[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
737
at least 1 month. Solutions in Sorenson buffer, pH 7.2, may be stored at 5 °, but the rate of inactivation has not been measured. The E. coli enzyme is reported to have high specificity for chloramphenicol. "~
Miscellaneous As a product of the enzymic reaction within the organism, p-nitrophenylserinol does not accumulate in large amounts, but is N - a c y l a t e d or metabolized to p-nitrobenzoic acid and other products. ~,~," The Streptomyces and bacterial enzymes are intracellular, except under autolytic conditions. -° In Streptomyces species 3022a substrate access to the enzyme appears to be prevented in cultures producing chloramphenicol, or in cultures adapted to grow under nonproducing conditions in the presence of the antibiotic? 6 G. N. Smith and C. S. Worrel, Arch. Biochem. 28, 232 (1950).
[57] Chloramphenicol Acetyltransferase from Chloramphenicol-Resistant Bacteria B y W. V. SHAW Chloramphenicol q- acetyl-S-CoA-~ chloramphenicol 3-acehtte + IIS-(?oA
(1)
H~.N/R2 OH FId. 1. Structure of compounds related to chloramphenicol. The asymmetric carboa atoms at C-1 and C-2 generate d~e four possible stereoisomers of which only the D-threo isomer has significant antibiotic activity. Chloramphenicol has the following functional groups: R, = --NO:; R: = --COCHCh ; and R~ = --OH. Chloramphenicol resistance is frequently encountered among many genera of bacteria. Although in some instances the underlying mechanism may be a relative impermeability to chloramphenicol (CM), the resistance phenotype is most commonly the result of inactivation of the antibiotic by the enzyme chloramphenicol acetyltransferase (CAT)?,'-' The 1W. V. Shaw, J. Biol. Chem. 241~, 687 (1967). : Y. Suzuki and S. Okamoio, J. Biol. Chem. 242, 4722 (1967).
738
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
O-acetoxy derivatives of CM are devoid of antibiotic activity since they do not bind to bacterial ribosomes and therefore fail to inhibit polypeptide elongation. The structural gene for CAT is commonly extraehromosomal among the Enterobacteriaceae, where it is carried by R factors conferring resistance to CM and is similarly associated with a plasmid in resistant staphylococci. CAT has also been detected in CM-resistant isolates of Streptococcus ]aecalis, Diplococcus pneumoniae, and Agrobacterium tume]aciens. Bacteria which synthesize CAT begin the exponential phase of growth only after the concentration of unmodified CM has fallen to a level that is no longer inhibitory. The duration of such an increment in the usual lag phase is roughly proportional to the concentration of CM at the time of innoculation. This relationship holds for gram-negative species harboring R factors or for any bacteria carrying plasmids which dictate the constitutive synthesis of CAT. A more complex situation exists in staphylococci, streptococci, pneumococci, and A. tume]aciens wherein CAT is an inducible enzyme. Chloramphenicol is an effective inducer of CAT in such strains, but it is also a potent inhibitor of bacterial protein synthesis. Because of these competing properties and the fact that the acetylated product is not an inducer, the kinetics of-CAT induction in bacteria with inducible CM resistance are complex. Levels of CAT activity can be maintained at a high level only by continued challenge with CM or by induction with a "gratuitous inducer" which is neither acetylated nor serves as an inhibitor of protein synthesis. Although all known R factors carrying the CAT gene in enteric bacteria mediate constitutive synthesi s of the enzyme, the specific activity of CAT in crude extracts is subject to wide variations due to gene dosage effects and also to "catabolite repression" by glucose and certain other growth substrates. Examples in the first instance are the "relaxed" replication of certain R factors in Proteus mirabilis ~ and the vegetative growth of bacteriophage P1 CM in Escherichia coli. ~ Both situations have in common a high multiplicity of plasmid-borne CAT gene copies. A less dramatic example is the occasional occurrence of strains of E. coli (or related enteric bacteria) which harbor two compatible plasmids, both of which may carry one copy of the CAT region2 Catabolite repression of CAT synthesis in E. coli is mediated by a mechanism involving cyclic adenosine 5'-monophosphate (cAMP), in common with the streptomycin adenylylating enzyme and the more gens R. Rownd, H. Kasamatsu, and S. Mickel, Ann. N . Y . Acad. Sci. 182, 188 (1971). ' E. Kondo, D. K. Haapala, and S. Falkow, Virology 40, 431 (1970). 5 W. V. Shaw, L. C. Sands, and N. Datta, Proc. Nat. Acad. Sci. U~S. 69, 3049 (1972).
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
739
eral cases of fl-galactosidase and gatactokinase. 6 The decrease in CAT levels seen with growth in glucose-containing media can be prevented by the addition of high concentrations of cAMP or by the substitution of glycerol or other nonrepressing energy sources for glucose. Requirements ]or CM Acetylation Techniques. More techniques for studying the enzymic acetylation of CM are described below than are likely to be of use for any given research objective, The thin-layer chromatographic method for detecting CM acetylation in cell suspensions is useful in screening presumptive CAT producing strains or for exploring other possible mechanisms of CM inactivation. The spectrophotometric methods excel for general purposes and can also be adapted for the measurement of either CM or acetyl coenzyme A (CoA). The very sensitive radioisotopic method appears cumbersome but is remarkably reliable in practice and very useful for detecting and quantitating v'ery low levels of CAT activity. It should be stressed that the availability of CAT as a reagent offers interesting possibilities for (a) the specific detection of the biologically active isomer of CM and its quantitation and (b) the instantaneous removal (by inactivation) of CM present in any in vitro biological system. A preliminary investigation of the use of CAT to measure CM in body fluids is encouraging as regards specificity, sensitivity, and speed: features of enzymic assay techniques which are usually lacking in many of the commonly used bacteriological assay methods for antibiotics. ~ An additional possible application of CAT is its use to inactivate CM in instances wherein the antibiotic has been added as a potentially reversible inhibitor of protein synthesis. Apart from the above considerations it should be noted that CAT may be of value to molecular biologists as it is a gene product which can be assayed with specificity and great sensitivity. Two temperate phages have been isolated which confer CM resistance on the host cell in the lysogenic state, and in both cases the phenotype is due to the synthesis of CAT. Phage P1 CM was isolated as a recombinant between an R factor and phage P1 and, more recently, a derivative of phage lambda (~ CM) has been constructed which also carries the CAT gene. ~ It has been possible to synthesize CAT in a cell-free system from E. coli using P1 CM template DNA, 9 and it seems likely that either or both ~J. Harwood and D. H. Smith. Biochem. Biophys. Res. Commun. 42, 57 (1971). ~P. Lietman, personal communication, 1973. 8j. R. Scott, Virology 53, 327 (1973). B. de Crombrugghe, I. Pastan, W. V. Shaw, and J. L. Rosner, Nature (Lo~don) New Biol. 241, 237 (1973).
740
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
of the CM phages will be useful for studies of the control of transcription and translation.
Assay Methods
Principles. Enzyme activity can be quantitated by either measuring (a) the CM-dependent disappearance of acetyl-S-CoA; (b) the appearance of 3-O-acetoxy derivative of CM; or (c) the formation of reduced (unesterified) CoA. In practice, the choice between these alternatives will be determined by the level of sensitivity required and by complications created by interfering substances. The 3-monoacetoxy derivative of CM is the initial product of the reaction and is devoid of significant antibiotic activity. A diacetoxy product appears at a rate two orders of magnitude slower than the monoacetylation reaction, and its formation is also catalyzed by CAT. Although the mechanism has not been studied in detail, there is circumstantial evidence favoring the following sequence of reactions: CM-3-acetate ~ CM-l-acetate CM-l-acetate + acetyl-S-CoA--~ CM-1,3-diacetate + HS-CoA
(2)
(3)
The reaction described by Eq. (2) appears to be a nonenzymic and pHdependent acyl migration. It seems likely that the slow overall rate of formation of the diacetate derivative is due both to an unfavorable equilibrium of the nonenzymic step and also to a low rate of 3-O-acetylation of CM-l-acetate by CAT. Although Eqs. (2) and (3) should be kept in mind in any kinetic or mechanistic studies of CAT, they do not in fact interfere significantly with measurements of CAT which utilize the stoichiometry of Eq. (1).
Chromatographic Detection o] Chloramphenicol Acetylation Although of historical interest as a means of assaying CAT, the thinlayer chromatographic separation and quantitation of CM and its acetoxy derivatives still has limited applications, especially for the detection of CM acetylation by bacterial cell suspensions. The chromatographic determination of the fate of CM when incubated with a culture of a resistant microorganism also can reveal (a) other mechanisms than acetylation if the latter is not involved or (b) no alteration of CM, thereby suggesting a relative impermeability to CM of the bacterium in question. The use of 14C-labeled CM is recommended because of the difficulties in detecting small amounts of products formed when the initial concentra-
[571
CHLORAMPHENICOL ACETYLTRA_NSFERASE
741
tion of nonradioactive CM may not exceed 10 uM (3.2 ~g/ml). Since no general protocol can be formulated, a typical example will be given to illustrate a possible application. A strain of E. coli which is resistant to CM (minimum inhibitory concentration in nutrient broth = 100 t,g/ml) is to be tested for the presence of CAT. An overnight culture is diluted 1:1000 into 5 ml of fresh growth medium (the same liquid medium used for the sensitivity testing) containing J'~C]chloramphenicol at a concentration of 32 /~gfml, (0.1 raM) and a specific activity of 1 mCi/mmole. At the conclusion of tile exponential phase of growth the culture tube is centrifuged and 1 ml of the culture supernatant is extracted with 1 ml of ethyl acetate by agitation on a vortex mixer followed by centrifugation. The process is repeated twice and the ethyl acetate supernatants are pooled in a conical centrifuge tube and evaporated to dryness in air stream or on a steam bath. The sample is then taken up to 0.1 ml of ethyl acetate and spotted by repeated applications with a capillary pipette at the origin of a thinlayer sheet alongside an extract of an uninoculated control culture which has been treated in similar fashion. The chromatograms are developed in ascending fashion with the appropriate solvent until the front is a few centimeters from the top of the sheet. After removal from the tank or beaker the sheets are air dried and subjected to radioautography overnight after appropriately marking the sheets with reference points made with radioactive ink. On inspection the film will reveal whether bacterial growth has been accompanied by conversion of CM to its monoand diacetyl derivatives. The choice of thin-layer supports and solvents is arbitrary, but the following have been useful~: (1) alumina in benzene-methanol (85:15, v/v}; (2) silica gel in chloroform-methanol (95:5, v/v). Commercially available thin sheets of both alumina and silica gel on inert supports are available from a number of suppliers. Those which contain a fluorescent additive (such as Ladd thin fihns, Packard) are especially useful, as CM and its products give a prominent "quench" when present as major components. The translucent Mylar sheets are useful since they can be aligned with the exposed X-ray fihn and viewed by transmitted light. The areas of the chromatogram are marked, cut out with scissors, and dropped into scintillation vials for counting. Quantitation of the percent conversion of CM to acetyl products can thus be determined directly, and if samples of the culture "tre taken at several points in time, the data can be plotted to show the kinetics of inactivation. The same approach may be utilized to follow the course of the enzyme reaction, but the methods described below have distinct advantages in convenience and precision.
742
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Spectrophotometric Assay The most convenient technique for quantitating the rate of CM acetylation takes advantage of the generation of a free CoA sulfhydryl group coincident with transfer of the acetyl group to CM. Reaction of the reduced CoA with 5,5"-dithiobis-2-nitrobenzoic acid (DTNB) yields the mixed disulfide of CoA and thionitrobenzoic acid and a molar equivalent of free 5-thio-2-nitrobenzoate. 1° The latter has a molar extinction coefficient of 13,600 at 412 nm. The assay is best carried out with a recording spectrophotometer equipped with a temperature-controlled cuvette chamber set at 37 ° .
Reagents Tris. hydrochloride, 1.0 M, pH 7.8 Acetyl-CoA, 5 mM Chloramphenicol (D-threo) 5 nlM 5,5'-Dithiobis-2-nitrobenzoic acid (DTNB) The only reagent solution that must be stored frozen is acetyl-CoA. The reaction mixture is freshly prepared from the individual reagents by dissolving 4 mg of DTNB in 1.0 ml of Tris.HC1 buffer, after which 0.2 ml of the acetyl-CoA stock solution is added and the total volume is made up to 10 ml. The final concentrations of each component are as follows: Tris.HC1 (100 mM), acetyl-CoA (0.1 mM), and DTNB (0.4 mg/ml). After the cuvette (1 cm light path) containing enzyme and the reaction mixture has been allowed to equilibrate with the waterbath, the reaction is started by the addition of CM at a final concentration of 0.1 mM. The rate of increase in absorption at 412 nM prior to the addition of CM is subtracted from the observed rate after the start of the reaction, and net change in extinction per minute is divided by 13.6 to give the result in micromoles per minute of CM-dependent DTNB reacted. Since the latter is equal to the rate of acetylation and since 1 unit of CAT = 1 ~mole of CM acetylated per minute (37°), the calculation also yields the number of units of enzyme in the cuvette. Two factors influence the precision of the DTNB assay for CAT. First, care must be taken to avoid very high concentrations of mercaptans in the enzyme solution. In practice the addition of 2-mercaptoethanol or dithiothreitol in excess of 1 mM to crude or even partially purified preparations of CAT presents problems when more than 10 ~l of enzyme must be added to a 1-ml cuvette to obtain a significant rate of CM acetylation. A more troublesome problem occurs with crude cell extracts prepared from certain genera of bacteria that contain high levels of thiolo A. F. S. A. Habeeb, this series, Vol. 25, p. 457.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
743
esterase activity. In such instances the control rate of increase in 412 nm absorbance due to acetyl-CoA hydrolysis prior to the addition of CM may approach or exceed the increment seen after adding the antibiotic. In some instances it may be necessary to resort to partial purification of CAT before the spectrophotometric assay can be used with confidence. The [l+C]acetate procedure which utilizes an acetyl-CoA generating system should also be considered as a possible solution to the problem of high nonspecific thioesterase background (see below). A serious limitation to the DTNB assay for CAT concerns the intrinsic high reactivity of essential cysteine thiol groups in certain variants of CAT. In the absence of protecting reduced mercaptans, enzyme activity decreases rapidly after addition of DTNB. Since the rate of CM acetylation is substantially greater than the rate of CAT inactivation, an approximation of the initial rate for CAT activity can be obtained by taking special care to add enzyme immediately prior to addition of CM. An alternative spectrophotometric method 1 can be used when a high concentration of competing mercaptans interferes with the DTNB assay. The loss of an acyl group from thioesters such as acetyl-CoA is accompanied by a decrease in absorption in the ultraviolet. The difference in molar extinction coefficients of acetyl-CoA and reduced CoA plus acetate is 4500 at 232 nm. 11 Special care must be taken to remove interfering ultraviolet absorbing material from the enzyme preparation by gel filtration or dialysis. The contribution of the absorption due to protein added to the euvette becomes a more serious obstacle in crude extracts, especially those with low levels of CAT activity. Apart from the inconvenience of measurements in the far ultraviolet region and the fact that the method is intrinsically less sensitive than the DTNB procedure, the assay of thioester cleavage at 232 nm suffers from being a difference method. The absolute decreases in absorbance per unit time due to the presence of CM and low levels of CAT may be impossible to quantitate without recourse to the use of a dual beam recording spectrophotometer.
Assay by Direct Measurement o] ["C ]Acetyl Chloramphenicol The need for a highly sensitive, rapid, and specific means of quantitating the synthesis of CAT in a complex E. coli extract 9 led to the development of a radioactive method which, unlike that described above, does not require chromatography. The principle of the method is that the neutral [14C]acetoxy-chloramphenicol derivatives can be quantitatively extracted into benzene at alkaline pH whereas ionized and polar species such as [14C]acetate and [14C]acetyl-CoA remain in the aqueous phase. 11E. R. Stadtman, this series, Vol. 3, p. 985.
744
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
The sensitivity of the method is limited only by the specific activity of [14C]acetate used. Specificity is achieved by measuring the increment in radioactivity extracted from an assay mixture containing CM as compared to that from a control incubation without CM. Should any doubt arise that the products are, in fact, the acetoxy derivative(s) of chloramphenicol, the chromatographic methods described previously may be used for confirmation. The most convenient means of terminating the assay incubation has been found to be the decomposition of acetyl-CoA catalyzed by phosphotransacetylase in the presence of 100 mM arsenate. The arsenolysis reaction obviates the need for extremes of temperature or pH and does not interfere with the extraction of products. Although the coupled assay requires numerous reagents, including three enzymes, all the components are readily available from commercial sources. A mixture of buffer, salts, and substrates may be stored frozen for convenience, but the reagent enzymes should be stored separately under the recommended conditions and added individually to the final incubation mixture immediately prior to the assay. The most convenient means of standardizing the radioactive assay to conform with the spectrophotometric unit of CAT activity (as defined above) is to assay a preparation of crude or purified CAT by both techniques. The two methods should agree within 5-10% when: (a) care is taken to ensure adequate temperature control in both instances; (b) the amount of CAT in each case is appropriate for the measurement of initial rates, and (c) the extractions are carried out with care. The experiments in which the radioactive assay have been used thus far have required that the method be specific, sensitive, and reproducible, rather than of a high absolute accuracy with respect to reference measurements. The maintenance of a saturating concentration of [14C]acetyl-CoA for CM acetylation by CAT is achieved by means of the following coupled reactions and the addition of an excess of pyruvic kinase, acetate kinase, and phosphotransacetylase: ADP -{- phosphoenolpyruvate ~ pyruvate T ATP ATP T [~4C]acetate ~ [~4C]acetyl phosphate T ADP [14C]acetyl phosphate + HS-CoA ~ [~4C]acetyl-S-CoA T phosphate
(4) (5) (6)
The pyruvic kinase system (Eq. 4) has been employed to generate a catalytic level of ATP since adenine nucleotides are competitive inhibitors (with respect to acetyl-CoA) of CAT. Although each of the reagent enzymes must be added in excess, it is usually not necessary to assay each one independently before its addition to the assay incubation. It is actually easier and more useful to ascertain that each is, in fact, present in excess than to assign a precise value to the activity of each component.
[57]
745
CHLORAMPHENICOL ACETYLTRANSFERASE
This is best accomplished by assuming the accuracy of the supplier's nominal activity specifications as a point of departure. The radioactive assay is then run with a sample of CAT with known activity by the DTNB method. Dilutions of each reagent enzyme are made, and aliquots of several such dilutions of each enzyme are tested while the other two are added at concentrations assumed to be 10-fold higher than the 1 unit required (1 umole/min) for each under the conditions of the assay. For each enzyme reagent so tested a limiting dilution shoukt be reached where the final amount of [~4C]acetyl CM radioactivity extracted is (a) lower than that with all less dilute samples of the enzyme in question and (t)~ less than that expected from the known activity of CAT present from the DTNB procedure. The assay conditions described below are a modification of those reported previously." The present procedure has evolved in order: (a) to avoid the inhibition of CAT by adenine nucleotides (see above), (b) to ensure that acetyl-CoA and CM are present at ~aturating concentrations, (el to be certain that the reagent enzymes are in excess and art present in a favorable environment for activity, and (d) to maximize the efficiency of the extraction procedure without introducing either high blank v'dues or artifacts2'-'
Reagents
Volume (ul) per 250 ul incubation
Final concentration
1 M T r i s . HCI (pH 7.8) 100 m M MgCI: 100 m M A T P 100 m M P h o s p h o e n o l p y r u v a t e 5 m M Coenzyme A (reduced; lithium salt) 10 m M [1-~4Cl Sodium :~eetate Acetate kinase P y r u v a t e kinase Phosphot r'msacet ylase
25 15 7.5 50 20 10 See text See lext See lext
100 m.'ll 6 mM 3 mM 20 m M 0.4 m M O. 4 m M
1 Unit 1 Unit I Unit
Each incubation is carried out in a conical centrifuge tube (nominal volume approximately 12-15 ml) to simplify the subsequent extraction t)rocedure. Tile final volume of 250 ul is achieved by the addition of tile ,2 Two other techniques have been tried unsuccessfully for the separation of P4C]acetoxy chloramphenicol from radioactive acetate and acetyl-CoA. Tile tatier should be absorbable by anion exchange resins (such as Dowex 1) whereas the nonionized product is not. An alternative approach is the selective absorption of C M and its P~C]acetyl derivatives to charcoal after arsenolysis of ["C]acetyl-CoA. In practice b o t h of these approaches have given high and variable blank values.
746
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
requisite volume of deionized water and the sample containing CAT, after which the components are thoroughly mixed. The blank incubation contains only water to volume, but no enzyme. In both instances the reaction is started by the addition of 5 ~l of chloramphenicol (5 mM) immediately prior to placing each Parafilm-sealed tube into a water bath at 37 °. After 20 rain of incubation the reaction is terminated by the addition of 25 ~l of 1.1 M sodium arsenate followed by thorough mixing. The extraction and processing of the product is carried out in the following manner. Two milliliters of benzene (analytical reagent grade) is added to each incubation tube, and the latter is agitated on a Vortex mixer for 20 sec. The tube is centrifuged in a nonrefrigerated clinical centrifuge for a few minutes to separate phases, and the upper benzene layer is carefully taken with a Pasteur pipette and placed in a glass scintillation vial. The extraction is repeated with a second 2 ml of benzene. The extracts are pooled, and 100 ~l of glacial acetic acid is added to each scintillation vial. After thorough mixing of the acetic acid and benzene, the samples are dried under a heat lamp in an air stream. After the samples are thoroughly dried, an appropriate scintillation counting solution is added (any convenient type is adequate since the product is soluble in most organic solvents and there is no nonvolatile residue to cause quenching) and each vial is assayed for 14C radioactivity. Blank incubations (in which CM has been added in the absence of CAT) or controls (containing CAT but no CM) should yield extracts with no more than 100-200 dpm above background when the specific activity of sodium acetate is of the order of 5 mCi per millimole. It should be apparent that initial CAT velocities will be measured accurately with this technique only if CAT is present in amounts such that both CM and acetyl-CoA are still at saturating concentrations after 20 m~n of incubation. In practice this means the addition of no more than approximately 0.0005 unit of CAT to any given incubation, an amount of enzyme sufficient to catalyze the acetylation of 10 nmole of CM in 20 min (or 40% of the 25 nmoles present initially). It is fortunate that the DTNB method begins to give reliable data at CAT levels near 0.0005 unit when a recording spectrophotometer with scale expansion (to 0 . 1 0 D units full scale) is employed. The choice of method will therefore usually favor the radioactive assay whenever the concentration of CAT is less than 0.005 unit/ml (0.005 unit per 100-~1 sample).
Purification of Chloramphenicol Acetyltransferase General Remarks. Conventional techniques of protein fractionation have been adequate for the purification of CAT from (1) R factor-bear-
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
747
ing strains of E. coli, (2) staphylococci harboring CM plasmids. (3) CMresistant mutants of Proteus mirabilis in which the CAT gene is probably chromosomal, and (4) selected strains of Agrobacterium tume]aciens. Although the following protocol describes a typical purification for an R factor type of CAT, the principles can be applied to any bacterial source of the enzyme. Since each variant of CAT examined thus far has been an acidic protein, the use of DEAE-cellulose absorption is common to all purifications and is an important step. The molarity of sodium chloride required to elute CAT in each instance will be found to roughly parallel the net charge as measured by the apparent isoelectric point (pK~; isoelectric focusing). The most acidic CAT purified thus far (Agrobacterium tume]aciens; pK~ = 3.93) is eluted at 0.21 M ~3 whereas all other species of CAT (pK~ = range of 4.8 to 5.7) have been recovered at lower ionic strengths (sodium chloride; 0.1(}-0.19 M). The procedure described gives a homogeneous CAT product from R factor-containing strains of E. coli and isolates of S. aureus harboring a CM plasmid. The ease with which such results can be achieved is due to the fact that fully induced S. aureus cultures and virtually all R ÷ E. coli cultures synthesize CAT to levels approximating 0.5-1% of the soluble cell protein in stationary phase (see Table I for summary of typical purification). It should be anticipated that other genera or species which synthesize CAT may do so at lower rates and that additional purification steps may be required to obtain a pure enzyme preparation. A useful maneuver in such instances is a second DEAE-eellulose step (after gel filtration) employing a different pH and salt gradient (for example, zero to 0.5 M sodium phosphate at pH 7.0).
Typical Purification o] CAT ]rom E. coli Growth o] Bacteria. A prototrophic strain of E. coli carrying an R factor for CM resistance (or a P1 CM lysogen of such a strain) is grown to the stationary phase of growth at 37 ° with vigorous shaking (or alternative means of aeration) in a well buffered nonrepressing growth mediam. The latter is conveniently and inexpensively provided by a medium containing per liter: K2HPO4, 14 g; KHP04, 6 g; (NH~).,SO~, 2 g; MgS(L, 0.2 g; easamino acids (Difco), 1 g; and glycerol, 5 g. A heavyduty orbital shaker platform capable of taking twelve 2-liter Erlenmeyer flasks will yield approximately 35-40 g (wet weight) of cells from 14 liters if each flask contains only 1200 ml of the above medium to ensure adequate aeration. The cells are harvested with a continuous flow centrifuge (Sharpies Supercentrifuge or Sorvall continuous-flow attachment for 1~L. C. Sands and W. V. Shaw, unpublished studies.
748
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
the RC-2B centrifuge) and suspended in a final volume of 200 ml of 50 mM Tris.HC1 (pH 7.8) containing 2-mercaptoethanol at a concentration of 50 ttM (TM buffer). Step 1. Crude Extract. The cells are broken by sonication or by extrusion in a French pressure cell (Aminco). If the latter method is used a few crystals of deoxyribonuclease should be added to reduce the viscosity of the suspension prior to centrifugation. The cell debris is then removed by centrifugation (30,000 g for 20 min), and the supernatant is taken for purification. Such crude preparations of CAT are stable for months at --20 ° . All subsequent steps are carried out at 0-4 ° . Step 2. Streptomycin Sul]ate Precipitation. Solid streptomycin sulfate is dissolved in the crude extract at a final concentration of 1%. The precipitate obtained after 30 min is collected by centrifugation and discarded. After this step, to remove the bulk of nucleic acids the concentration of pH 7.8 Tris. HC1 buffer is increased to 100 mM prior to precipitation of CAT with ammonium sulfate. Step 3. First Ammonium Sul]ate Step. CAT is precipitated from the buffered supernatant of the streptomycin step by the addition of finely ground ammonium sulfate (enzyme grade) to a final concentration equivalent to 50% saturation. 14 The ammonium sulfate is added slowly with stirring and the preparation is allowed to stand for 30 min in an ice bath before centrifugation at 30,000 g for 20 rain. The supernatant is decanted carefully and put aside until it has been determined that it contains less than approximately 10% of the total activity in the precipitate after the latter is dissolved in 50 ml of TM buffer containing 0.2 mM chloramphenicol (TCM buffer). In the event that CAT has not been precipitated effectively, additional ammonium sulfate should be added to the supernatant to achieve 55% saturation and the centrifugation repeated. It is imperative that the purification proceed to the heat step directly and especially that freezing of ammonium sulfate containing solutions of CAT be avoided. Step 4. Heat Step. All naturally occurring variants of R-factor CAT have been found to be sufficiently thermostable to permit the use of a heat step in the purification. 5,1~ After the precipitate from the ammonium sulfate step is dissolved in TCM buffer the enzyme preparation is allowed to reach room temperature and is then placed in a heated water bath set at 60% The extract is stirred slowly for 10 min, cooled, and centrifuged, and the precipitate is discarded. Step 5. Second Ammonium Sul]ate Step. The supernatant fluid from the heat step is brought to 50% saturation with ammonium sulfate, and ~4A. A. Green and W. L. Hughes, this series, Vol. 1, p. 67. 15W. V. Shaw and R. F. Brodsky, J. Bacteriol. 95, 28 (1968).
[571
CHLORAMPHENICOL ACETYLTRANSFERASE
749
the CAT-containing precipitate is collected as in the earlier precipitation step. The precipitate is dissolved in a small volume (15-25 ml) of TCM buffer and either (a) dialyzed against the latter to remove residual ammonium sulfate, or (b) desalted on a Sephadex G-25 column of suitable size which has been equilibrated with TCM buffer. In practice the gel filtration method is more convenient since it permits completion of all steps up to and including the loading of the DEAE column in a normal working day. Step 6. DEAE-Cellulose Chromatography. The usual precautions are taken to ensure that the chromatography medium contains particles of near uniform size and that the column is well packed and completely equilibrated with the TCM buffer. Microgranular (DE-52) Whatman DEAE-cellulose (dry or prewetted) should be satisfactory in all respects, so long as the manufacturer's preeyeling instructions are followed. A packed column bed of dimensions 2.5 X 40 em should be adequate for a preparation of the size described above and can be developed with a l-liter gradient of SaC1 (0-0.4 M) in TCM buffer after the desalted sample has been applied to the top of the eohmm and the latter washed with several column volumes of TCM buffer. CAT activity should be eluted iust before the midpoint of the NaC1 gradient (0.15-0.20 M in most instanees~ and can be easily detected by the speetrophotometrie (DTNB) method. The strategy for pooling the CAT containing tubes will depend on the goal of the overall purification. Since the enzyme is stable at 0-4 ° for at least 24 hr there is much to be said for determining the CATspecific activity for each tube in the peak and for surveying the number and types of contaminants by polyaerylamide electrophoresis before pooling tubes. When the DEAE step is to be followed by gel filtration, ttw pooled peak tubes can be concentrated by membrane ultrafiltration (Amicon or equivalent types) to a volmne suitable for the column step to follow. Step 7. Gel Filtration. In principle, any gel filtration support which will include CAT (molecular weight: 80,000t should be useful for size separations. In practice, Sephadex G-100 proved satisfactory and has been the only method used. A volume (approximately 5-10 roll of concentrated enzyme solution from the prior step is applied to the bottom of such a column (2.5)< 100 era) equilibrated with TCM buffer containing 0.2 M NaC1 and the elution is continued in upward fashion using th~ same buffer. As in the prior step the peak tubes are pooled judiciously on the basis of specific activity data and/or electrophoretic purity and concentrated if required. The activity of CAT in the elution buffer is stable at --20 ° for at least one year. Step 8. Additional or Alternate Steps. Although in most instances the
750
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
TABLE I PURIFICATION OF CHLORAMPHENICOL ACETYLTRANSFERASEa
Step 1. 2. 3. 4. 5. 6.
Crude extract Streptomycin sulfate First a m m o n i u m sulfate H e a t (60 °) Second a m m o n i u m sulfate DEAE-cellulose chromatography 7. Gel filtration (Sephadex G-100)
Protein (rag total)
Enzyme activity (units total)
Specific activity (units/ rag)
Purification (fold)
Yield (%)
5400 4150 2800 900 640 51
4900 4650 4400 4150 4050 2150
0.9 1.1 1.6 4.6 6.3 42 1
(1) 1.2 1.8 5.1 7.0 47
(100) 95 90 85 83 44
14
1750
125
139
36
CAT obtained from the peak tubes of G-100 column (Step 7) was homogeneous by disc gel electrophoresis.
above procedures will yield electrophoretically homogeneous CAT, it m a y be necessary to carry out an additional step. In the past this has taken the form of either a batchwise adsorption procedure with Alumina C~ gel immediately following the heat step 15 or a terminal step involving a second D E A E column run at a different pH a n d / o r with a different buffer system than that used in step 6. The arguments in favor of the alumina C~ gel step are that it is relatively straightforward, quite effective, and does not preclude the second D E A E procedure if the latter is required. Conversely, since homogeneous CAT can often be obtained without the alumina C~ gel step, it is convenient to "wait and see" before adding an additional procedure. A totally different strategy from the conventional one described above should be mentioned, namely, that of affinity chromatography. 16 Initial experiments with CM-substituted agarose as a specific adsorbant for CAT have been encouraging as regards specificity, but the elution behavior of the enzyme has been unsatisfactory in some respects. The approach employed has been to attach the free amine of CM (where R2 = H in Fig. 1) to a solid support via the formation of an amide bond with the free carboxyl group of CH-Sepharose (Pharmacia) using a water-soluble carbodiimide reagent. The rationale for the use of CM base as ligand is that the nature of the N-acyl (R2) substituent of the CM skeleton is a less critical determinant of substrate affinity for CAT than the 1,3~ P. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
751
propanediol side chain or p-phenyl substituent. ~7 Current studies are aimed at avoiding the extremes of ionic strength, and CM concentration which are both required to quantitatively elute CAT from the adsorbant described23
Purification o] CAT ]rom Other Bacterial Species Staphylococcus sp. Since staphylococci are more fastidious in their growth requirements and CAT does not appear to be repressed by glucose in a representative collection of strains examined,13 the preferred growth medium is commercially available complex media such as Penassay Broth (Difco). All wild-type isolates of staphylococci harbor the genes for an inducible CAT is and, hence, careful attention to the details of induction (derepression) is necessary for optimum yields of enzyme. Conditions for "gratuitous" induction have been described previously 19 for the 3deoxy analog of CM (where R3 = H), but since this compound is neither available commercially nor readily synthesized, an alternative procedure will be described. A CM-resistant isolate of Staphylococcus sp. is tested for CM resistance on nutrient agar containing 50 t~g of CM per milliliter, and a single colony is picked for overnight growth in Penassay (PA) broth containing the same concentration of CM (PA-CM). Growth on a preparative scale is begun in PA-CM medium with an inoculum from the overnight starter culture equal to approximately 5% of the total volume of the large-scale culture, and the process is repeated to yield a suitable volume of inoculum (e.g., 800 ml) for a total final preparative volmne of 14 liters. By analogy with the earlier protocol for E. coli, this can be accomplished by inoculating each of 12 Erlenmeyer flasks (nominal volume of 2 liters) containing 1200 ml of PA-CM with approximately 60 ml of starter culture. Growth is then allowed to proceed at 37 ° on a rotary shaker. After each doubling of the cell mass (as approximated by turbidity measurements) fresh sterile CM is added to bring the final concentration to 50 ~g/ml. When the cultures have reached the final stationary phase of growth a final addition of CM is made and the cells are allowed to shake for an additional period of 20-30 rain to ensure maximum induction before harvest. Cells are best collected by centrifugation in large (polypropylene or stainless steel) bottles to avoid the hazardous aerosol obtained with use of an "open" centrifuge such as the conventional Sharples instrument. The yield of cells should be of the order of 40-50 g (wet weight). 1TW. V. Shaw and R. F. Brodsky, Antimicrob. Ag. Chemother. 1967, 257 (1968). ~SL. C. Sands and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 299 (1973). 19E. Winshell and W. V. Shaw, J. Bacteriol. 98, 1248 (1969).
752
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Cell lysis is accomplished by the use of Lysostaphin (Schwarz-Mann) as follows. 19 The pooled cell pellets are washed by suspension in 1 liter of ice cold Tris-saline (TS) buffer (50 mM Tris.HC1, pH 7.5, and 145 mM sodium chloride) and collected by centrifugation. The cells are resuspended in approximately 1 liter of TS buffer containing Lysostaphin (10 units/ml) and deoxyribonuclease (50 ~g/ml) and incubated at 37 ° with agitation using a magnetic stirrer. Cell lysis is nlonitored by observing the decrease in turbidity at 660 nm of diluted aliquots of the suspension and is usually complete after 1 hr. The supernatant fluid (crude extract) obtained after centrifugation (10,000 g for 20 min) to remove cell debris should contain approximately 0.2 unit of CAT per milligram of soluble protein. The purification of staphylococcal CAT can be achieved by a straightforward 4-step procedure which omits the preliminary streptomycin sulfate precipitation and the second ammonium sulfate step of the E. coli protocol. Since the overall purification of CAT from staphylococci has been described in detail elsewhere 1~,19 and is similar to that outlined for the R factor enzyme (see above), only the ammonium sulfate precipitation step will be described here. The crude extract obtained after treatment with Lysostaphin is buffered to 100 mM with pH 7.8 Tris.HC1 and made 50 mM for 2-mercaptoethanol prior to the addition of sufficient finely ground ammonium sulfate to achieve 70% of saturation. For most variants of staphylococcal CAT, the supernatant after centrifugation of the precipitate obtained should contain more than 90% of the total enzyme activity present in the crude extract. The supernatant is then brought up to a theoretical 90% saturation with further addition of ammonium sulfate, and the precipitate is collected by centrifugation and dissolved in TCM buffer for desalting and DEAE chromatography, as described for the E. coli variants of CAT.
Properties of Chloramphenicol Acetyltransferase General Comments. Although there is no single type of CAT which
can be chosen as representative, the variants studied to date possess the following properties: pH optimum of 7.8; native molecular weight of 80,000 (and quaternary structure, of four identical subunits of 20,000 each) ; and apparent isoelectric point between 5.4 and 4.0. All CAT variants therefore behave on polyacrylamide gel electrophoresis as typical globular acidic proteins of varying net charge and a corresponding broad range of electrophoretic mobilities when examined at alkaline pH. Each variant is specific for the D-threo stereoisomer of chloramphenicol (or con-
[57l
CHLORAMPHENICOL ACETYLTRANSFERASE
753
geners) as the acyl acceptor and a marked preference for acetyl-S-CoA as the aeyl donor as compared with other acyl-S-CoA homologs. Most of the important differences observed to date between the enzymes themselves (as opposed to their mode of synthesis and its control) have been detected by kinetic or immunological means. The most easily tabulated properties are summarized in Table II. Specificity for Acyl Aeceptor. A large number of analogs and isomers of C3I have been screened for their ability to serve as substrates for O-acylation by acetyl-S-CoA. ',~5,~,~'~,~° All compounds resulting from changes in the configuration or carbon skeleton of the substituted 1,3propanediol side chain are virtually inactive as substrates for CAT. The D-erythro isomer of C3I and the pair of L isomers fall into this category as do the analogs of D-threo-CM with R~ substitutions which lack a free primary hydroxyl group or those in which the carbon skeleton is extended beyond that of the preferred 1,3-propanediol. The "3-methyl analog" of CM which preserves the 1,3-diol configuration, but introduces the complication of a secondary 3-hydroxyl substituent is illustrative of compounds of the latter class. The effect of variations in the substituent at the 2-amino position (R2 in Fig. 1) on acyl acceptor activity has been studied in some detail for the CAT from both R + E. eoli and from S. aureus. In general, the free amine of CM (R~ = H) is virtually inactive whereas any N-acyl substitution leads to measurable activity. Replacement of the nitro group at the para position (R~ in Fig. 1) of the 1-phenyl substituent with a variety of functional groups has led to analogs with a wide range of acyl acceptor activity. ~ SpeeiI%itg ]or the Aeyl Donor. Although no systematic study of donors in the acylation of CM by CAT has been made, a few compounds have been screened for activity. Whereas the propionyl and butyryl thioesters of CoA are less active than acetyl-S-CoA, the acidic acyl thioesters such as succinyl-S-CoA and malonyl-S-CoA do not serve as acyl donors. Taking all the available data into account the requisite structure for the best acyl donor is that of acetyl-S-CoA. The importance of the presence of the complete structure of CoA is apparent both from the inactivity of acetyl-S-dephospho-CoA, acetyl-S-pantetheine, and the acetyl derivative of acyl carrier protein and from the observation that adenine imeleotides are inhibitors of the CAT reaction and are competitive with respect to acetyl-S-CoA. 2~ The apparent Km for acetyl-S-CoA from an average of measurements carried out on several independently isolated vari'ults of R factor CAT is approximately 0.05 raM. ':~ '-'°W. V. Shaw, D. W. Bentley, and L. Sands, J. B(zcteHol, 104, 10% (1970). '~ N. (~arber and J. Zipser, Biochim. Biopt~,ys. Actc~ 220, 343 (1970).
754
[57]
ANTIBIOTIC INACTIVATION AND MODIFICATION
Ng.r~ o
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[58]
CLINDAMYCIN PHOSPHOTRANSFERASE
755
Activators and Inhibitors. CAT does not require any known cofactors. It is not inactivated by EDTA. The most important inhibitors are those known to be specific for essential thiol groups. Iodoacetate, p-mercuribenzoate, and N-ethylmaleimide inhibit CAT irrespective of source, 2° and at least one variant of R-factor CAT is uniquely susceptible to inactivation by DTNB. 2~ Stability. Preparations of purified CAT have been stored at --20 ° for more than two years with less than 20% loss of activity. The considerable stability of the enzyme has been an asset in purification and in attempts to obtain crystals suitable for X-ray diffraction. Crystals grown at room temperature in 25% saturated solutions of ammonium sulfate (pH 7) containing dithiothreitol (0.5 raM) and chloramphenicol (0.1 raM) were dissolved and found to yield at least 60% of the initial activity after 4 weeks? 3 Reversible Denaturation and Hybridization o/Subunits. Native tetrameric CAT can be recovered after dialysis of preparations treated with 6 M guanidinium HC1 under reducing conditions. Any two homologous variants (a and fl) of CAT will hybridize with one another to yield a heteromeric species of the general structure a~fl2, but no evidence has been obtained for the asymmetric ~fl:~ and ~3fl hybrids. ~'ls The S. aureus and R-factor variants are sufficiently different in structure to preclude detectable hybrid formation./3 :2 T. J. Foster and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 99 (1973).
[58] Clindamycin Phosphotransferase By JOHN H. COATS CH5
J
/N~, 5'~C3H7 ~ '
CH5
CH3
[a H--C--Cl 17
p H--C--Cl
/N~, + ATe
.o o SCH5 OH CLINDAMYCIN
CH5
I
=, 5'~c5H 7 "~2'
[7
+ ADP
.o o CH5 OPOsH2
~"
CLINDAMYCIN 3-PHOSPHATE
Several species of streptomycetes have been found to phosphorylate clindamycin, the semisynthetic antibiotic produced by chlorination of
CHLORAMPHENICOL ACETYLTRANSFERASE
737
at least 1 month. Solutions in Sorenson buffer, pH 7.2, may be stored at 5 °, but the rate of inactivation has not been measured. The E. coli enzyme is reported to have high specificity for chloramphenicol. "~
Miscellaneous As a product of the enzymic reaction within the organism, p-nitrophenylserinol does not accumulate in large amounts, but is N - a c y l a t e d or metabolized to p-nitrobenzoic acid and other products. ~,~," The Streptomyces and bacterial enzymes are intracellular, except under autolytic conditions. -° In Streptomyces species 3022a substrate access to the enzyme appears to be prevented in cultures producing chloramphenicol, or in cultures adapted to grow under nonproducing conditions in the presence of the antibiotic? 6 G. N. Smith and C. S. Worrel, Arch. Biochem. 28, 232 (1950).
[57] Chloramphenicol Acetyltransferase from Chloramphenicol-Resistant Bacteria B y W. V. SHAW Chloramphenicol q- acetyl-S-CoA-~ chloramphenicol 3-acehtte + IIS-(?oA
(1)
H~.N/R2 OH FId. 1. Structure of compounds related to chloramphenicol. The asymmetric carboa atoms at C-1 and C-2 generate d~e four possible stereoisomers of which only the D-threo isomer has significant antibiotic activity. Chloramphenicol has the following functional groups: R, = --NO:; R: = --COCHCh ; and R~ = --OH. Chloramphenicol resistance is frequently encountered among many genera of bacteria. Although in some instances the underlying mechanism may be a relative impermeability to chloramphenicol (CM), the resistance phenotype is most commonly the result of inactivation of the antibiotic by the enzyme chloramphenicol acetyltransferase (CAT)?,'-' The 1W. V. Shaw, J. Biol. Chem. 241~, 687 (1967). : Y. Suzuki and S. Okamoio, J. Biol. Chem. 242, 4722 (1967).
738
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
O-acetoxy derivatives of CM are devoid of antibiotic activity since they do not bind to bacterial ribosomes and therefore fail to inhibit polypeptide elongation. The structural gene for CAT is commonly extraehromosomal among the Enterobacteriaceae, where it is carried by R factors conferring resistance to CM and is similarly associated with a plasmid in resistant staphylococci. CAT has also been detected in CM-resistant isolates of Streptococcus ]aecalis, Diplococcus pneumoniae, and Agrobacterium tume]aciens. Bacteria which synthesize CAT begin the exponential phase of growth only after the concentration of unmodified CM has fallen to a level that is no longer inhibitory. The duration of such an increment in the usual lag phase is roughly proportional to the concentration of CM at the time of innoculation. This relationship holds for gram-negative species harboring R factors or for any bacteria carrying plasmids which dictate the constitutive synthesis of CAT. A more complex situation exists in staphylococci, streptococci, pneumococci, and A. tume]aciens wherein CAT is an inducible enzyme. Chloramphenicol is an effective inducer of CAT in such strains, but it is also a potent inhibitor of bacterial protein synthesis. Because of these competing properties and the fact that the acetylated product is not an inducer, the kinetics of-CAT induction in bacteria with inducible CM resistance are complex. Levels of CAT activity can be maintained at a high level only by continued challenge with CM or by induction with a "gratuitous inducer" which is neither acetylated nor serves as an inhibitor of protein synthesis. Although all known R factors carrying the CAT gene in enteric bacteria mediate constitutive synthesi s of the enzyme, the specific activity of CAT in crude extracts is subject to wide variations due to gene dosage effects and also to "catabolite repression" by glucose and certain other growth substrates. Examples in the first instance are the "relaxed" replication of certain R factors in Proteus mirabilis ~ and the vegetative growth of bacteriophage P1 CM in Escherichia coli. ~ Both situations have in common a high multiplicity of plasmid-borne CAT gene copies. A less dramatic example is the occasional occurrence of strains of E. coli (or related enteric bacteria) which harbor two compatible plasmids, both of which may carry one copy of the CAT region2 Catabolite repression of CAT synthesis in E. coli is mediated by a mechanism involving cyclic adenosine 5'-monophosphate (cAMP), in common with the streptomycin adenylylating enzyme and the more gens R. Rownd, H. Kasamatsu, and S. Mickel, Ann. N . Y . Acad. Sci. 182, 188 (1971). ' E. Kondo, D. K. Haapala, and S. Falkow, Virology 40, 431 (1970). 5 W. V. Shaw, L. C. Sands, and N. Datta, Proc. Nat. Acad. Sci. U~S. 69, 3049 (1972).
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
739
eral cases of fl-galactosidase and gatactokinase. 6 The decrease in CAT levels seen with growth in glucose-containing media can be prevented by the addition of high concentrations of cAMP or by the substitution of glycerol or other nonrepressing energy sources for glucose. Requirements ]or CM Acetylation Techniques. More techniques for studying the enzymic acetylation of CM are described below than are likely to be of use for any given research objective, The thin-layer chromatographic method for detecting CM acetylation in cell suspensions is useful in screening presumptive CAT producing strains or for exploring other possible mechanisms of CM inactivation. The spectrophotometric methods excel for general purposes and can also be adapted for the measurement of either CM or acetyl coenzyme A (CoA). The very sensitive radioisotopic method appears cumbersome but is remarkably reliable in practice and very useful for detecting and quantitating v'ery low levels of CAT activity. It should be stressed that the availability of CAT as a reagent offers interesting possibilities for (a) the specific detection of the biologically active isomer of CM and its quantitation and (b) the instantaneous removal (by inactivation) of CM present in any in vitro biological system. A preliminary investigation of the use of CAT to measure CM in body fluids is encouraging as regards specificity, sensitivity, and speed: features of enzymic assay techniques which are usually lacking in many of the commonly used bacteriological assay methods for antibiotics. ~ An additional possible application of CAT is its use to inactivate CM in instances wherein the antibiotic has been added as a potentially reversible inhibitor of protein synthesis. Apart from the above considerations it should be noted that CAT may be of value to molecular biologists as it is a gene product which can be assayed with specificity and great sensitivity. Two temperate phages have been isolated which confer CM resistance on the host cell in the lysogenic state, and in both cases the phenotype is due to the synthesis of CAT. Phage P1 CM was isolated as a recombinant between an R factor and phage P1 and, more recently, a derivative of phage lambda (~ CM) has been constructed which also carries the CAT gene. ~ It has been possible to synthesize CAT in a cell-free system from E. coli using P1 CM template DNA, 9 and it seems likely that either or both ~J. Harwood and D. H. Smith. Biochem. Biophys. Res. Commun. 42, 57 (1971). ~P. Lietman, personal communication, 1973. 8j. R. Scott, Virology 53, 327 (1973). B. de Crombrugghe, I. Pastan, W. V. Shaw, and J. L. Rosner, Nature (Lo~don) New Biol. 241, 237 (1973).
740
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
of the CM phages will be useful for studies of the control of transcription and translation.
Assay Methods
Principles. Enzyme activity can be quantitated by either measuring (a) the CM-dependent disappearance of acetyl-S-CoA; (b) the appearance of 3-O-acetoxy derivative of CM; or (c) the formation of reduced (unesterified) CoA. In practice, the choice between these alternatives will be determined by the level of sensitivity required and by complications created by interfering substances. The 3-monoacetoxy derivative of CM is the initial product of the reaction and is devoid of significant antibiotic activity. A diacetoxy product appears at a rate two orders of magnitude slower than the monoacetylation reaction, and its formation is also catalyzed by CAT. Although the mechanism has not been studied in detail, there is circumstantial evidence favoring the following sequence of reactions: CM-3-acetate ~ CM-l-acetate CM-l-acetate + acetyl-S-CoA--~ CM-1,3-diacetate + HS-CoA
(2)
(3)
The reaction described by Eq. (2) appears to be a nonenzymic and pHdependent acyl migration. It seems likely that the slow overall rate of formation of the diacetate derivative is due both to an unfavorable equilibrium of the nonenzymic step and also to a low rate of 3-O-acetylation of CM-l-acetate by CAT. Although Eqs. (2) and (3) should be kept in mind in any kinetic or mechanistic studies of CAT, they do not in fact interfere significantly with measurements of CAT which utilize the stoichiometry of Eq. (1).
Chromatographic Detection o] Chloramphenicol Acetylation Although of historical interest as a means of assaying CAT, the thinlayer chromatographic separation and quantitation of CM and its acetoxy derivatives still has limited applications, especially for the detection of CM acetylation by bacterial cell suspensions. The chromatographic determination of the fate of CM when incubated with a culture of a resistant microorganism also can reveal (a) other mechanisms than acetylation if the latter is not involved or (b) no alteration of CM, thereby suggesting a relative impermeability to CM of the bacterium in question. The use of 14C-labeled CM is recommended because of the difficulties in detecting small amounts of products formed when the initial concentra-
[571
CHLORAMPHENICOL ACETYLTRA_NSFERASE
741
tion of nonradioactive CM may not exceed 10 uM (3.2 ~g/ml). Since no general protocol can be formulated, a typical example will be given to illustrate a possible application. A strain of E. coli which is resistant to CM (minimum inhibitory concentration in nutrient broth = 100 t,g/ml) is to be tested for the presence of CAT. An overnight culture is diluted 1:1000 into 5 ml of fresh growth medium (the same liquid medium used for the sensitivity testing) containing J'~C]chloramphenicol at a concentration of 32 /~gfml, (0.1 raM) and a specific activity of 1 mCi/mmole. At the conclusion of tile exponential phase of growth the culture tube is centrifuged and 1 ml of the culture supernatant is extracted with 1 ml of ethyl acetate by agitation on a vortex mixer followed by centrifugation. The process is repeated twice and the ethyl acetate supernatants are pooled in a conical centrifuge tube and evaporated to dryness in air stream or on a steam bath. The sample is then taken up to 0.1 ml of ethyl acetate and spotted by repeated applications with a capillary pipette at the origin of a thinlayer sheet alongside an extract of an uninoculated control culture which has been treated in similar fashion. The chromatograms are developed in ascending fashion with the appropriate solvent until the front is a few centimeters from the top of the sheet. After removal from the tank or beaker the sheets are air dried and subjected to radioautography overnight after appropriately marking the sheets with reference points made with radioactive ink. On inspection the film will reveal whether bacterial growth has been accompanied by conversion of CM to its monoand diacetyl derivatives. The choice of thin-layer supports and solvents is arbitrary, but the following have been useful~: (1) alumina in benzene-methanol (85:15, v/v}; (2) silica gel in chloroform-methanol (95:5, v/v). Commercially available thin sheets of both alumina and silica gel on inert supports are available from a number of suppliers. Those which contain a fluorescent additive (such as Ladd thin fihns, Packard) are especially useful, as CM and its products give a prominent "quench" when present as major components. The translucent Mylar sheets are useful since they can be aligned with the exposed X-ray fihn and viewed by transmitted light. The areas of the chromatogram are marked, cut out with scissors, and dropped into scintillation vials for counting. Quantitation of the percent conversion of CM to acetyl products can thus be determined directly, and if samples of the culture "tre taken at several points in time, the data can be plotted to show the kinetics of inactivation. The same approach may be utilized to follow the course of the enzyme reaction, but the methods described below have distinct advantages in convenience and precision.
742
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Spectrophotometric Assay The most convenient technique for quantitating the rate of CM acetylation takes advantage of the generation of a free CoA sulfhydryl group coincident with transfer of the acetyl group to CM. Reaction of the reduced CoA with 5,5"-dithiobis-2-nitrobenzoic acid (DTNB) yields the mixed disulfide of CoA and thionitrobenzoic acid and a molar equivalent of free 5-thio-2-nitrobenzoate. 1° The latter has a molar extinction coefficient of 13,600 at 412 nm. The assay is best carried out with a recording spectrophotometer equipped with a temperature-controlled cuvette chamber set at 37 ° .
Reagents Tris. hydrochloride, 1.0 M, pH 7.8 Acetyl-CoA, 5 mM Chloramphenicol (D-threo) 5 nlM 5,5'-Dithiobis-2-nitrobenzoic acid (DTNB) The only reagent solution that must be stored frozen is acetyl-CoA. The reaction mixture is freshly prepared from the individual reagents by dissolving 4 mg of DTNB in 1.0 ml of Tris.HC1 buffer, after which 0.2 ml of the acetyl-CoA stock solution is added and the total volume is made up to 10 ml. The final concentrations of each component are as follows: Tris.HC1 (100 mM), acetyl-CoA (0.1 mM), and DTNB (0.4 mg/ml). After the cuvette (1 cm light path) containing enzyme and the reaction mixture has been allowed to equilibrate with the waterbath, the reaction is started by the addition of CM at a final concentration of 0.1 mM. The rate of increase in absorption at 412 nM prior to the addition of CM is subtracted from the observed rate after the start of the reaction, and net change in extinction per minute is divided by 13.6 to give the result in micromoles per minute of CM-dependent DTNB reacted. Since the latter is equal to the rate of acetylation and since 1 unit of CAT = 1 ~mole of CM acetylated per minute (37°), the calculation also yields the number of units of enzyme in the cuvette. Two factors influence the precision of the DTNB assay for CAT. First, care must be taken to avoid very high concentrations of mercaptans in the enzyme solution. In practice the addition of 2-mercaptoethanol or dithiothreitol in excess of 1 mM to crude or even partially purified preparations of CAT presents problems when more than 10 ~l of enzyme must be added to a 1-ml cuvette to obtain a significant rate of CM acetylation. A more troublesome problem occurs with crude cell extracts prepared from certain genera of bacteria that contain high levels of thiolo A. F. S. A. Habeeb, this series, Vol. 25, p. 457.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
743
esterase activity. In such instances the control rate of increase in 412 nm absorbance due to acetyl-CoA hydrolysis prior to the addition of CM may approach or exceed the increment seen after adding the antibiotic. In some instances it may be necessary to resort to partial purification of CAT before the spectrophotometric assay can be used with confidence. The [l+C]acetate procedure which utilizes an acetyl-CoA generating system should also be considered as a possible solution to the problem of high nonspecific thioesterase background (see below). A serious limitation to the DTNB assay for CAT concerns the intrinsic high reactivity of essential cysteine thiol groups in certain variants of CAT. In the absence of protecting reduced mercaptans, enzyme activity decreases rapidly after addition of DTNB. Since the rate of CM acetylation is substantially greater than the rate of CAT inactivation, an approximation of the initial rate for CAT activity can be obtained by taking special care to add enzyme immediately prior to addition of CM. An alternative spectrophotometric method 1 can be used when a high concentration of competing mercaptans interferes with the DTNB assay. The loss of an acyl group from thioesters such as acetyl-CoA is accompanied by a decrease in absorption in the ultraviolet. The difference in molar extinction coefficients of acetyl-CoA and reduced CoA plus acetate is 4500 at 232 nm. 11 Special care must be taken to remove interfering ultraviolet absorbing material from the enzyme preparation by gel filtration or dialysis. The contribution of the absorption due to protein added to the euvette becomes a more serious obstacle in crude extracts, especially those with low levels of CAT activity. Apart from the inconvenience of measurements in the far ultraviolet region and the fact that the method is intrinsically less sensitive than the DTNB procedure, the assay of thioester cleavage at 232 nm suffers from being a difference method. The absolute decreases in absorbance per unit time due to the presence of CM and low levels of CAT may be impossible to quantitate without recourse to the use of a dual beam recording spectrophotometer.
Assay by Direct Measurement o] ["C ]Acetyl Chloramphenicol The need for a highly sensitive, rapid, and specific means of quantitating the synthesis of CAT in a complex E. coli extract 9 led to the development of a radioactive method which, unlike that described above, does not require chromatography. The principle of the method is that the neutral [14C]acetoxy-chloramphenicol derivatives can be quantitatively extracted into benzene at alkaline pH whereas ionized and polar species such as [14C]acetate and [14C]acetyl-CoA remain in the aqueous phase. 11E. R. Stadtman, this series, Vol. 3, p. 985.
744
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
The sensitivity of the method is limited only by the specific activity of [14C]acetate used. Specificity is achieved by measuring the increment in radioactivity extracted from an assay mixture containing CM as compared to that from a control incubation without CM. Should any doubt arise that the products are, in fact, the acetoxy derivative(s) of chloramphenicol, the chromatographic methods described previously may be used for confirmation. The most convenient means of terminating the assay incubation has been found to be the decomposition of acetyl-CoA catalyzed by phosphotransacetylase in the presence of 100 mM arsenate. The arsenolysis reaction obviates the need for extremes of temperature or pH and does not interfere with the extraction of products. Although the coupled assay requires numerous reagents, including three enzymes, all the components are readily available from commercial sources. A mixture of buffer, salts, and substrates may be stored frozen for convenience, but the reagent enzymes should be stored separately under the recommended conditions and added individually to the final incubation mixture immediately prior to the assay. The most convenient means of standardizing the radioactive assay to conform with the spectrophotometric unit of CAT activity (as defined above) is to assay a preparation of crude or purified CAT by both techniques. The two methods should agree within 5-10% when: (a) care is taken to ensure adequate temperature control in both instances; (b) the amount of CAT in each case is appropriate for the measurement of initial rates, and (c) the extractions are carried out with care. The experiments in which the radioactive assay have been used thus far have required that the method be specific, sensitive, and reproducible, rather than of a high absolute accuracy with respect to reference measurements. The maintenance of a saturating concentration of [14C]acetyl-CoA for CM acetylation by CAT is achieved by means of the following coupled reactions and the addition of an excess of pyruvic kinase, acetate kinase, and phosphotransacetylase: ADP -{- phosphoenolpyruvate ~ pyruvate T ATP ATP T [~4C]acetate ~ [~4C]acetyl phosphate T ADP [14C]acetyl phosphate + HS-CoA ~ [~4C]acetyl-S-CoA T phosphate
(4) (5) (6)
The pyruvic kinase system (Eq. 4) has been employed to generate a catalytic level of ATP since adenine nucleotides are competitive inhibitors (with respect to acetyl-CoA) of CAT. Although each of the reagent enzymes must be added in excess, it is usually not necessary to assay each one independently before its addition to the assay incubation. It is actually easier and more useful to ascertain that each is, in fact, present in excess than to assign a precise value to the activity of each component.
[57]
745
CHLORAMPHENICOL ACETYLTRANSFERASE
This is best accomplished by assuming the accuracy of the supplier's nominal activity specifications as a point of departure. The radioactive assay is then run with a sample of CAT with known activity by the DTNB method. Dilutions of each reagent enzyme are made, and aliquots of several such dilutions of each enzyme are tested while the other two are added at concentrations assumed to be 10-fold higher than the 1 unit required (1 umole/min) for each under the conditions of the assay. For each enzyme reagent so tested a limiting dilution shoukt be reached where the final amount of [~4C]acetyl CM radioactivity extracted is (a) lower than that with all less dilute samples of the enzyme in question and (t)~ less than that expected from the known activity of CAT present from the DTNB procedure. The assay conditions described below are a modification of those reported previously." The present procedure has evolved in order: (a) to avoid the inhibition of CAT by adenine nucleotides (see above), (b) to ensure that acetyl-CoA and CM are present at ~aturating concentrations, (el to be certain that the reagent enzymes are in excess and art present in a favorable environment for activity, and (d) to maximize the efficiency of the extraction procedure without introducing either high blank v'dues or artifacts2'-'
Reagents
Volume (ul) per 250 ul incubation
Final concentration
1 M T r i s . HCI (pH 7.8) 100 m M MgCI: 100 m M A T P 100 m M P h o s p h o e n o l p y r u v a t e 5 m M Coenzyme A (reduced; lithium salt) 10 m M [1-~4Cl Sodium :~eetate Acetate kinase P y r u v a t e kinase Phosphot r'msacet ylase
25 15 7.5 50 20 10 See text See lext See lext
100 m.'ll 6 mM 3 mM 20 m M 0.4 m M O. 4 m M
1 Unit 1 Unit I Unit
Each incubation is carried out in a conical centrifuge tube (nominal volume approximately 12-15 ml) to simplify the subsequent extraction t)rocedure. Tile final volume of 250 ul is achieved by the addition of tile ,2 Two other techniques have been tried unsuccessfully for the separation of P4C]acetoxy chloramphenicol from radioactive acetate and acetyl-CoA. Tile tatier should be absorbable by anion exchange resins (such as Dowex 1) whereas the nonionized product is not. An alternative approach is the selective absorption of C M and its P~C]acetyl derivatives to charcoal after arsenolysis of ["C]acetyl-CoA. In practice b o t h of these approaches have given high and variable blank values.
746
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
requisite volume of deionized water and the sample containing CAT, after which the components are thoroughly mixed. The blank incubation contains only water to volume, but no enzyme. In both instances the reaction is started by the addition of 5 ~l of chloramphenicol (5 mM) immediately prior to placing each Parafilm-sealed tube into a water bath at 37 °. After 20 rain of incubation the reaction is terminated by the addition of 25 ~l of 1.1 M sodium arsenate followed by thorough mixing. The extraction and processing of the product is carried out in the following manner. Two milliliters of benzene (analytical reagent grade) is added to each incubation tube, and the latter is agitated on a Vortex mixer for 20 sec. The tube is centrifuged in a nonrefrigerated clinical centrifuge for a few minutes to separate phases, and the upper benzene layer is carefully taken with a Pasteur pipette and placed in a glass scintillation vial. The extraction is repeated with a second 2 ml of benzene. The extracts are pooled, and 100 ~l of glacial acetic acid is added to each scintillation vial. After thorough mixing of the acetic acid and benzene, the samples are dried under a heat lamp in an air stream. After the samples are thoroughly dried, an appropriate scintillation counting solution is added (any convenient type is adequate since the product is soluble in most organic solvents and there is no nonvolatile residue to cause quenching) and each vial is assayed for 14C radioactivity. Blank incubations (in which CM has been added in the absence of CAT) or controls (containing CAT but no CM) should yield extracts with no more than 100-200 dpm above background when the specific activity of sodium acetate is of the order of 5 mCi per millimole. It should be apparent that initial CAT velocities will be measured accurately with this technique only if CAT is present in amounts such that both CM and acetyl-CoA are still at saturating concentrations after 20 m~n of incubation. In practice this means the addition of no more than approximately 0.0005 unit of CAT to any given incubation, an amount of enzyme sufficient to catalyze the acetylation of 10 nmole of CM in 20 min (or 40% of the 25 nmoles present initially). It is fortunate that the DTNB method begins to give reliable data at CAT levels near 0.0005 unit when a recording spectrophotometer with scale expansion (to 0 . 1 0 D units full scale) is employed. The choice of method will therefore usually favor the radioactive assay whenever the concentration of CAT is less than 0.005 unit/ml (0.005 unit per 100-~1 sample).
Purification of Chloramphenicol Acetyltransferase General Remarks. Conventional techniques of protein fractionation have been adequate for the purification of CAT from (1) R factor-bear-
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
747
ing strains of E. coli, (2) staphylococci harboring CM plasmids. (3) CMresistant mutants of Proteus mirabilis in which the CAT gene is probably chromosomal, and (4) selected strains of Agrobacterium tume]aciens. Although the following protocol describes a typical purification for an R factor type of CAT, the principles can be applied to any bacterial source of the enzyme. Since each variant of CAT examined thus far has been an acidic protein, the use of DEAE-cellulose absorption is common to all purifications and is an important step. The molarity of sodium chloride required to elute CAT in each instance will be found to roughly parallel the net charge as measured by the apparent isoelectric point (pK~; isoelectric focusing). The most acidic CAT purified thus far (Agrobacterium tume]aciens; pK~ = 3.93) is eluted at 0.21 M ~3 whereas all other species of CAT (pK~ = range of 4.8 to 5.7) have been recovered at lower ionic strengths (sodium chloride; 0.1(}-0.19 M). The procedure described gives a homogeneous CAT product from R factor-containing strains of E. coli and isolates of S. aureus harboring a CM plasmid. The ease with which such results can be achieved is due to the fact that fully induced S. aureus cultures and virtually all R ÷ E. coli cultures synthesize CAT to levels approximating 0.5-1% of the soluble cell protein in stationary phase (see Table I for summary of typical purification). It should be anticipated that other genera or species which synthesize CAT may do so at lower rates and that additional purification steps may be required to obtain a pure enzyme preparation. A useful maneuver in such instances is a second DEAE-eellulose step (after gel filtration) employing a different pH and salt gradient (for example, zero to 0.5 M sodium phosphate at pH 7.0).
Typical Purification o] CAT ]rom E. coli Growth o] Bacteria. A prototrophic strain of E. coli carrying an R factor for CM resistance (or a P1 CM lysogen of such a strain) is grown to the stationary phase of growth at 37 ° with vigorous shaking (or alternative means of aeration) in a well buffered nonrepressing growth mediam. The latter is conveniently and inexpensively provided by a medium containing per liter: K2HPO4, 14 g; KHP04, 6 g; (NH~).,SO~, 2 g; MgS(L, 0.2 g; easamino acids (Difco), 1 g; and glycerol, 5 g. A heavyduty orbital shaker platform capable of taking twelve 2-liter Erlenmeyer flasks will yield approximately 35-40 g (wet weight) of cells from 14 liters if each flask contains only 1200 ml of the above medium to ensure adequate aeration. The cells are harvested with a continuous flow centrifuge (Sharpies Supercentrifuge or Sorvall continuous-flow attachment for 1~L. C. Sands and W. V. Shaw, unpublished studies.
748
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
the RC-2B centrifuge) and suspended in a final volume of 200 ml of 50 mM Tris.HC1 (pH 7.8) containing 2-mercaptoethanol at a concentration of 50 ttM (TM buffer). Step 1. Crude Extract. The cells are broken by sonication or by extrusion in a French pressure cell (Aminco). If the latter method is used a few crystals of deoxyribonuclease should be added to reduce the viscosity of the suspension prior to centrifugation. The cell debris is then removed by centrifugation (30,000 g for 20 min), and the supernatant is taken for purification. Such crude preparations of CAT are stable for months at --20 ° . All subsequent steps are carried out at 0-4 ° . Step 2. Streptomycin Sul]ate Precipitation. Solid streptomycin sulfate is dissolved in the crude extract at a final concentration of 1%. The precipitate obtained after 30 min is collected by centrifugation and discarded. After this step, to remove the bulk of nucleic acids the concentration of pH 7.8 Tris. HC1 buffer is increased to 100 mM prior to precipitation of CAT with ammonium sulfate. Step 3. First Ammonium Sul]ate Step. CAT is precipitated from the buffered supernatant of the streptomycin step by the addition of finely ground ammonium sulfate (enzyme grade) to a final concentration equivalent to 50% saturation. 14 The ammonium sulfate is added slowly with stirring and the preparation is allowed to stand for 30 min in an ice bath before centrifugation at 30,000 g for 20 rain. The supernatant is decanted carefully and put aside until it has been determined that it contains less than approximately 10% of the total activity in the precipitate after the latter is dissolved in 50 ml of TM buffer containing 0.2 mM chloramphenicol (TCM buffer). In the event that CAT has not been precipitated effectively, additional ammonium sulfate should be added to the supernatant to achieve 55% saturation and the centrifugation repeated. It is imperative that the purification proceed to the heat step directly and especially that freezing of ammonium sulfate containing solutions of CAT be avoided. Step 4. Heat Step. All naturally occurring variants of R-factor CAT have been found to be sufficiently thermostable to permit the use of a heat step in the purification. 5,1~ After the precipitate from the ammonium sulfate step is dissolved in TCM buffer the enzyme preparation is allowed to reach room temperature and is then placed in a heated water bath set at 60% The extract is stirred slowly for 10 min, cooled, and centrifuged, and the precipitate is discarded. Step 5. Second Ammonium Sul]ate Step. The supernatant fluid from the heat step is brought to 50% saturation with ammonium sulfate, and ~4A. A. Green and W. L. Hughes, this series, Vol. 1, p. 67. 15W. V. Shaw and R. F. Brodsky, J. Bacteriol. 95, 28 (1968).
[571
CHLORAMPHENICOL ACETYLTRANSFERASE
749
the CAT-containing precipitate is collected as in the earlier precipitation step. The precipitate is dissolved in a small volume (15-25 ml) of TCM buffer and either (a) dialyzed against the latter to remove residual ammonium sulfate, or (b) desalted on a Sephadex G-25 column of suitable size which has been equilibrated with TCM buffer. In practice the gel filtration method is more convenient since it permits completion of all steps up to and including the loading of the DEAE column in a normal working day. Step 6. DEAE-Cellulose Chromatography. The usual precautions are taken to ensure that the chromatography medium contains particles of near uniform size and that the column is well packed and completely equilibrated with the TCM buffer. Microgranular (DE-52) Whatman DEAE-cellulose (dry or prewetted) should be satisfactory in all respects, so long as the manufacturer's preeyeling instructions are followed. A packed column bed of dimensions 2.5 X 40 em should be adequate for a preparation of the size described above and can be developed with a l-liter gradient of SaC1 (0-0.4 M) in TCM buffer after the desalted sample has been applied to the top of the eohmm and the latter washed with several column volumes of TCM buffer. CAT activity should be eluted iust before the midpoint of the NaC1 gradient (0.15-0.20 M in most instanees~ and can be easily detected by the speetrophotometrie (DTNB) method. The strategy for pooling the CAT containing tubes will depend on the goal of the overall purification. Since the enzyme is stable at 0-4 ° for at least 24 hr there is much to be said for determining the CATspecific activity for each tube in the peak and for surveying the number and types of contaminants by polyaerylamide electrophoresis before pooling tubes. When the DEAE step is to be followed by gel filtration, ttw pooled peak tubes can be concentrated by membrane ultrafiltration (Amicon or equivalent types) to a volmne suitable for the column step to follow. Step 7. Gel Filtration. In principle, any gel filtration support which will include CAT (molecular weight: 80,000t should be useful for size separations. In practice, Sephadex G-100 proved satisfactory and has been the only method used. A volume (approximately 5-10 roll of concentrated enzyme solution from the prior step is applied to the bottom of such a column (2.5)< 100 era) equilibrated with TCM buffer containing 0.2 M NaC1 and the elution is continued in upward fashion using th~ same buffer. As in the prior step the peak tubes are pooled judiciously on the basis of specific activity data and/or electrophoretic purity and concentrated if required. The activity of CAT in the elution buffer is stable at --20 ° for at least one year. Step 8. Additional or Alternate Steps. Although in most instances the
750
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
TABLE I PURIFICATION OF CHLORAMPHENICOL ACETYLTRANSFERASEa
Step 1. 2. 3. 4. 5. 6.
Crude extract Streptomycin sulfate First a m m o n i u m sulfate H e a t (60 °) Second a m m o n i u m sulfate DEAE-cellulose chromatography 7. Gel filtration (Sephadex G-100)
Protein (rag total)
Enzyme activity (units total)
Specific activity (units/ rag)
Purification (fold)
Yield (%)
5400 4150 2800 900 640 51
4900 4650 4400 4150 4050 2150
0.9 1.1 1.6 4.6 6.3 42 1
(1) 1.2 1.8 5.1 7.0 47
(100) 95 90 85 83 44
14
1750
125
139
36
CAT obtained from the peak tubes of G-100 column (Step 7) was homogeneous by disc gel electrophoresis.
above procedures will yield electrophoretically homogeneous CAT, it m a y be necessary to carry out an additional step. In the past this has taken the form of either a batchwise adsorption procedure with Alumina C~ gel immediately following the heat step 15 or a terminal step involving a second D E A E column run at a different pH a n d / o r with a different buffer system than that used in step 6. The arguments in favor of the alumina C~ gel step are that it is relatively straightforward, quite effective, and does not preclude the second D E A E procedure if the latter is required. Conversely, since homogeneous CAT can often be obtained without the alumina C~ gel step, it is convenient to "wait and see" before adding an additional procedure. A totally different strategy from the conventional one described above should be mentioned, namely, that of affinity chromatography. 16 Initial experiments with CM-substituted agarose as a specific adsorbant for CAT have been encouraging as regards specificity, but the elution behavior of the enzyme has been unsatisfactory in some respects. The approach employed has been to attach the free amine of CM (where R2 = H in Fig. 1) to a solid support via the formation of an amide bond with the free carboxyl group of CH-Sepharose (Pharmacia) using a water-soluble carbodiimide reagent. The rationale for the use of CM base as ligand is that the nature of the N-acyl (R2) substituent of the CM skeleton is a less critical determinant of substrate affinity for CAT than the 1,3~ P. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
751
propanediol side chain or p-phenyl substituent. ~7 Current studies are aimed at avoiding the extremes of ionic strength, and CM concentration which are both required to quantitatively elute CAT from the adsorbant described23
Purification o] CAT ]rom Other Bacterial Species Staphylococcus sp. Since staphylococci are more fastidious in their growth requirements and CAT does not appear to be repressed by glucose in a representative collection of strains examined,13 the preferred growth medium is commercially available complex media such as Penassay Broth (Difco). All wild-type isolates of staphylococci harbor the genes for an inducible CAT is and, hence, careful attention to the details of induction (derepression) is necessary for optimum yields of enzyme. Conditions for "gratuitous" induction have been described previously 19 for the 3deoxy analog of CM (where R3 = H), but since this compound is neither available commercially nor readily synthesized, an alternative procedure will be described. A CM-resistant isolate of Staphylococcus sp. is tested for CM resistance on nutrient agar containing 50 t~g of CM per milliliter, and a single colony is picked for overnight growth in Penassay (PA) broth containing the same concentration of CM (PA-CM). Growth on a preparative scale is begun in PA-CM medium with an inoculum from the overnight starter culture equal to approximately 5% of the total volume of the large-scale culture, and the process is repeated to yield a suitable volume of inoculum (e.g., 800 ml) for a total final preparative volmne of 14 liters. By analogy with the earlier protocol for E. coli, this can be accomplished by inoculating each of 12 Erlenmeyer flasks (nominal volume of 2 liters) containing 1200 ml of PA-CM with approximately 60 ml of starter culture. Growth is then allowed to proceed at 37 ° on a rotary shaker. After each doubling of the cell mass (as approximated by turbidity measurements) fresh sterile CM is added to bring the final concentration to 50 ~g/ml. When the cultures have reached the final stationary phase of growth a final addition of CM is made and the cells are allowed to shake for an additional period of 20-30 rain to ensure maximum induction before harvest. Cells are best collected by centrifugation in large (polypropylene or stainless steel) bottles to avoid the hazardous aerosol obtained with use of an "open" centrifuge such as the conventional Sharples instrument. The yield of cells should be of the order of 40-50 g (wet weight). 1TW. V. Shaw and R. F. Brodsky, Antimicrob. Ag. Chemother. 1967, 257 (1968). ~SL. C. Sands and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 299 (1973). 19E. Winshell and W. V. Shaw, J. Bacteriol. 98, 1248 (1969).
752
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Cell lysis is accomplished by the use of Lysostaphin (Schwarz-Mann) as follows. 19 The pooled cell pellets are washed by suspension in 1 liter of ice cold Tris-saline (TS) buffer (50 mM Tris.HC1, pH 7.5, and 145 mM sodium chloride) and collected by centrifugation. The cells are resuspended in approximately 1 liter of TS buffer containing Lysostaphin (10 units/ml) and deoxyribonuclease (50 ~g/ml) and incubated at 37 ° with agitation using a magnetic stirrer. Cell lysis is nlonitored by observing the decrease in turbidity at 660 nm of diluted aliquots of the suspension and is usually complete after 1 hr. The supernatant fluid (crude extract) obtained after centrifugation (10,000 g for 20 min) to remove cell debris should contain approximately 0.2 unit of CAT per milligram of soluble protein. The purification of staphylococcal CAT can be achieved by a straightforward 4-step procedure which omits the preliminary streptomycin sulfate precipitation and the second ammonium sulfate step of the E. coli protocol. Since the overall purification of CAT from staphylococci has been described in detail elsewhere 1~,19 and is similar to that outlined for the R factor enzyme (see above), only the ammonium sulfate precipitation step will be described here. The crude extract obtained after treatment with Lysostaphin is buffered to 100 mM with pH 7.8 Tris.HC1 and made 50 mM for 2-mercaptoethanol prior to the addition of sufficient finely ground ammonium sulfate to achieve 70% of saturation. For most variants of staphylococcal CAT, the supernatant after centrifugation of the precipitate obtained should contain more than 90% of the total enzyme activity present in the crude extract. The supernatant is then brought up to a theoretical 90% saturation with further addition of ammonium sulfate, and the precipitate is collected by centrifugation and dissolved in TCM buffer for desalting and DEAE chromatography, as described for the E. coli variants of CAT.
Properties of Chloramphenicol Acetyltransferase General Comments. Although there is no single type of CAT which
can be chosen as representative, the variants studied to date possess the following properties: pH optimum of 7.8; native molecular weight of 80,000 (and quaternary structure, of four identical subunits of 20,000 each) ; and apparent isoelectric point between 5.4 and 4.0. All CAT variants therefore behave on polyacrylamide gel electrophoresis as typical globular acidic proteins of varying net charge and a corresponding broad range of electrophoretic mobilities when examined at alkaline pH. Each variant is specific for the D-threo stereoisomer of chloramphenicol (or con-
[57l
CHLORAMPHENICOL ACETYLTRANSFERASE
753
geners) as the acyl acceptor and a marked preference for acetyl-S-CoA as the aeyl donor as compared with other acyl-S-CoA homologs. Most of the important differences observed to date between the enzymes themselves (as opposed to their mode of synthesis and its control) have been detected by kinetic or immunological means. The most easily tabulated properties are summarized in Table II. Specificity for Acyl Aeceptor. A large number of analogs and isomers of C3I have been screened for their ability to serve as substrates for O-acylation by acetyl-S-CoA. ',~5,~,~'~,~° All compounds resulting from changes in the configuration or carbon skeleton of the substituted 1,3propanediol side chain are virtually inactive as substrates for CAT. The D-erythro isomer of C3I and the pair of L isomers fall into this category as do the analogs of D-threo-CM with R~ substitutions which lack a free primary hydroxyl group or those in which the carbon skeleton is extended beyond that of the preferred 1,3-propanediol. The "3-methyl analog" of CM which preserves the 1,3-diol configuration, but introduces the complication of a secondary 3-hydroxyl substituent is illustrative of compounds of the latter class. The effect of variations in the substituent at the 2-amino position (R2 in Fig. 1) on acyl acceptor activity has been studied in some detail for the CAT from both R + E. eoli and from S. aureus. In general, the free amine of CM (R~ = H) is virtually inactive whereas any N-acyl substitution leads to measurable activity. Replacement of the nitro group at the para position (R~ in Fig. 1) of the 1-phenyl substituent with a variety of functional groups has led to analogs with a wide range of acyl acceptor activity. ~ SpeeiI%itg ]or the Aeyl Donor. Although no systematic study of donors in the acylation of CM by CAT has been made, a few compounds have been screened for activity. Whereas the propionyl and butyryl thioesters of CoA are less active than acetyl-S-CoA, the acidic acyl thioesters such as succinyl-S-CoA and malonyl-S-CoA do not serve as acyl donors. Taking all the available data into account the requisite structure for the best acyl donor is that of acetyl-S-CoA. The importance of the presence of the complete structure of CoA is apparent both from the inactivity of acetyl-S-dephospho-CoA, acetyl-S-pantetheine, and the acetyl derivative of acyl carrier protein and from the observation that adenine imeleotides are inhibitors of the CAT reaction and are competitive with respect to acetyl-S-CoA. 2~ The apparent Km for acetyl-S-CoA from an average of measurements carried out on several independently isolated vari'ults of R factor CAT is approximately 0.05 raM. ':~ '-'°W. V. Shaw, D. W. Bentley, and L. Sands, J. B(zcteHol, 104, 10% (1970). '~ N. (~arber and J. Zipser, Biochim. Biopt~,ys. Actc~ 220, 343 (1970).
754
[57]
ANTIBIOTIC INACTIVATION AND MODIFICATION
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[58]
CLINDAMYCIN PHOSPHOTRANSFERASE
755
Activators and Inhibitors. CAT does not require any known cofactors. It is not inactivated by EDTA. The most important inhibitors are those known to be specific for essential thiol groups. Iodoacetate, p-mercuribenzoate, and N-ethylmaleimide inhibit CAT irrespective of source, 2° and at least one variant of R-factor CAT is uniquely susceptible to inactivation by DTNB. 2~ Stability. Preparations of purified CAT have been stored at --20 ° for more than two years with less than 20% loss of activity. The considerable stability of the enzyme has been an asset in purification and in attempts to obtain crystals suitable for X-ray diffraction. Crystals grown at room temperature in 25% saturated solutions of ammonium sulfate (pH 7) containing dithiothreitol (0.5 raM) and chloramphenicol (0.1 raM) were dissolved and found to yield at least 60% of the initial activity after 4 weeks? 3 Reversible Denaturation and Hybridization o/Subunits. Native tetrameric CAT can be recovered after dialysis of preparations treated with 6 M guanidinium HC1 under reducing conditions. Any two homologous variants (a and fl) of CAT will hybridize with one another to yield a heteromeric species of the general structure a~fl2, but no evidence has been obtained for the asymmetric ~fl:~ and ~3fl hybrids. ~'ls The S. aureus and R-factor variants are sufficiently different in structure to preclude detectable hybrid formation./3 :2 T. J. Foster and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 99 (1973).
[58] Clindamycin Phosphotransferase By JOHN H. COATS CH5
J
/N~, 5'~C3H7 ~ '
CH5
CH3
[a H--C--Cl 17
p H--C--Cl
/N~, + ATe
.o o SCH5 OH CLINDAMYCIN
CH5
I
=, 5'~c5H 7 "~2'
[7
+ ADP
.o o CH5 OPOsH2
~"
CLINDAMYCIN 3-PHOSPHATE
Several species of streptomycetes have been found to phosphorylate clindamycin, the semisynthetic antibiotic produced by chlorination of
