THE JOURNAL OF h1o~o~1c.4~ CHEYISTRY Vol. 242, No. 20, Issue of October 25,~~. 4722-4730,1967

Printed in U.S.B.

The Enzymatic Acetylation of Chloramphenicol by the

Multiple Drug-resistant I!Lwherichia coli

Carrying R Factor

(Received for publication, April 17, 1967)

YOSHIAKI SUZUKI AND SUYEHIKO OKAMOTO

From the Laboratory of Radiation Research and the Department of Chemistry, National Institute of He&h of Japan, Kamiosaki, Shinagawa-ku, Tokyo, Japan

SUMMARY

The chloramphenicol-inactivating enzyme in the multiple drug-resistant Escherichia coli carrying R factor produced two products from chloramphenicol in a cell-free system as well as in whole cells.

Thin layer chromatography on silica gel, infrared spectros- copy, gas chromatography, and nuclear magnetic resonance analysis were carried out with the products. These products were identified as D-fhero-Z-dichloroacetamido-l-(p-nitro- phenyl)-1-hydroxy-3-acetoxypropane and D-fhreo-2-dichlo- roacetamido-l-(p-nitrophenyl)-l,3-diacetoxypropane.

It was shown that a chloramphenicol-resistant E. coli B selected in vitro and a sensitive strain of E. coli K-12 have no capacity to produce acetyl derivatives of chloramphenicol.

Studies of the substrate specificity, heat stability, pH optimum, and chromatographic behavior of the chloram- phenicol-acetylating enzyme from the multiple drug-re- sistant E. coli were carried out.

The chloramphenicol-acetylating enzyme from the multi- ple drug-resistant E. coli produces a 3-0-acetyl derivative of chloramphenicol and converts the former to a 1,3- 0, O- diacetyl derivative. The enzyme which produces the mono- acetyl derivative was purified 120-fold, while the enzyme which produces the diacetyl derivative from the monoacetyl derivative was purified about SO-fold by the same purifica- tion procedures. It was not possible to decide whether one or two enzymes participate in the acetylation of chloram- phenicol.

When the multiple drug-resistant E. coli cells were con- verted to spheroplasts by lysozyme and ethylenediamine- tetraacetate, the chloramphenicol-acetylating enzyme was not released into the medium under conditions in which the bulk of ribonuclease was released but P-galactosidase was not.

In our previous paper it was reported that the multiple drug- resistant Escherichia coli carrying R factor has drug-inactivating

enzymes for chloramphenicol, dihydrostreptomycin, and kana- mycin (1). We have also reported that the chloramphenicol- resistant strains of Xtaphylococcus aureus isolated from clinical materials have a similar chloramphenicol-inactivating enzyme (2). Based on the requirement of acetyl coenzyme A as a co- factor for inactivation of chloramphenicol, acetylation of the drug was proposed as the mechanism of inactivation (1). The present paper deals with the confirmation of the proposed mechanism by identifying the products of chloramphenicol inactivation, and reports on the purification and characterization of the chloram- phenicol-inactivating enzyme from E. coli carrying R factor. A similar independent experiment on the identification of the prod- ucts has been reported recently by W. V. Shaw (3).

EXPERIMENTAL PROCEDURE

Bacterial Strains-Details of E. coli K-12 CSH (4) and E. co&i

K-12 R5, a derivative of K-12 CSH made resistant to five drugs by transmission of an R factor from a naturally isolated drug- resistant strain of Shigella sonnei (5), have been described (1). E. coli B CM’, a chloramphenicol-resistant strain, was selected in vitro (6).

Culture Xedium-The peptone-glucose medium contained 10 g of poly-peptone, 1.0 g of glucose, 3.0 g of NaCl, 0.1 mmole of CaClz 1.0 mmole of MgCl*, and 0.32 mmole of KH2P04 in 1000 ml (pH 7.0). Medium A (7) was used in the experiment involving spheroplast formation. This medium contained 0.12 M Tris- HCl buffer, 0.08 M NaCl, 0.02 M NH&l, 0.003 M Na2S04, 0.001 M

MgCl*, 2 X 10e4 M CaC&, 2 X 1OF M ZnSOa, 0.5% glucose, and 0.5% poly-peptone (Daigo Eiyo Company, Osaka, Japan) in a total volume of 1000 ml (pH 7.5).

Cultural Conditions-E. coli cells were grown in the peptone- glucose medium for 18 hours with shaking at 37”; the culture was diluted about 50-fold with the fresh peptone-glucose medium, and the cells were grown with forced aeration to the late loga- rithmic phase. The yield of cells was 2 to 3 g per liter, wet weight.

Buglers-The standard buffer contained 0.02 M Tris-HCl (pH 7.8), 0.01 M magnesium acetate, 0.06 M KCl, and 0.006 M 2- mercaptoethanol. Elution buffers contained 0.02 M Tris-HCl

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(pH 7.8), lop4 M EDTA, and appropriate amounts of KC1 as indicated with or without 0.006 ILI 2-mercaptoethanol.

Procedures for Preparation of Enzyme Fractions-The culture of E. coli was chilled rapidly by pouring it onto crushed ice and collecting it by centrifugation. Cells were washed twice with the standard buffer, homogenized in an equal volume of the same buffer, and disrupted by passage through a French pressure cell at 420 kg per cm2. To the disrupted suspensions, DNase (5 pg per ml) was added, and the suspensions were centrifuged twice at 30,000 x g for 20 min. The 30,000 g supernatant u~as dialyzed overnight and further cent,rifuged twice at 105,000 x g for 90 min to obtain a supernatant fraction (the S 100 fraction). For purification of the chloramphenicol-acetylating enzyme, the S 100 fraction was used as the starting material. The fraction was heated at 65” for 5 min in the presence of chloramphenicol (2 MM), denatured protein was removed by centrifugation, and the supernatant was designated the heated fraction. The heated fraction was treated with ammonium sulfate at 30% saturation, the precipitate was discarded, and t,he supernatant was precipi- tated with ammonium sulfate at 70y0 saturation. The precipi- tate was collected, dissolved in the standard buffer, and dialyzed against the same buffer. This will be referred to as ammonium sulfate fraction. This fraction was treated with ethanol by means of salt-ice cooling, and the precipit,ate formed at a final ethanol concentration of 45oj, was collected. The precipitate was dissolved in the standard buffer and dialyzed against the elution buffer containing 0.12 M KC1 and 0.006 M 2-mercapto- ethanol (ethanol fraction). The ethanol fraction 12.7 mg of protein was loaded on a DEAE-Sephadex A-50 column 1 x 15 cm and was fractionated with 100 ml of the elution buffer containing a linear gradient concentrationof KC1 from 0.12 to 0.30 M. Four- milliliter fractions were collected. The fraction which was ac- tive in chloramphenicol acetylation was designated the A-50 fraction, and the peak fraction was called the A-50 peak frac- tion.

TheOto30~o,30t040%,40t050%,50t065~,andOto70~0 ammonium sulfate fractions were obtained by precipitation of the S 100 fraction with ammonium sulfate (the 50 to 65 y0 ammonium sulfate fraction, for example, is one which was precipitated at 65% saturation of ammonium sulfate after removal of the pre- cipitate at 50 To saturation).

Enzymatic Inactivation of Chloramphenicol-The complete sys- tem contained 100 pmoles of Tris-HCl buffer (pH 7.8), 10 pmoles of magnesium acetate, 60 pmoles of KCl, 3 pmoles of ATP, 0.04 pmoles of coenzyme A, various amounts of chloramphenicol, JYLhloramphenicol (about 3 x lo4 cpm), and various amounts of the K-12 R5 enzyme fraction in a total volume of 0.5 ml. When the partially purified R5 enzyme fractions were used, the S 100 fraction of K-12 CSH (about 0.8 mg of protein), which did not show any chloramphenicol-inactivating activity (1) or chloramphenicol-acetylating activity measured in the complete system mentioned above, was supplied as a source of an acetyl- CoA-generating system. The mixture was incubated at 37” for an appropriate period of time; a O.lO-ml aliquot was pipetted into 0.40 ml of preheated water and heated for 5 min at 95” to termi- nate the reaction.

Assay Methods of Chloramphenicol-acetylating Activity-En- zyme activity which produces a monoacetyl derivative of chloram- phenicol from chloramphenicol was assayed by a benzene ex- traction method. The heat-denatured sample mentioned above was extracted three times with benzene (0.5 ml), the extracts

were combined, and a 0.5-ml portion was pipetted onto a stain. less steel planchet, which was dried. The radioactivity was measured by a windowless, gas flow counter. By this method intact chloramphenicol was extracted by about lo%, while the fully inactivated products w-ere extracted by about 90%. There- fore, a correction was carried out as follows.

p=9B-H 10B - (B + HI 1OB - T -= zz-

8 8 8

where P is radioactive counts of inactivated products, B is the benzene-extractable counts ( = counts measured x 3 ), H is counts remaining in t’he reaction mixture after the benzene extraction, and T is the total counts contained in the O.l-ml aliquot of the reaction mixture.

Since the rate of diacetyl deriv&ive formation by the chloram- phenicol-acetylating enzyme is only 1 to 2% of that of mono- acetyl derivative formation (see “Results”), at an early period in the reaction woe could assume the activity obtained to be that of monoacetyl derivative formation. Enzyme activity which con- verts t,he monoacetyl derivative to the diitcetyl derivative (see “Results”) was determined by either a paper or silica gel thin layer chromatographic technique. Ethyl acetate extraction (1.0 ml) from the heat-denatured sample was carried out twice. About 98 y0 of both intact chloramphenicol and inactivated prod- ucts was extracted by this procedure. The e&act was spotted on a paper strip (Toyo Roshi No. 51A, 2 X 40 cm) or silica gel thin layer plate (Eastman Chromagram Sheet), and was devel- oped with the solvents described below. After scanning of the radioactive peaks, the enzyme activity that produced the di- acetyl derivative of chloramphenicol was determined by meas- suring the area under each radioactive peak.

Experiments following the incorporation of ‘Y-acetate into chloramphenicol and other substrates were carried out as fol- lows. The reaction mixture, containing %-acetic acid, was incubated at 37” with other cofactors, denatured by heat, and extracted with ethyl acetate. The extracted radioactivity was measured by a liquid scintillation counter, or fractionated by means of paper or silica gel thin layer chromatography, and scanned with the radiochromatogram scanner.

Assay of Antibiotic Activity of Chlcramphenicol-This method has been described in a previous paper (1).

Preparation of Spheroplasts-Spheroplast formation was car- ried out by the method of Neu and Heppel (8) with a slight modification. An overnight culture of E. coli K-12 R5, 0.6 ml, was inoculated into 30 ml of fresh Medium A and incubated at 37” with shaking for 3 hours. Thirty minutes before harvesting, isopropyl-/3-n-thiogalactopyranoside was added to a concentra- tion of 5 x 10h4 M. Cells were harvested by centrifugation in the cold, washed twice with 4 ml of 0.01 M Tris-HCl buffer (pH 7.8), and suspended in 6 ml of 0.03 M Tris-HCl buffer (pH 7.8) con- taining 8 y0 polyethylene glycol (average molecular weight, 1000) at 17” (1.3 x lOlo cells per ml). (These fractions are designated the supernatant, Washing 1, and Washing 2, respectively.) The suspension was supplemented with EDTA, pH 7.5, to a concen- trat.ion of 0.001 ~VI, followed immediately by sufficient 0.5% lysozyme to give 10 Kg per ml. The suspension was kept stand- ing for 25 min at 17” and centrifuged at 10,000 X g for 10 min. The resultant supernatant (the released fraction) was kept, and the pellet was suspended in 6 ml of water, shaken vigorously to cause lysis of the spheroplasts. The lysed spheroplast suspen- sion was centrifuged, the supernatant (the spheroplast fraction)

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4724 Enzymatic Acetylation of Chloramphenicol Vol. 242, Ko. 20

was saved, and the pellet nas suspended in 2 ml of water and sonically disrupted at 10 kc for 5 min (the residue fraction). Each fraction was assayed for ribonuclease, /3-galactosidase, and chloramphenicol-acetylating activity.

Assay of Ribonuclease-The method described by Neu and Heppel (9) was used.

Assay of fi-Galactosidase-The activity of this enzyme was measured according to the method of Horiuchi, Tomizawa, and Novick (10).

Determination of Protein Concentration-Protein concentration was determined by the biuret method of Gornall, Bardawill, and David (II), and by the Folin method of Lowry et al. (12).

Column Chromatography-DEAE-Sephadex i\-50 ( Pharmacia) wassuspended in water, and washed successively with 0.5 N NaOH solution, l\~ater, 0.5 N HCl solution, water, 0.5 N NaOH solution, water, and elution buffer. After the pH of the suspension buffer was adjusted and t,he column was packed, the column was washed with the elution buffer for 3 to 4 hours.

Inactivation of Chloramphenicol by Intact Cells-E. coli K-12 R5 cells or B CMr cells (1 x lOa cells per ml) were inoculated into the peptone-glucose medium, which contained 5 pg of chloramphenicol per ml (14C-chloramphenicol, 3 x lo4 cpm), and the culture was shaken for 18 hours at 37”. The cultures were sonically disrupted for 5 min at 10 kc, and extraction was carried out with ethyl acetate. The extract was chromatographed on a thin layer of silica gel or paper.

Thin Layer Chromatography on Silica Gel-The silica gel thin layer plate used R-as an Eastman Chromagram Sheet (100 I*), type K301R (silica gel with fluorescent indicator). The plate was activat.ed by heating at 100” for 15 min, and the extract from the reaction mixture was spotted on it. Development was carried out M?-ith benzene-methanol-water(60 : 35 : 5, lower phase) for about 30 min. The chromatogram was surveyed for ultra- violet absorption and radioactivity.

Paper Chromatography-The paper strip used was Toyo Roshi No. 51 A (2 x 40 cm). The ethyl acetate extract from the reaction mixture was spotted on the paper and developed with a benzene-methanol-water (98 : 2 : 2, upper phase) for about 90 min. With this system, some cautions should be taken: all the pro- cedures must be carried out at about 20”, since at higher tem- perature the monoacetyl derivative of chloramphenicol shows a very high RF value; immediate use of the shaken solvent must be avoided, since it causes a forward tailing of int’act. chlorampheni- col. The developed paper was scanned for radioactivity accord- ing to the method described in the following section.

Scanning of Radioactivity on Chromatogram and Estimation of Amount of Products-The chromatogram obtained by thin layer or paper chromatography was scanned for radioactivity with a radiochromatogram scanner of the windowless gas flow type. The percentages of intact chloramphenicol, monoacetyl deriva- tive, and diacetyl derivative were estimated by measuring the area under each peak.

Radioactivity Measurement with Liquid Scintillation Counter- The solvent consisted of 9 g of 2,5-diphenyloxazole (PPO), 0.2 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP), 90 g of naphthalene, 100 ml of toluene, and dioxane in a total volume of 1000 ml. Into 12 ml of the solvent, 0.1 to 0.3 ml of sample was dissolved and counted in a liquid scintillation counter.

Analysis of Monoactyl Derivative by Infrared Spectroscopy, Gas Chromatography, and Nuclear Magnetic Resonance Xpectros- copy-As reported under “Results,” an enzyme fraction A

from DEAE-Sephadex column chromatography, Fig. 5b was obtained which has an ability to produce only the monoacetyl derivative from chloramphenicol. Five hundred milligrams of chloramphenicol were inactivated m-ith this enzyme fraction in the complete system for chloramphenicol inactivation described above, and the reaction mixture was extracted with ethyl acetate. The extract was washed successively with 0.5% sulfuric acid solution, 0.5% sodium carbonate solution, and water, and dried over sodium sulfate. The residue obtained upon evaporation of the solvent was dissolved in water, and the solution was extracted with benzene. The benzene extract was washed with water and evaporated. By this procedure residual chloramphenicol that escaped inactivation was completely removed, and the residue was considered to be the monoacet,yl derivative of chlorampheni- col. Crystallization of the monoacetyl derivative was tried but was unsuccessful. With this sample infrared spectroscopy, gas chromatography, and nuclear magnetic resonance spectroscopy were carried out. Infrared absorption spectroscopy was carried out by the Nujol method in a I\ippon Bunko apparatus. Gas chromatography was carried out by Yamamoto, Iguchi, and Aoyama according to their method (13). ,4 Varian apparatus was used for nuclear magnetic resonance spectroscopy.

Chemicals-Chloramphenicol, I)-three-2-dichloroacetamido-l- (p-nitrophenyl)-1 ,3-propanediol, was generously supplied by Dr. T. Osono, Yamnouchi Pharmaceutical Company; it had a melting point of 150”. The stereoisomer of chloramphenicol, DL-erythro- 2-dichloroacetamido-l-(p-nitrophenyl)-l , 3-propanediol, was gen- erously supplied by Dr. M. Nagawa, Sankyo Pharmaceutical Company; it had a melting point of 172”. The 3-0-acetyl derivative of chloramphenicol, I)-threo-2-dichloroacetamido-l- (p-nitrophenyl)-l-hydroxy-3-acetosypropane, was generously supplied by Dr. K. Kasuya, Sankyo Pharmaceutical Com- pany; its melting point was 80”; CX~~ +3.48 (5yo in dioxane). The 1,3-O, 0-diacetyl derivative of chloramphenicol, u-threo-2- dichloroacetamido-1-(p-nitrophenyl)-1 ,3-diacetoxypropane, was generously prepared and supplied by Dr. Osono and Mr. K. Murakami, Yamanouchi Pharmaceutical Company; its melting point was 140.0-140.5”.

Calculated: C 44.24, H 3.96, N 6.88 Found : C 44.51, H 3.90, N 6.82

Dextro Sulphenidol, n-three-l-(p-methylsulfonylphenyl) -2-di- chloroacetamido-1 ,3-propanediol, was supplied by Eizai Pharma- ceutical Company; it had a melting range of 166-167.5”. n-three-1-(p-Nitrophenyl)-2-amino- 1,3-propanediol, D- threo- l- (p-nitrophenyl) -2. acetamido - 1,3 - propanediol, and D - three - 2- amino-3-(p-nitrophenyl)-3-hydroxypropionic acid were generously supplied by Dr. M. Kawashima, Takeda Pharmaceutical Com-

paw. Coenzyme A and ATP were purchased from Sigma. Acetyl-CoA was prepared according to Simon and Shemin (14). I)-threo-2-Dichloroacetamido-l-(p-nitrolhenyl)-lf 3 -propanediol- 3-14C, 7.75 and 18.0 mC per mmole, was purchased from The Radiochemical Centre, Amersham, England. Sodium acetate- l-l%, 22.8 mC per mmole, and sodium acetate-2-14C, 33 mC per mmole, were also purchased from The Radiochemical Centre.

RESULTS

Identification of Acetylated Products

Reversion of Inactivated Products to Antibiotically Active Sub- stance by Hydrolysis-Based on the acetyl-CoA requirement of

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the chloramphenicol-inactivating enzyme from E. coli K-12 R5, we supposed the mechanism of inactivation to be acetyldtion of the drug (1). I f the supposition were true, we could expect the recovery of antibiotic activity by hydrolysis of the inactivated products.

Chloramphenicol (1 mg) was inactivated by the S 100 frac- tion of K-12 R5 in the complete system described in “Experimen- tal Procedure.” The residual chloramphenicol estimated by the assay of antibiotic activity was shown to be below 9% of the original activity. The inactivated products, together with the residual chloramphenicol, were extracted with ethyl acetate, and the solvent nas evaporated. The residue obtained was dis- solved in acetone. According to t,he method of Kunz and Hud- son (15), the inactivated products mere hydrolyzed, and anti- biotic activity in the hydrolysate was measured. About 70% of the original antibiotic activity was restored. This result sug- gests that the inactivated products are mainly the 0-acetyl derivative of chloramphenicol if the inactivation occurs through acetylation of the drug, since the N-acetyl derivative should be more resistant than the 0-acetyl derivative to hydrolysis under the above conditions.

Separation of Inactivated Products from Chloramphenicol-14C- Chloramphenicol was incubated with the R5 S 100 fraction and other cofactors, and extracted nith ethyl acetate. The extract was fractiotated by thin layer chromatography on silica gel. The chromatographic profile obtained displays t.hree radioactive peaks (Fig. 1). The peak labeled CrM in Fig. 1 represents re- sidual chloramphenicol and sho1v-s antibiotic activity. Peak Z and II do not show any antibiotic activity and represent in- activated products, which, as shown below, coincided with the authentic samples, the 3-O-acetyl- and 1,3-O, 0-diacetyl derivatives of chloramphenicol, respectively.

Determination of Amount of Jcetyl Residue Incorporated into Inactivated Products-Under the same experimental conditions, two separate experiments were carried out. One system con- tained 14C-chloramphenicol and W-acetic acid with ot,her co-

loool zz CM I II

; 500 -

0 3 6 9

CM FROM ORIGIN

‘FTR. 1. Chromatographic profile of the extract from the re- action mixture. The reaction mixture contained ‘ZC- and I%- chloramphenicol (300 pg, 1.2 X lo4 cpm), 1.2 mg of protein of K-12 R5 S 100 fraction, and other cofactors described in “Experimental Procedure” in a total volume of 0.5 ml. The mixture was incu- bated for 30 min at 37” and extracted with ethyl acetate, and the extract was fractionated by thin layer chromatography as de- scribed in the test.

TABLE 1

Determination of amount of acetyl ~esiclues incorporated into acet$ation products

The reaction mixtures contained 100 pmoles of Tris-HCI buffer (pH 7.8), 10 Mmoles of magnesium acetate, 60 pmoles of KCl, 6 pmoles of ATP, 0.08 Mmole of CoA, 5.0 mg of protein of R5 S 100 fraction, 1.0 rmole of chloramphenicol, and either 14C-acetic acid (2.27 X lo6 cpm, for the ‘%-acetic acid system) or 14C-chloram- phenicol (3.0 X lo4 cpm, for the 14C-chloramphenicol system) in a total volume of 1.0 ml. The mixtrlres were incubated at 37” for 60 min; O&ml aliquots were taken, and the reaction was termi- nated by heating. The ethyl acetate extracts were counted with a liquid scintillation counter (‘T-acetic acid system) or fraction- ated by thin layer chromatography and scanned for radioactive

peaks (‘4C-chloramphenicol system). Calculation was carried out as described in “Experimental Procedure.”

Systems Chlor;$phmi-/ Product I 1 Product II

p?zoles

‘%-Acetic acid. 0.349 1.29

~4C-Chloramphenicol. 0.046 0.341 0.613

Molar ratio. 1.0 2.1

factors in the reaction mixture, \\hile the other contained l’Vchloramphenico1 and Y-acetic acid.

After incubation at 37”, the reaction was terminated by heating, and extraction was carried out with ethyl acetate. The extractable counts from the latter system were assumed to be the counts incorporated into the inactivated products, since no significant radioactivity was detected in the extract when the reaction was carried out uithout substrate chloramphenicol or with the CSH S 100 fraction instead of the R5 S 100 fraction. This last assumption n-as supported by the following result, obtained with thin layer chromat,ography of the extract. The extract from the complete system containing chloramphenicol, R5 enzyme fraction, and 14C-acet,ic acid showed only two peaks which coincided with those (1 and 11 in Fig. 1) of inactivated products.

The extract from the system n hich contained 14C-chloram- phenicol was fractionated by thin layer chromatography, and the amounts of each product formed were calculated ( Table I ). The amounts of acetyl residue incorporated into each product were calculated and are shown in Table I.

These data lead us t,o the conclusion that the amount of acetyl residue incorporated into 1 mole of Product I is 1 mole, and that incorporated into 1 mole of Producb II is 2 moles, indicating that Product I is a monoacetyl derivative and Product II is a diacetyl derivative of chloramphenicol.

I3oth the carbon atoms of acetic acid were incorporated into the chloramphenicol molecule, since experiments n ith either l-14C- or 2-14C-acetic acid gave identical results.

Kinetic Studies of Formation of Monoacetyl and Diacetyl De- rivatives of Chloramphenicol by Acetylating Enzyme-The enzyme from E. coli K-12 R5 cells produces both a monoacetyl and a diacetyl derivative of chloramphenicol. The relationship be- tween the two products was investigated through kinetic studies of chloramphenicol acetylation.

Chloramphenicol was incubated in the complete system, and the time course of the product formation was followed by paper chromatography. The results are shown in Fig. 2. Under the

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4726 Enzymatic Acetylation of Chloramphenicol Vol. 242, No. 20

experimental condit,ions, chloramphenicol was converted to the showed an additional band representing a carbonyl group at monoacetyl derivative within 1 min by the acetylating enzyme. 1700 cm-1 compared to that of chloramphenicol. The over-all Then the amount of monoacetyl derivative decreased, and the pattern was similar to that of the authentic 3-0-acetyl derivative diacetyl derivative of chloramphenicol increased with time. The of chloramphenicol. Although the result is not itself conclu- results indicate the following process: chloramphenicol ---t mono- sive for the identification of the product, it is consistent with the acetyl derivative ---f diacetyl derivative. above mentioned mechanism of the acetylation of the drug.

Another kinetic study showed that the rate of the reaction producing the monoacetyl derivative was 50 to 100 times greater than that producing the diacetyl derivative (see, for example, the specific activities of Line 1 in Table II).

ilnalysis of Morwacetyl Derivative by Infrared Absorption Spectroscopy, Gas Chromatography, and Nuclear Magnetic Res- onance Spectroscopy-The monoacetyl derivative was prepared as described in “Experimental Procedure” and subjected to each analysis. The infrared absorption spectrum of the sample

Gas chromatography showed that the product coincided with the authentic 3-0-acetyl derivative. The nuclear magnetic resonance spectrum showed an additional 3-proton signal at 7.9 ppm (7) compared to that of chloramphenicol, and all the signals observed were essentially the same as that of the authentic 3-O-acetyl derivative of chloramphenicol.

Detailed Analysis of Products with Thin Layer Chromatog- raphy-using the thin layer chromatographic technique, we carried out a detailed investigation of the acetylated products. Fig. 3, c, d, and f, represents authentic samples of chloram- phenicol, 3-0-acetyl derivative, and 1,3-G, 0-diacetyl derivative, respectively. The 3-0-acetyl derivative gives one major and two trace spots; one of the two traces corresponds to chloram- phenicol, and the other is unknown. When the authentic 3-0- acetyl derivative is incubated in 0.10 M Tris-HCl buffer (pH 7.8) at 37”, the unknown spot increases nonenzymatically with time (Fig. 3, d and e) and takes about 5 min to reach a plateau level (about 20% of the major spot, measured spectrophotometri- tally). The chromatograms of the extract from the complete inactivation system (Fig 3, a and b) show four spots, three of which correspond to chloramphenicol, 3-0-acetyl derivative, and 1,3-O, 0-diacetyl derivative. The remaining one corresponds to the above mentioned unknown spot. The unknown spot is inferred to be the 1-O-acetyl derivative of chloramphenicol, and the satellite peak shown in Fig. 1 (as a shoulder on the left side of Peak I) represents this unknown spot. As unfortunately we did not have an authentic sample of the I-O-acetyl derivative, hydrolysis of 1,3-O, 0-diacetyl derivative was carried out accord- ing to the method of Kunz and Hudson (15), and the resultant chromatogram is shown as g in Fig. 3. It gives four spots, three of which correspond to chloramphenicol and its 3-O-acetyl and 1,3-O, 0-diacetyl derivatives. The remaining one was assumed to be the I-O-acetyl derivative of chloramphenicol and coincided

with the unknown spot. With the use of a large amount of the acetylating enzyme, acetylation of chloramphenicol was carried out; within a very short time (20 set) appreciable formation of acetylated products occurred, but the amount of the unknown spot remained as a trace (Fig. 3a). The percentage of the m- known spot increased but did not esceed 20% of the 3-O-acetyl

0. 15

03 IJJ 0.18 -I

0 I

7 0.05

FIG. 2. Kir Let ;ic studies of the formation of monoacetyl- and

I I I I I

0 1 0 20 50 90

MINUTES

diacetyl derivatives of chloramphenicol. The reaction mixture contained 50 pg of chloramphenicol (3 X lo4 cpm), 1.3 mg of pro- tein of R5 S 100 fraction, and other cofactors as described in “Experimental Procedure” in a total volume of 0.5 ml. The reaction mixture was incubated at 37”, and 0.05.ml aliquots were taken for assay at the indicated times. The ethyl acetate extracts were fractionated by paper chromatography. The chromato- grams were scanned for the radioactive peaks and the amounts of chloramphenicol and monoacetyl and diacetyl derivative were estimated. q , chloramphenicol; 0, monoacetyl derivative of chloramphenicol; l , diacetyl derivative of chloramphenicol.

TABLE II

PuriJication of chloramphenicol-acetylating enzyme

Fraction

Protein -

?ng

l.SlOO 252 2. Heated 63.4

3. Ammonium sul- fate............. 34.7

4. Ethanol.. 17.4

5. A-50 6.7 G. A-50 peak.. 0.2

-4ctivity forming the first product Activity forming the second product

Specific activity

/mdes/min/ mg protein

0.241 0.870

1.71 98 7.1 34.7 23.2 68 4.9 3.37 97 14 17.4 38.1 56 8.1 9.85 109 41 3.7 74.6 23 16

29.3 9.6 121 0.2 223 3.8 47

%

100 91

Purification Protein

-fold mg

1.0 252

3.6 63.4

Specific activity

PlZplZOltY/+?Zi?&/ mg pot&z

4.71 13.4

Recovery Purification

% -fold

100 1.0 72 a.9

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derivative after prolonged incubation. The unknown spot is therefore thought to be a nonenzymatic product formed by incu- bation in the buffer.

From these results and the data described previously, we can conclude that the first product formed by the chloramphenicol- inactivating enzyme is n-three-2-dichloroacetamido-l-(p-nitro- phenyl)-1-hydroxy-3-acetoxypropane and the second is D-k?O-

2 - dichloroacetamido - 1 - (p - nitrophenyl) - 1,3 - diacetoxypropane produced by way of the first.

Lack of Chloramphenicol-acetylating Enzyme in E. coli K-12 CSH-Various attempts to detect the chloramphenicol-acetylat- ing activity in a cell-free preparation of E. coli K-12 CSH with- out R factor gave completely negative results.

Products Formed by Intact E. coli K-12 R5, K-12 CSH, or B CM7 Cells-The R5 cells were incubated with X-chloramphenicol under the condition described in “Experimental Procedure.” The ethyl acetate extract from a sonically disrupted culture was fractionated by paper chromatography. The result is shown in Fig. 4. There are two products which correspond to the 3-O- acetyl and 1,3-O, 0-diacetyl derivatives of chloramphenicol. The second product observed on inactivation by the enzyme fraction is therefore not an artificial product produced only in a cell-free system.

The results of a similar experiment with a chloramphenicol- sensitive strain of K-12 (0.14 /.~g of I%-chloramphenicol and 1 x lo* or 1 x 10 lo cells per ml) and the resistant B CM’ cells selected in vitro did not show any significant acetylated products. Therefore, these strains lack the chloramphenicol-acetylating enzyme and the mechanism of the drug resistance of B CM’ is different from that of R5 cells.

Purijication and Characterization of Acetylating Enzyme

All treatment of the enzyme was performed at l-4” unless otherwise noted.

Simple Assay Conditions for Enzyme Activity That Produces Diacetyl Derivative from Monoacetyl Derivative-From the results of the kinetic studies mentioned above, the substrate of the enzyme which produces the diacetyl derivative is assumed to be the monoacetyl derivative of chloramphenicol. As we did not have a labeled monoacetyl derivative, r4C-chloramphenicol and an enzyme fraction which produces the monoacetyl derivative only were utilized to supply the labeled monoacetyl derivative. Such an enzyme fraction was obtained by ammonium sulfate fractionation (50 to 65% fraction) or column chromatography in the absence of 2-mercaptoethanol (Fraction A of Fig. 5b). With this system, an assay of the activity producing the diacetyl derivative was possible. \

Partial Puri$cation of Chloramphenicol-acetylating Enzyme- By using the technique described in “Experimental Procedure,” partial purification of the chloramphenicol-acetylating enzyme was carried out with the S 100 fraction from K-12 R5 as a start- ing material. The degree of purification at each step is shown in Table II.

The recovery of the activity forming the first product (mono- acetyl derivative) remained constant throughout the purifica- tion procedure, while that of the activity forming the second (diacetyl derivative) decreased gradually. We could not, how- ever, separate both activities even during the last column chro- matography. The A-50 peak fraction of the last step in the purification showed a 121- and 47-fold increase in specific activ-

0 3 6 9

CM FROM ORIGIN

FIG. 3. Chromatogram of the enzymatically acetylated prod- ucts and the authentic samples observed by ultraviolet absorp- tion. In a, 30 pg of chloramphenicol were incubated for 20 set at 37” in the complete system described in “Experimental Procedure” (2.3 mg of protein of the R5 S 100 fraction were used). The ethyl acetate extract was chromatographed. Several trace spots ob- served in the absence of chloramphenicol in the incubation mixture have been omitted from the chromatogram. In b, 60 rg of chlor- amphenicol were incubated for 30 min at 37” in the complete system (0.58 mg of protein of the R5 S 100 fraction). In c and d, 20 pg of authentic chloramphenicol and d, 30 pg of authentic 3- 0-acetyl derivative of chloramphenicol were used, respectively. In e, 30 pg of authent,ic 3-0-acetyl derivative of chloramphenicol were incubated for 15 min at 37” in 0.10 M Tris-HCl buffer; pH 7.8. In f. 30 UUP of authentic 1.3-O.O-diacetvl derivative of chloram- phenicol’were used. In i, 4O’pg of aithentic 1,3-O,O-diacetyl derivative of chloramphenicol were partially hydrolyzed with KOH.

0 5 10 15 20 c rvl FROM ORIGIN

FIG. 4. Products formed by the whole cells of E. coli R5. The arrows represent the positions of the authentic samples, chlor- amphenicol, 3-O-acetyl derivative, and 1,3-O,O-diacetyl deriva- tive, from left to Tight, respectively.

ities of the first and second product formation, respectively, over the S 100 fraction.

The pH Optimum of Acetylating Enzyme-The pH optimum of the acetylating enzyme was investigated in an acetylating sys- tem containing the R5 S 100 fraction, ATP, CoA, and an excess of acetyl-CoA generator (CSH S 100 fraction), which was used in most of the experiments in this paper. The enzyme activity that produces the first product showed a broad pH optimum, whereas that producing the second has a rather narrow optimum, both showing a peak at pH 7.8. The same result was obtained in experiments with acetyl-CoA as a cofactor and a purified enzyme fraction (A-50 peak fraction). The purified fraction

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4728 Enzymatic Acetylation of Chloramphenicol Vol. 242, No. 20

did not require Mg++ in the presence of acetyl-CoA for the acetylation of chloramphenicol.

Heat Stability of Acetylating Enzyme-The activity producing the first product of the 30 to 40% ammonium sulfate fraction (in the standard buffer) was stable up to 60” for 5 min, dimin- ished considerably at 70” for 5 min, and was abolished completely at 80” for 5 min. The enzyme activity was stabilized against heating at 10” by the addition of the substrate chloramphenicol (2 rnM).

In order to assay the enzyme activity producing the diacetyl derivative, the 50 to 65% ammonium sulfate fraction was sup- plied in addition to the CSH S 100 fraction and other required reaction components that were added to the heated enzyme fraction. We could not find any significant difference between the heat, stability of the monoacetylating and diacetylating activities.

Substrate Speci$city of Acetylating Enzyme-The reaction sys- tem is described in “Experimental Procedure.” The radio- activity extracted from the complete reaction system containing ‘4C-acetic acid was considered to be radioactivity incorporated into the substrate, since no significant radioactivity was ex- tracted in the absence of the acetylating enzyme or a substrate (for example, chloramphenicol). The results of experiments of this type are shown in Table III and are expressed as a rough estimation of strongly positive, positive, or negative and as percentage of control (chloramphenicol) when possible, because the extraction efficiency of the various acetylated products of each substrate may be different.

The acetylating enzyme showed comparable activity toward chloramphenicol (D- three) , D - threo - I- (p -nitrophenyl) - 2 - acet- amido-1,3-propanediol, and n-fhreo-l-(p-methylsulfonylphenyl)-

TABLE III Substrate specificity of acetylating enzyme

The reaction mixture contained 1 /*mole of the compound to be tested, 2.2 X 105 cpm of ‘%-acetic acid, 1.0 mg of protein of R5 S 100 fraction, and other cofactors described in “Experimental Procedure.” After incubation at 37”, ethyl acetate extraction and other procedures were carried ollt as described in the text. The estimation of the extraction efficiency was also carried out by the same method.

Compound

Chloramphenicol, D-threo-l-(p-nitro- phenyl).2.dichloroacetamido-1,3-pro- panediol.

nL-erythro Isomer of chloramphenicol. D-three-l-(p-Nitrophenyl).2-acetamido-1,3

propanediol. D-lhreo-1-(p-Methylsulfonylphenyl)-2-di-

chloracetamido-1,3-propanediol . d-O-Acetyl derivative of chloramphenicol. n-three-1-(p-Nit,rophenyl)-2-amino-1,3-

propanediol D-three-2-Amino-3-(p-nitrophenyl)-3-hy-

droxypropionic acid. . Phenethyl alcohol. 1,3-Propanediol.

Degree of lcorporatior L

,f ‘4C-acetyl widues into

the compound’=

+++ ++

+++

+++ +

+

- - -

Percentage of control (chloram- phenicol)

100 55

75

1-2

<5 (2)

fi +++, strongly positive; ++, positive; -, negative.

2-dichloroacetamido-l , 3-propanediol (Table III). The ster- eoisomer of chloramphenicol, m-erythro-1 -(p-nitrophenyl) -2 - dichloroacetamido-1,3-propanediol, was found to be a less active substrate of the acetylating enzyme than chloramphenicol (Table III). The 3-0-acetyl derivative of chloramphenicol was also found to be a substrate; however, the rate of incorporation of the acetyl residue into the compound was faster than expected from the result of the kinetic studies of the acetylation of chloramphen- icol. Furthermore, the chromatographic profile of the ethyl acetate extract showed two peaks, one in the location of the 3-0- acetyl derivative and the other in that of 1,3-O, 0-diacetyl de- rivative. These results were considered to mean that there were some contamination of chloramphenicol in the substrate, deacety- lation and reacetylation of the substrate, or nonenzymatic mi- gration of an acetyl residue from position 3 to 1 of the pro- panediol moiety of the substrate.

The radioactive acetyl residue was incorporated into D-threo-l- (p-nitrophenyl)-2-amino-l, 3-propanediol at a rate of about 2 % of that of chloramphenicol. We measured the ethyl acetate ex- traction efficiency of the substrate instead of the acetylated prod- ucts, which we could not do, and found it to be about 40% under the condition, described in “Experimental Procedure” at pH 7.8. Therefore, even after maximal correction, the data mentioned above (2 %) will not, exceed 5 y. ( =2 x 100/40’%), because we can expect that the acetylated products of the substrate would be more effectively extracted with ethyl acetate than would be the substrate. Thus, w-e can conclude that the activity of the acetylating enzyme for this substrate is below 5% of that for chloramphenicol (Table III).

wthreoS-Amino - 3 - (p - nitrophenyl) - 3 -hydroxypropionic acid, phenethyl alcohol, and 1,3-propanediol did not show any sign that they are substrates of the acetylating enzyme (Table III). As for the first of these three compounds, paper chromatography (1-butanol-acetic acid-water, 4:2: 1) was carried out after incuba- tion in order to detect any acetylation of the compound in the reaction mixture itself, but we could not find any radioactive peak which was specific for t&e presence of the acetylating enzyme in the reaction mixture. Further negative checks were not carried out on the remaining two compounds, because it seemed unlikely that acetylated products of these compounds could not, be ex- tracted with et,hyl acetate.

From these results w-e assume that the acetylating enzyme shows a considerable specificity for the chloramphenicol molecule. We think it appropriate to call the enzyme that produces the 3-0-acetvl derivative of chloramphenicol acetyl-COA : chloram- phenicol 0-acetyltransferase.

Column Chromatographic Behavior qf the Acetylating Enzyme on DEAE-Sephadex A-50 in Presence or Absence of %Mercaptoetha- no&The 30 to 65 y. ammonium sulfate fraction dialyzed against the elution buffer containing 0.05 RI KC1 and 0.006 M 2-mercapto- ethanol was loaded on a DEAE-Sephadex A-50 column. The enzyme was fractionated by linear gradient elution of KC1 from 0.05 to 0.35 M in the presence of 2.mercaptoethanol. The result- ant profile is shown in Fig. 5a. In the presence of 2-mercapto- ethanol, both the mono- and diacetylating activities completely overlap each other.

However, in the absence of 2-mercaptoethanol the monoacety- lating activity showed two peaks (Fractions A and B of Fig. 5b), of which only the latter (Fraction B) showed diacetylating ac- tivity (Fig. 5b). Separate rechromatography of Fractions A and B was carried out also in the absence of 2-mercaptoethanol.

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Issue of October 25, 1967 Y. Suxuki and X. Okamoto 4729

Fraction B did not produce a new fraction corresponding to Frac- tion A in this rechromatography, and vice versa. Several trials to obtain Fraction B from Fraction A (and vice versa) were un- successful. The results of the gel filtration through Sephadex G-100 suggested that Fractions -1 and B have a similar molecular size.

Localization of Chloramphenicol-acetylating Enzyme-The sur- face localization of certain enzymes has been reported by Malamy and Horecker (16) and Neu and Heppel (8, 9, 17, 18). It w-as of interest to know if the chloramphenicol-acetylating enzyme is also located on the cell surface. Spheroplasts were prepared from K-12 R5 cells according to the method of Neu and Heppel (8).

I I I I I

; - t <

IO 20 30

FRACT I ON No.

- I I I I = z 3 n 0 (L -JO- & E

rnE z d -\

I IO 20 30

FRACTION NO.

FIG. 5. Column chromatographic behavior of the acetylating enzyme in the presence and absence of Z-mercaptoethanol. a, chromatographic profile of the acetylating enzyme in the presence of 2-mercaptoethanol (0.006 M). The 30 to 65oj, ammonium sulfate fraction (30 mg of protein) was loaded on a DEAE-Sephadex A-50 column (1 X 16 cm) and fractionated by the elution buffer con- taining 2-mercaptoethanol (0.006 M) and a linear gradient concen- tration of KC1 from 0.05 to 0.35 M (total volume, 200 ml); 7-ml fractions were collected. b, chromatographic profile of the acety- lating enzyme in the absence of 2-mercaptoethanol. The 30 to 65% ammonium sulfate fraction (60 mg of protein) was loaded on a DEAE-Sephadex A-50 column (1 X 18 cm) and fractionated by the elution buffer containing a linear gradient concentration of KC1 from 0.12 to 0.30 M (t)otal volume, 150 ml) ; 5-ml fractions were collected. 0, activity producing the monoacetyl derivative of chloramphenicol from chloramphenicol; l , activity producing the diacetyl derivative of chloramphenicol from the monoacetyl derivative.

TABLE IV

Localization of acetylaling enzyme

Fraction Rh-ase p-Galactosidase Chloramphenicol- acetylating enzyme

y? of told actidy recovered

Supernatant 0 0 0 Washing 1. 0 0 1 Washing 2. 2 0 1 Released. 76 8 13 Spheroplast. 18 82 79 Residue. 4 10 6

The results are shown in Table IV. About 80% of the ribonu- clease was released into medium from spheroplasts, while only 8 y0 of the P-galactosidase and 13 ‘% of the chloramphenicol-acety- lating enzyme was released. About 80% of these latter two en- zymes were retained in spheroplasts. These results indicate that the chloramphenicol-acetglating enzyme is not localized on the cell surface.

DISCUSSION

The products formed by the enzyme from the multiple drug- resistant E. coli carrying R factor were identified as the 3-O-acetyl and 1,3-O, 0-diacetyl derivatives of chloramphenicol. We could also find the 1-0-acetyl derivative of chloramphenicol in the prod- ucts. These findings are consistent with those of Shaw (3). We may conclude that the I-O-acetyl derivative is a nonenzymatic product, because an authentic sample of 3-O-acetyl derivative pro- duced 1-O-acetyl derivative when the incubation was carried out in Tris-HCl buffer, and the formation of 1-O-acetyl derivative in an enzymatic system appeared to be independent of the amount of the acetylating enzyme but dependent on the length of the in- cubation time. When the nonradioactive 3-0-acetyl derivative was incubated with the acetylating enzyme and labeled acetyl donor, the radioactive 3-0-acetyl derivative was produced as well as the expected 1,3-O, 0-diacetyl derivative (see “Results” and Reference 3). The results suggest that there is an acyl migration between the positions 3 and 1 of monoacetyl derivative, or a deacylation of the monoacetyl or diacetyl derivative. The ob- servation by Shaw (3) that 1-O-acetyl derivative did not accept acetyl residues makes the mechanism of over-all reaction dif- ficult to understand.

We could not find a remarkable amount of other producbs, for example, a product formed by the reduction of the aromatic nit,ro group (19); however, we occasionally observed a trace amount of unidentified product, which appeared very near to the origin of the thin layer chromatograms.

The biological reason why the monoacetyl derivative is con- verted to the diacetyl derivative by the enzyme from E. ccli R5 is not yet known. The enzyme activity producing the second product is not essential for the expression of drug rrsistance. At present we cannot conclude whether one or two enzymes produce the two products in E. coli K-12 R5.

The acetylating enzyme showed about 50% specificity toward the DL-erythro isomer of chloramphenicol. According to Shaw (3), the enzyme showed only 1% specificity toward the D-erythro

isomer. Thus we can assume that the enzyme has comparable specificity toward chloramphenicol and the L-erythro isomer. Investigations of other substrates have indicated that the sub-

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4730 Enzymatic Acetylation of Chloramph.enicol Vol. 242, No. 20

skate specificity of the acetylating enzyme is very specific for Acknowledgments-We wish to express our gratitude to Drs. J. the chloramphenicol molecule (this paper and Reference 3). Tomizawa, T. Komai, and D. Mizuno for their advice and dis-

We could not find any acetylated products either in cell-free cussion throughout the course of this work. We wish to thank preparations or in whole cells of the chloramphenicol-sensitive Dr. T. Osono and Mr. K. Murakami for their preparation and strain E. coli K-12 CSH, or in whole cells of B CM’ under con- supply of authentic samples, Dr. S. Nakazawa and Miss A. ditions which allowed the K-12 R5 cells to produce detectable Furuchi for their help with the infrared absorption spectroscopy, amounts of the acetylated products. Shaw (3) reported an im- Dr. M. Natsumefor his help with the nuclear magnetic resonance nortant finding that a chloramphenicol-sensitive strain of E. coli spectroscopy, and Mrs. N. Okamoto for her technical assistance. K-10 showed aweak acetylating activity. It will be worthwhile to clarify the situation regarding this point, since it may be rele- vant to the problem of the origin and mode of expression of the resistance genes of R factor.

It has long been known that E. coli produces another enzyme which can inactivate chloramphenicol by reducing the nitro group (19). This nitroreductase is not specific for chloramphenicol, its capacity to inactivate chloramphenicol is poor, and there is no definite correlation between the enzyme activity and chloram- phenicol resistance. In contrast, chloramphenicol is rapidly in-

activated by the cell extract as well as by whole cells of K-12 R5. This chloramphenicol-acetylating enzyme is specific for chlor-

amphenicol. According to our observations, the enzyme is com- pletely absent in K-12 CSH and is only produced by cells with R factor (see Reference 1). Even in the observations of Shaw (3),

the correlation of the enzyme activity and chloramphenicol re- sistance is quite clear. Therefore, it is probable that the chlor-

amphenicol-acetylating enzyme is the basis of chloramphenicol resistance in cells carrying R factor. The results with B CM’ indicate that the mechanism of chloramphenicol resistance of E. coli select,ed in vitro is quite different from that of E. coli K-12 carrying R factor.

The substrate specificity of the chloramphenicol-acetylating enzyme suggests that the origin of this enzyme may be related to the presence of chloramphenicol in natural circumstances, as suggested for penicillinase (20, 21).’

1 Note Added in Proof-It has been shown from our recent study on a temperature-sensitive mutant R factor that the chloram- phenicol resistance gene of the R factor controls the synthesis of the chloramphenicol-acetylating enzyme directly and this enzyme is the cause of chloramphenicol resistance of the cell. The enzyme preparation obtained from a strain of E. cola carrymg a mutant H. (1950). factor, which showed temperature-sensitive resistance to chloram- 20. SMITH, J. M. B., AND MARPLES, M. J., Nature, 201, 844 (1964). phenicol, also showed a heat-labile characteristic (K. Mise and Y. 21. STEWART, G., The penicillin yroup of drugs, American Elsevier Suzuki, in preparation). Publishing Company, Inc., New York, 1965, p. 148.

1. 2.

3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. (19G5).

19. SMITH, G. N., AND WORREL, C. S., Arch. Biochem.. 28, 232

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Yoshiaki Suzuki and Suyehiko Okamoto Carrying R FactorEscherichia coli

The Enzymatic Acetylation of Chloramphenicol by the Multiple Drug-resistant

1967, 242:4722-4730.J. Biol. Chem. 

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