated at 37C for 15 min with added amino acids and adenosinetriphosphate (ATP) to exhaust endogenous messen-ger] supernatant fluid by methods previously described(5, 9). Incubations of polynucleotide-directed, cell-free protein synthetic reaction mixtures were preparedand assayed for radioactivity as described (6, 9).Sm inactivation mixtures. Crude extracts employed

as a source of Sm-adenylating enzyme were preparedas an IS,oo fraction and were dialyzed three times, 8 to10 hr each in no. 20 D.C. dialysis tubing (Visking Co.,Chicago, Ill.) at 0 C against 100 volumes of buffer[50 mm tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4)-6 mm j3-mercaptoethanol-12.5mM magnesium acetate]. The reaction mixtures con-sisted of, except where otherwise noted, 350 ,umoles ofSm, 2.1 mmoles of ATP or other nucleoside phos-phates, 5 ,umoles of magnesium acetate or chloride,6 ;moles of ,B-mercaptoethanol, and 2 to 8 mg of pro-tein extract of E. coli B or B/RE130 per ml of mixture.

These mixtures were incubated at 37 C for variousintervals, at which time the reaction was stopped byheating at 80 to 95 C for 5 min. The resulting precipi-tate was removed by centrifugation twice at 3,000 X gfor 15 min, and the supernatant fluid was collected forfurther study. [Heating at 100 C for 5 min had no unto-ward effect on either biological or colorimetric (16)assays for Sm. Furthermore, heat precipitation of theprotein in the mixtures did not lead to nonspecific

binding of Sm in the precipitate, since solutions of Smat equal concentration in water or in a heated reactionmixture had identical biological activity.]

Chromatography. Thin-layer chromatography wasperformed on plates of PEI cellulose and diethyl-aminoethyl cellulose prepared according to Randerath(13). After ascending chromatography to 15 to 16 cmin 0.1 M or 0.8 M LiCl, the plates were divided into 15to 16 blocks, 1.0 cm long by 1.3 cm wide, which weretaken up with a razor blade to scintillation vials.Radioactivity was counted in aqueous Bray's solution(2) or in a scintillation solution containing 4 g of 2,5-diphenyloxazole and 50 mg of p-bis[2-(5-phenyloxa-zolyl)]-benzene (Pilot Chemicals, Inc., Watertown,Mass.) per liter of toluene in either a Nuclear Chicagoscintillation counter model 724 or a Packard countermodel 3375.

RESULTS

Basis for the R factor resistance. Two observa-tions suggested that R factors mediate resistanceto Sm by a mechanism different from that of theclassic chromosomal locus which alters the 30Sribosome. (i) In R factor infected strains, resistanceis dominant,whereas in strains diploid for sensitivean( resistant chromosomal loci Sm sensitivity isdominant (20a). (ii) The level of R factor resist-ance, but not high-level resistance, varies with theconcentration of inoculated bacteria when testedby a tube dilution method. The minimal inhibitoryconcentration (MIC) for E. coli B/RE130 (theR factor-containing strain), measured in trypticdigest broth, was 15 to 30 ,g/ml with an inoculumof 5 X 102 cells/ml, but 1,000 ,g/ml with aninoculum of S X 105 cells/ml. The MIC for E.coli BR, a classic Sm-resistant mutant with ribo-somes insensitive to Sm, was 10,000 ,ug/ml withinocula of both sizes. Since the inoculum effecthas been associated with extracellular drug-destroying enzymes (14), these results suggestedthat R factors do not code simply for SmR ribo-somes but that they might mediate resistance toSm by means of a drug-inactivating enzyme. Todistinguish the role of ribosomes from that of thesoluble fraction, the following series of experi-ments was conducted.

Comparison of ribosomes and ISMoo from E. coli Band B/RE130. Extracts of E. coli B and B/RE130were tested for their ability to incorporate 14C-phenylalanine and 14C-isoleucine into proteinunder in vitro conditions in which Sm pro-motes misreading of messenger ribonucleic acid(mRNA). The results presented in Table 1 indi-cate that 10 ,g of Sm per ml inhibited the in-corporation of phenylalanine by 30%. The sameconcentration of Sm stimulated isoleucine in-corporation ninefold in a polyuridylic acid (polyU)-directed system employing E. coli B ribosomesand an ISoo supernatant fraction from E. coli B.

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TABLE 1. Sm-induced misreading with extracts of E. coli B and B/RE130a

4C-amino14C-am.no acid Drug Source of Source of RNA acid incor-

supernatant ribosomes m potacino

Phenylalanine..................... 0 B B 0 8Phenylalanine..................... OB B polyJ 367Phenylalanine..................... + B B poly U 262Isoleucine......................... 0 B B poly U 129Isoleucine......................... + B B poly U 1,180Phenylalanine..................... 0 B/RE130 B poly U 189Phenylalanine..................... + B/RE130 B poly U 212Isoleucine......................... 0 B/RE130 B poly U 123Isoleucine......................... + B/RE130 B poly U 134

Phenylalanine..................... 0 B B/RE130 poly U 388Phenylalanine..................... + B B/RE130 poly U 185Isoleucine......................... 0 B B/RE130 poly U 84Isoleucine......................... + B B/RE130 poly U 734

a Each 100-,uliter incubation contained 'IC-amino acid and the other 19 amino acids at 10-1 M, mag-nesium acetate at 3.2 X 10-i M, ammonium acetate at 1.1 X 10-l M, j-mercaptoethanol at 6 X 10- M,Tris-hydrochloride buffer (pH 7.4) at 5 X 10-' M, phosphoenolpyruvate at 5 X 10-3 M, pyruvate kinaseat 30,ug/ml, ATP at 10-' M, guanosine triphosphate at 5 X 10-' M, poly U at 100,ug/ml, streptomycinat 10 pg/ml, supernatant fluid at 7.5 mg of protein/ml, and ribosomes at 2.2 mg/ml. Streptomycin wasincubated in the complete system minus ribosomes and mRNA for 60 min at 37 C; ribosomes and mRNAwere then added as indicated, and reaction mixtures were incubated for 30 min at 37 C. Thereafter,samples were collected and counted as described (9).

Similar results were obtained when E. coli B/RE130 ribosomes were substituted for E. coli Bribosomes. However, substitution of E. coli B/RE130 supernatant fluid for that of E. coli Bvirtually eliminated the effects of Sm on proteinsynthesis and mRNA misreading. These datasuggested that (i) the supernatant fraction of theE. coli B/RE130 extract contained some factor(s)which eliminated the biological activity of Smand (ii) E. coli B/RE130 ribosomes were sensitiveto Sm.

Since the R factor RE130 also conferred resist-ance to Blue and Spec but not to Neo or Kana,E. coli B/RE130 extract was tested for the specific-ity of its ability to inactivate these aminoglyco-sides in vitro (Tables 2, 3). The E. coli B/RE130extract did not affect the capacity of Kana orNeo, nor did it significantly affect the ability ofSpec to inhibit cell-free protein synthesis. How-ever, the extract completely eliminated the effectof Blue on protein synthesis and largely removedmisreading caused by Blue (Table 4).Drug inactivation measured by bioassay. The

products from drug-inactivating incubations weretested for their effect on drug-sensitive bacteria.The extract of E. coli B did not alter the growthinhibitory effect of Sm, Blue, or Spec on the indi-cator strain. On the other hand, experiments per-

TABLE 2. Inhibition of protein synthesis by Sm,Kana, and Neo in extracts of

E. coli B and B/RE130O

Source of PhnlaieGroup Drug supernatant incorporationtncoportiopmoles/min

1 0 B 4182 Sm B 933 0 B/RE130 4704 Sm B/RE130 476

5 0 B 2656 0 B/RE130 2617 Neo B 2108 Neo B/RE130 1239 Kana B 20210 Kana B/RE130 170

a Incubation conditions were similar to thosedescribed in Table 1, with the following excep-tions: magnesium acetate was used at 10-' M,ammonium acetate at 6 X 1Or-M, supernatantfraction of E. coli B at 3.5 mg of protein/ml,supernatant fraction of E. coli B/RE130 at 3.75 mg ofprotein/ml, and all antibiotics at 10 pg/ml. Ribo-somes from E. coli B were used in all groups: forgroups 1 to 4 the concentration was 3.9 mg/ml;for groups 5 to 10, 1.95 mg/ml. Antibiotics wereadded at the onset of incubation.

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TABLE 3. Inhibition of protein synthesis by Spec inextracts of E. coli B and B/REJ3OG

Drug Bacterial mRNA Valineconcn extract incorporation

g/ml fpmoles/min

0 B 0 28O B poly lb 3680.1 B poly I 3511.0 B poly I 258

10.0 B poly I 51

0 B/RE130 0 70 B/RE130 poly I 2980.1 B/RE130 poly I 2941.0 B/RE130 poly I 231

10.0 B/RE130 poly I 100

a Bacterial extracts were in the form of IS3ofractions. Incubation conditions were similar tothose of Table 2, except for the following: mag-nesium acetate was used at 2.7 X 10-' M, poly-inosinic acid at 100 ug/ml, and IS3. fraction inamounts sufficient to yield 2.5 mg of ribo-somes/ml. Spec was added at the beginning of a30-min incubation period at 37 C. At the end ofthat period, specimens were collected and countedas before.

b Polyinosinic acid.

formed as in Table 5 at pH 8.6 showed that E.coli B/RE130 extract eliminated the inhibitoryeffect of Sm, Blue, and, surprisingly, Spec. Allthree rates of inactivation were 28 nmoles ofdrug per mg of protein crude extract per hr ofincubation. Identical experiments showed noeffect of E. coli B/RE130 extract on the biologicalactivity of other aminoglycosides, such as Neo,

Kana, gentamycin, paromomycin, and kasuga-mycin, or of viomycin to all of which strain RE130does not confer resistance. These results demon-strate that E. coli B/RE130 extract specificallyinactivates only Sm, Blue, and Spec but not all ofthe antibiotics of similar structure and function.Here, as throughout the experiments, Sm anddihydrostreptomycin behaved indistinguishablyin both biological and chromatographic studies.The results also support previous studies whichindicated that separate R factor loci mediate theresistances to Sm, Neo, and Kana but that the locimediating resistance to Sm and Blue cannot beseparated by genetic techniques (D. H. Smith,unpublished data).

Cofactor requirements for Sm inactivation. Todefine the requirements for inactivation of Sm,the system employed for in vitro protein synthesiswas simplified, and the reaction products weretested by bioassay. Inactivation of Sm by thiscrude extract required Mg+- and ATP; buffer and(3-mercaptoethanol enhanced the degree of in-activation. The optimal Mg- concentration wasapproximately 8 to 10 mM; monovalent cationsand salts of nickel, cobalt, calcium, zinc, andmanganese could not replace Mg+ in this reac-tion mixture. The optimal pH for inactivationwas approximately 8.3.The requirement for ATP could not be replaced

by equimolar concentrations of adenosine di-phosphate (ADP), adenosine monophosphate(AMP), adenosine, adenine, other nucleosidetriphosphates, or other deoxyadenine-containingcompounds. Deoxyadenosine triphosphate(dATP), however, could replace ATP (Table 5).This finding cannot be explained by ATP contam-

TABLE 4. Blue-induced inhibition and misreading with extracts of E. coli B and B/RE130a

Source of Suc f'Caioai"IC-amino acid Drug supernatant Source of mRNA |aino acidr~action ribosomes incorporation

pmoks/minPhenylalanine..................... 0 B B 0 5.5Phenylalanine..................... 0 B B poly U 284Phenylalanine..................... + B B poly U 225Isoleucine......................... 0 B J poly U 101Isoleucine......................... + B B poly U 346

Phenylalanine..................... 0 B/RE130 B poly U 206Phenylalanine...................... + B/RE130 B polyU 202Isoleucine......................... 0 B/RE130 B poly U 104Isoleucine.+ B/RE130 B poly U 182

a Incubation conditions were similar to those described in Table 1 with the following exceptions:magnesium acetate was used at 3 X 10-' M, ammonium acetate at 8.6 X 10-' M, supernatant fraction ofE. coli B at 3.5 mg of protein/ml, supernatant fraction of B/RE130 at 7.5 mg of protein/ml, E. coli Bribosomes at 2 mg of ribosomes/ml, and Blue at 10,ug/ml. Antibiotic was added at the outset of incuba-tion.

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TABLE 5. In vitro inactivation of Sm bycrude cell extracts"

Amt of SmContents of mixture6 inactivatedper m.#of

proteic

jig

E. coli B extract, complete system <2E. coli B/RE130 extract, complete

system 16-extract........................... <2-ATP........................... <2-Mg+........................... <2-NH4 ........................... 16-Tris-hydrochloride............... 10-,8-Mercaptoethanol .............. 13-ATP+UTP..................... <2-ATP + GTP..................... 2-3-ATP+CTP..................... <2-ATP+TTP..................... <2-ATP + adenine, adenosine,dAMP, S-adenosyl methionine,dADP, AMP, ADP, ADPG (per-formed separately)............... <2

-ATP + dATP.................... 16

a Rates of inactivation are based on the bio-assay described in Materials and Methods. Mg-+was present in either its acetate or chloride form.Nucleotide triphosphates and adenine-containingcompounds were at a concentration of 1.87 mm inthe mixtures, except where otherwise noted.

b Abbreviations: UTP, uridine triphosphate;GTP, guanosine triphosphate; CTP, cytosine tri-phosphate; TTP, thymidine triphosphate; dAMP,deoxyadenosine monophosphate; ADPG, adenosinediphosphate-glucose.

c In crude extract per hour of incubation.

ination of dATP for the following reason: no ATPwas observed when 50 nmoles of dATP was chro-matographed by a thin-layer technique which sep-arates dATP from ATP and allows detection of aslittle as 2 nmoles of ATP. A level of less than 5%contaminating ATP in the dATP would not pro-vide sufficient ATP to inactivate all of the Sm inthese incubations. Furthermore, isotopic studieswith labeled dATP (Fig. 4), described below, in-dicate participation of dATP in the inactivationof Sm.The results of parallel experiments established

that the inactivation of Spec (and of Blue) alsorequired ATP and Mg++ and raised the questionof whether one or more than one enzyme is in-volved in the inactivation of Spec and Sm. Sub-strate competition experiments were thereforeperformed (Table 6) to help resolve this question.These experiments demonstrated that increasingconcentrations of Spec in the reaction mixtureinterfered with the enzymatic inactivation of aconstant concentration of Sm. Conversely, Sm

interfered with the inactivation of Spec. The de-gree of inhibition is not proportional, however, tothe concentration of competing aminoglycosidesubstrate added.

Product of the Sm inactivation reaction. Radio-active substrates and thin-layer chromatographywere employed to define the characteristics of theproduct of the Sm inactivation reaction. Samplesof a reaction mixture containing "4C-Sm werechromatographed on PEI cellulose in neutrallithium salts after 0 and 4 hr of incubation, atwhich time no biologically active Sm was detect-able in the mixture. The inactivated Sm had anRp of 0.75 compared to 0.95 for Sm (Fig. 1). Sincethis system of chromatography also separatesnucleotides, nucleosides, and bases, it was em-ployed in all of the following experiments.The ATP requirement for inactivation per-

mitted the use of ATP labeled variously to deter-mine in what manner ATP was involved in thereaction. Figure 2 depicts the results of an experi-ment in which a,fB,y-32P-ATP was added to areaction mixture. The chromatogram of a sampleremoved from the mixture before incubationindicated that ATP remains near the origin (peak1). In the absence of Sm, the ATP was nonspecifi-cally degraded by this crude extract to products(identified by comigration of various known ade-nine-containing compounds in parallel experi-ments) which had the Rp of peaks 1 and 2 (see

TABLE 6. Inhibition of Sm and Spec inactivation"

Amt of Spec or SmRatio of 2 drugs in complete system inactivated per mg of

protein crude extractb

Sm/Spec pg

1/0.................... 16 (Sm)1/1.................... 13 (Sm)1/3.................... 7 (Sm)1/10.................... <2 (Sm)

Spec/Sm1/0.................... 16 (Spec)1/1.................... 14.5 (Spec)1/3.................... 12 (Spec)1/10.................... 10 (Spec)a These experiments were performed exactly as

those in Table 5, except that Spec interferencewith Sm inactivation was measured on plates con-taining E. coli BspecR as the indicator strain. Sminterference with Spec inactivation was measuredon plates containing E. coli AB 1157BmR as theindicator strain. A ratio of "1/0" indicates thatone drug was present in the incubation at 200pg/ml, the other at 0 ;g/ml; "1/1" indicates thatboth drugs were present at 200 jg/ml, etc. Themolecular weight of streptomycin is 580, and thatof spectinomycin is 502.

b Per hour of incubation.

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S Streptomycin

Eg InactivatedStreptomycin

5 10

The complete reaction mixture contained 1,000nmoles of a-32P-ATP and 150 nmoles of 3H-Sm.There were 242,000 32p counts/min on the chro-matogram, and 32,250 were associated with the3H-Sm in peak 3. This established that inactivatedproduct contained equimolar amounts of Sm andthe a-phosphate of ATP.Although inactivation of an antibiotic by

adenylation is unprecedented, it seemed unlikelythat the a-phosphate ofATP would be transferredto Sm without the adjacent nucleoside. The chro-matograms depicted in Fig. 4 illustrate that this isthe case. Samples were removed from four reac-tion mixtures containing the indicated nucleotidesafter 4 hr of incubation and chromatographed in0.8 M LiCl. (ATP degradation products, but notSm or inactivated Sm, have higher RF values inthis solvent than in that used in Fig. 3.) "4C-Smincubated without ATP ran with the solvent front(peak 4); 3H of the ATP (or dATP) ran in peaks1 and 2 when incubated without Sm, but it wasfound superimposed on the "YC-Sm in a new peak(peak 3) when incubated in a complete reaction;vias-oTho ro^e%fe*Nha 314 rrsinrit'1^nf xvth

FRACrIONFIG. 1. Thin-layer chromatography of 14C-Sm in its

normal and inactivated forms. Samples were removedfrom a reaction mixture after no incubation (Strepto-mycin) and after 4 hr of incubation (Inactivated Strep-tomycin). The samples were chromatographed on thesame thin-layer chromatography plate in 0.8 M LiClbut were assayed for radioactivity independently.

also Fig. 4). The chromatogram of a sample re-moved from an incubation in which the Sm wascompletely inactivated revealed a new 32P peak(peak 3) which had an Rr identical with inacti-vated Sm. The reaction mixture contained 2,070nmoles ofATP and 345 nmoles of Sm. There were64,000 32p counts/min on the chromatogram, and3,160 were at an RP of 0.77 (counts/min at 12,13, 14, and 15 cm from origin were pooled). Thesefindings suggested that 1 mole of phosphate hadbeen transferred from ATP to 1 mole ofSm in thecourse of the inactivation.

This did not, however, distinguish which of thethree phosphates from ATP was involved in theinactivation. Therefore, the same experiment wasrepeated with a-32P-ATP, with or without 3H-Sm(Fig. 3). The chromatogram of the complete re-action mixture with no incubation showed thatATP remained at the origin (peak 1), whereas Smran with the solvent front (peak 4). The 82P in amixture containing no Sm and incubated for 4 hrwas found in peaks 1 and 2 (ATP degradationproducts), whereas in a complete mixture incu-bated for 4 hr it was found in these two peaks andin a new peak (peak 3) coincident with the 3H-Sm.

mixAtUre. i nex prcen'ltage; Ul -rl VuLICIUCLL WILII

the "4C-Sm, calculated as above, suggests that theproduct of the reaction contains equimolaramounts of Sm and adenosine. These results, and

25

20

ft)

Q~

KII

10

5

0

4 Hr INCUBATION15k

1O-

5

05 10 15

FRACTION NUMBER

FIG. 2. Thin-layer chromatography of a reactionmixture containing a,j3--yJ'P-ATP. Samples were

removed after 0 and 4 hr of incubation, processed as inFig. 1, and chromatographed in 0.1 M LiCl.

4Qa.

Kj'4JK,

0 Hr INCUBATION

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ATP * STREPTOMYCIN 18 ~(4hr/Incubation) 4

t4t2 u3

121- ATrP(4hr Incubation)

5 lo 15FRACTION

FIG. 3. Thin-layer chromatography of a reactionmixture containing a-'i P-ATP with or without 'H-Sm.Samples were removed from the reaction mixture after0 (top) and 4 hr (middle) of incubation and chromato-graphed as in Fig. 2. A parallel reaction mixture con-taining a-as P-ATP and no Sm (bottom) was alsosampled after a 4-hr incubation and was chromato-graphed similarly.

those of the above experiments, indicate that Smis inactivated by adenylation.As noted earlier, dATP could replace the re-

quirement for ATP in reaction mixtures. Whenchromatographically purified 3H-dATP replacedATP in an incubation, 1 mole of dATP was foundcoupled to 1 mole ofSm in the product, providingfurther evidence for an active cofactor role fordATP in this reaction.To define further the nature of the product,

3H-ATP was incubated with the streptidine andstreptobiosamine degradation products of the Smmolecule (Fig. 5). Streptobiosamine, but notstreptidine, can be adenylated (peak 3) by theenzyme (Fig. 6).

Lastly, mixtures of Spec and Blue in completesystems containing 3H-ATP were sampled after 0and 4 hr of incubation and were chromatographedby the thin-layer technique. Comparison of the'H distributions at 0 and 4 hr revealed the appear-ance at 4 hr of a new 3H peak, RF = 0.75 to 0.80,in the chromatograms of both Spec- and Blue-containing reaction mixtures. In accord with theATP dependence of both Spec and Blue inactiva-tion, this finding further suggests that these twodrugs are also inactivated by adenylation.

Location and regulation of the enzyme. Growthof E. coli B/RE130 to stationary phase in mediumcontaining 15 ,ug of Sm per ml decreased the con-centration of biologically active Sm in the medium(measured by bioassay) by more than 80%.The first suggestion that the enzyme might be

located in the "periplasmic space" came from ex-periments in which cultures of E. coli B/RE130were osmotically shocked, and the medium("shock fluid") and cell extract prepared fromshocked, sonically treated cells were assayed fortheir ability to inactivate Sm. Morethan 50%of theenzymatic activity (evaluated by bioassay) wasreleased into the shock fluid in experiments withcells in the logarithmic phase. In these experi-ments, viable counts done on the cell suspensionsimmediately before and after the resuspension andincubation of the cell pellet in hypotonic solutionassured us that more than 70% of the cells sur-vived osmotic shock. Accordingly, ,3-galactosi-

t4i

(31

8-ATP 2

Bk 2

+ATp

STREPTOMTC/NSrSREPTOMYrCIN6 dATP 2

SrREPTOMYCIN

5

U

0:IV

10 15

FRACTION

FiG. 4. Thin-layer chromatography of reactionmixtures containing 14C-Sm with and without ring-labeled 'H-nucleotide triphosphates. From top tobottom, the reaction mixtures contained: (i) 3H-ATPalone, sampled after 4 hr of incubation; (ii) 'H-ATPand 14C-Sm, sampled after 4 hr, (iii) 3H-dATP and4C-Sm, sampled after 4 hr; and (iv) 14C-Sm in a reac-tion mixture containing no ATP or dATP and sampledafter 4 hr. The methods were identical to those in Fig.1.

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STRETPrOMYC/N - 0

(Bluensomycin) J HI H~~~~~~~~~~

LSrREPrOB/OSAMINE - o

HOH 0HO CO

OH H

FIG. 5. Molecular structures of Sm and Blue andtheir degradation products. No stereospecificity is in-

Htended. For Sm, R1 and R2 = -N-C = NH and

INH2

Rs = -CHO. For Blue, the R1 and R2 positions areH

occupied by -NH2 and -N--C = NH, but which

NH2functional group is at R1 and which at R2 is not known.For Blue, R3 = -CH20H.

explained by assuming the substrate specificity ofthe enzyme to be broad enough such that it canuse both nucleoside triphosphates to adenylateSm. Precedents for this situation exist amongcertain ATP-requiring enzymes of glycolyticpathways which phosphorylate their substratesnearly as well with dATP (for example, yeasthexokinase).With respect to aminoglycoside specificity, the

fact that the adenylating enzyme was ineffectiveagainst Kana or Neo under the conditions usedfor Sm inactivation is of interest in light of thework of Umezawa et al. (11, 22). They reportedthe existence of Kana-, dihydrostreptomycin-,and paromamine-inactivating activity in the solu-ble fraction of a single R factor-bearing strain(resistant to these three drugs) but could notdistinguish whether this activity was attributableto one or to more than one enzyme. Our resultsprove that drug resistance-mediating enzymesdirected against Sm need not also mediate resist-ance to Kana or Neo.

Similarly, Spec, an aminoglycoside antibioticconsiderably different in structure from Sm (Fig.7), appeared not to be (or only slightly) inacti-vated atpH 7.4 in the Nirenberg system, but it was

dase assays performed on portions of cell suspen-sion sampled before and after the osmotic shockprocedure revealed approximately 92% retentionof the activity of this intracellular enzyme by theshocked cells.To determine if the enzyme is produced con-

stitutively or is induced by Sm, inactivation po-tency was studied in the shock fluid of culturesof E. coli B/RE130 grown with and without 20jig of Sm per ml. The shock fluid of cells grownunder both conditions had the same specific ac-tivity when measured against Sm in bioassay.

DISCUSSIONThe simplest model which can account for the

data presented is that the R factor RE130 codesfor the constitutive synthesis of a periplasmicenzyme which adenylates Sm.The dialysis conditions employed should have

permitted a substantial percentage of the mole-cules with molecular weights of about 10,000daltons to dialyze out of the extract (3). There-fore, the fact that our thoroughly dialyzed extractswere even slightly more active in inactivating Smthan were the undialyzed extracts makes it un-likely that any necessary cofactors other thanATP or dATP were present in the reaction mix-tures.The apparent interchangeability of ATP and

dATP as required cofactors in the reaction may be

14J

"I4

50

40 F

30-

201

I o

STREPTOM/OSAM/NE

- 2

5 10 15

STREPrTDINE

2

FRACTION

FIG. 6. Thin-layer chromatography of reactionmixtures containing Sm or its degradation products asadenyl acceptors. All mixtures contained 14C-ATP andeither Sm, nothing, streptobiosamine, or streptidine.All mixtures were incubatedfor 4 hr, and samples wereremoved andprocessed as in Fig. 1.

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HARWOOD AND SMITH

SPECTINOMYCIN

H OHH ;H

H3C - N C H3

HO 0

NH O

OH3

FIG. 7. Molecular structure of spectinomycin.

inactivated in the simpler incubations evaluatedby bioassay. These latter experiments were per-formed at pH 8.6, which could explain the dis-crepancy. This also suggests that if one enzyme isresponsible for both Sm and Spec resistances, itsactivity against Sm and Spec is not identical atallpH values. The lack of proportionality betweenthe degree of interference and the concentrationof inhibitor (Table 6) does not, unfortunately,distinguish between three of the conceivablemodels for Sm or Spec adenylation, or both: oneenzyme-one polypeptide; one enzyme-two poly-peptides; or two enzymes, each of which inacti-vates one drug but is subject to interference viabinding of the other drug. Certain epidemiologicalfindings support, but do not prove, the concept oftwo enzymes (or at least of two different poly-peptides): occasional natural E. coli isolates havebeen found (i) to be SmR Spec' as well as Sm9SpecR and (ii) to confer a wide range of levels ofSpec resistance while retaining Sm resistance at afixed level. The converse is also found. A geneticanalysis of this question employing Sm- or Spec-sensitive mutants, or both, of the R factor REI 30is now underway in this laboratory and will bepublished subsequently.The fact that the Sm-adenylating enzyme does,

however, inactivate (and probably adenylate)Blue under all conditions tried with Sm is ofinterest for two reasons. First, of the aminoglyco-side molecules whose structures are known, Blueis the most nearly identical to Sm (Fig. 5). Second,all natural isolates of R factor-bearing entericbacteria found resistant to either Sm or Blue havealways been resistant to the other drug as well(D. H. Smith, unpublished data). Attempts toseparate Blue resistance from Sm resistancethrough spontaneous mutations, segregation, ortransduction of R factor fragments have consist-ently failed. This genetic evidence plus in vitroinactivation of Blue in the systems employed herestrongly suggest that both of these R factor resist-ances are coded for by the same cistron.

Host strain and various culture conditions havebeen commonly observed to influence the level ofSm resistance conferred by R factors. Further-more, mutations in R-SmR-containing strainsleading to higher-level Sm resistance are occa-sionally transferable (D. H. Smith, unpublisheddata). It would be of theoretical and practicalinterest to determine if the sole basis for thesevarying levels of Sm resistance is the specific ac-tivity of the Sm-adenylating enzyme in such cells.Such studies are currently in progress and will bereported elsewhere.

In accord with the findings of Yamada et al.(27), who determined that the 3' OH group of theN-methyl-L-glucosamine moiety of Sm is adenyl-ated, our results, obtained by different methods,suggest that the streptobiosamine moiety, and notthe streptidine ring of the molecule, is adenylated.Recently, Takasawa et al. (21) confirmed thatadenylation of Sm occurs at this 3' OH group.Similarly, all of the other presently establishedsites of R factor-mediated enzymatic alteration ofaminoglycoside molecules [Kana acetylation (23),Kana phosphorylation (11, 23), and paromaminephosphorylation (22)] are on either deoxyglucoseor N-methyl-L-glucosamine rings and not on thestreptose, deoxystreptamine, or streptidine ringsof the molecules. If alteration at these sites, partic-ularly as small a change as the addition of aphosphate group, removes the capacity of thedrug to inhibit protein synthesis or cause mis-reading, or both, such enzymes should prove tobe useful tools for future studies of the interactionbetween active sites on the drug molecule and theribosome.To our knowledge, before the discovery of the

adenylation of Sm (24), nothing had been re-ported concerning enzymatic adenylation of eitherantibiotics or sugars. In fact, reactions involvingadenylation of any class of substrate are relativelyrare in nature and seem to occur only (i) wherethe adenylated molecule is an intermediate in areaction which is subsequently broken down toform the product (e.g., amino acid activation andcoenzyme A biosynthesis) or (ii) when activationof enzymes occurs through covalent bonding ofAMP (e.g., muscle phosphorylase, glutamine syn-thetase). Thus, the reaction catalyzed here wouldappear to be of a highly unusual type, and the"origin" or "evolution" of this episome-coded,Sm-inactivating enzyme remains obscure.

ACKNOWLEDGMENTS

We thank J. Janjigian for excelient technical assistance andP. F. Sparling, H. Amos, and D. G. Fraenkel for helpful discussion.

This investigation was supported by Public Health Servicegrants AI-0836 from the National Institute of Allergy and Infec-tious Diseases and GM-14119 from the National Institute ofGeneral Medical Sciences. D. H. S. is a recipient of Career De-

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R FACTOR-MEDIATED STREPTOMYCIN RESISTANCE

velopment Award A120376 from the National Institute of Allergyand Infectious Diseases. J. H. is a predoctoral fellow supportedby Public Health Service Training Grant GM00177 from theNational Institute of General Medical Sciences.

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24. Umezawa, H., S. Takasawa, M. Okanishi, and R. Utahara1968. Adenylylstreptomycin, a product of streptomycininactivated by E. colt carrying R factor. J. Antibiotics(Tokyo) Ser. A 21:81-82.

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