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Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases
PLASMID
2,334-346
Trimethoprim
(1979)
Resistance Plasmids of Different Origin Encode Different Drug-Resistant Dihydrofolate Reductases BIRGITTATENNHAMMAR-EKMAN
Department
of Microbiology,
Faculty
of Pharmacy,
AND OLA SK~LD University
of Uppsala,
Uppsala,
Sweden
Received June 27, 1978 Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofoiate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R7.51 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a lO- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.
Plasmids mediating resistance to trimethoprim have been known for several years (Hedges et al., 1972) and the molecular mechanism of plasmid-borne trimethoprim resistance has been investigated. This was done with one of the R factors isolated first, R483 (Hedges, et al., 1972). It was found that this plasmid possessed the ability to induce the formation of a drugresistant target enzyme, dihydrofolate reductase (SkGld and Widh, 1974). This extrachromosomally coded enzyme was shown to be distinct from the chromosomal enzyme of E. cofi by several chemical criteria. Several different R factors carrying trimethoprim resistance have been shown to code for aberrant dihydrofolate reductases (Amyes and Smith, 1974; Amyes and Smith, 1976; Pattishall et al., 1977). These drugresistant enzymes were shown to have 0147-619X179/030334-13$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
molecular weights about twice as large as that of the chromosomal enzyme (PattishalI et al., 1977). The latter authors also clearly demonstrated the existence of two classes of these large plasmid-borne enzymes, which showed remarkable differences in their folic acid analog sensitivity. The aim of the work presented here was to compare plasmid enzymes from several, earlier characterized R factors and from plasmids newly isolated from clinical material. Clear characteristics of different plasmid-coded enzymes could give a clue to their origin and to their relationship to different transposons mediating trimethoprim resistance (Barth et al., 1976; Shapiro and Spom, 1977). By several techniques, including gel electrophoresis, at least three different plasmid-borne dihydrofolate reductases could be discerned.
334
PLASMID-BORNE
DIHYDROFOLATE
MATERIALS AND METHODS Bacterial
strains. The E. coli K-12 strain
EC1005 met-, nal’ was supplied by K. Nordstrom, Odense, Denmark (cf. Table 1). The R factors R388, R483, R721, and R751 were supplied by N. Datta, London (cf. Table 1). Bacterial strains 6275, 22259, 4891, and 1810 were trimethoprim-resistant isolates from clinical material obtained from hospitals in the area of Stockholm, Sweden. They were characterized as E. cofi by routine tests. Materials. Dihydrofolic acid was purchased from Sigma Chemical Co. (St. Louis, MO.) or prepared by the method of Blakley (l%O). NADPH, 3-(4,5-dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide (MTT), ribonuclease-A (type I-A), lysozyme (grade I), and trypsin were bought from Sigma Chemical Co. Methotrexate was obtained from Nutritional Biochemicals Corporation (Cleveland, Ohio) and aminopterin from KochLight and Co. Ltd. (Colnbrook, England). Trimethoprim lactate was a gift from the Wellcome Research laboratories (England) and nalidixic acid was donated by Winthrop AB (Sweden). [methyl-3H]Thymidine was purchased from the Radiochemical Centre (Amersham, England). Sephadex G-100 was from Pharmacia (Uppsala, Sweden).
335
REDUCTASES
Media. The mineral salts medium used contained per liter: Tris(hydroxymethyl)aminomethane (Tris) 12.0 g, KCl, 2.0 g, NH&l 2.0 g, MgCl,*6H,O 0.5 g, NqHPO,. 2H20 0.178 g, Na$O, 0.05 g, and casamino acids (Difco, certified) 0.5 g; the pH was adjusted to 7.2 with HCl and, after autoclaving, 5 g of glucose and FeCl, to 1O-5M was added. Required methionine was added to 40 pg/ml. Solid media were obtained by the addition of 2% agar. The rich medium TY-2 used in the fermentor contained per liter: tryptone 30.0 g, yeast extract 10.0 g, NH&l 2.5 g, N%HPO,. 2H,O 7.5 g, KH,POI 3.0 g, Na.#O,* 10HzO 2.5 g, glucose 40.0 g, MgS04.7H,0 0.2 g, and trace elements (Holme et al., 1970). Buffer A was 0.05 M in Tris-HCl, pH 7.2, 0.05 M in KCl, 0.01 M in mercaptoethanol, and 0.001 M in EDTA. Drug resistance transfer. Analysis for R factors was performed by mixing the presumptive donor with a nalidixic acid resistant recipient strain (EC1005 met-, naf’) as described earlier (Skold, 1976). Bacteria were grown in mineral salts medium supplemented with casamino acids (0.05%) and mixed at 2 x 1OBcells/ml of donor and 2 x lo9 cells/ml of recipient. After 2 h of incubation, recombinants were sought by plating the mixture on mineral salts medium
TABLE
1
CHARACTERISTICSOFBACTERIALSTRAINSHIGHLY RESISTANTTOTRIMETHOPRIM MIC, trimethoprim strain EC1005 EC1005 EC1005 EClOO5 3048 1810 EClOO5 EC1005 EC1005 EC1005
Resistance
markew
ham
(R627J) (R22259) (R4891)
Nd Nd,Tp,Sm Nal,Tp,Su.Tc, Cap Nd,Tp,Su, Sm
(R483) (R721) (R751) (R388)
Tp, Tp, Nal, NaI, N=L Nal,
Su, Su, Tp, Tp, Tp Tp,
Tc, Nitro, Sm Sm Sm Su
Sm
I >looo >lCKlO >locm ZlcoO >lcKw >lOCWl ZlooO >looo >lOOil
Dihydrofobate reductase contentb 0.3-0.6 6.8 6.1 8.3 5.8 77.3 8.2 13.9 0.9 0.3
Plasmid classification’
I F I I I ILZ 16 P W
Reference Grinsted et 01. (1972) This report This report This report This report This report Hedges er al. (1972) Jobanputra and Datta ( 1974) Jobanputra and Datta (1974) Data and Hedges ( 1972)
a Antibiotic sensitivity testing was perfomxd by the paper disc diffusion method. Abbreviations: Sm. streptomycin; Nal, nalidixic acid; Tp, trimethoprim; Su, sulfonamide; Tc, tetracyline; Cap, chloramphenicol: Nitro, nitrofurantoin. * Activity expressed as nmol of dibydrofolate reduced per min per 10s cells. c PiIus type of clinical isolates was determined by phage sensitivity as referred to by Meynell et a/. (196@. The phages used were ,.~2 and iFi. Compatibility groups are as described in the given references.
336
TENNHAMMAR-EKMANANDSKijLD
containing methionine (40 pg/ml), nalidixic acid (50 pg/ml) , and trimethoprim ( 15 pgrnl) . Gradient centrifugation analysis. Alkaline sucrose gradient centrifugation was performed as described by Grinstedet al. (1972). Bacterial cells growing logarithmically in mineral salts medium supplemented with deoxyadenosine, 250 pg/ml, were labeled for two generations with [3H]thymidine, 5 &i/ml (specific activity 15.8 Ci/mmol). The labeled culture was chilled on ice, centrifuged, and washed twice with ice-cold TES buffer (0.05 M Tris-HCl, pH 8.0, 0.005 M EDTA, 0.05 M NaCl). The pellet (about 8 x log cells) was resuspended in TES buffer containing lysozyme (1 mg/ml) and RNase A (500 I.Lg/ml). After incubation at 37°C for 15 min, sodium dodecyl sarcosinate (1% w/w) was added to give a clear, viscous lysate, which was sheared by passage through a 50-mm-long 21-gauge hypodermic needle five to six times at about 0.1 ml/s. Of the sheared lysate 0.1-0.2 ml was layered onto a 5-ml, 5-20% (w/w) linear sucrose gradient, in 0.3 M NaOH and 0.01 M EDTA. Centrifugation was for 60 min at 4°C and at 35,000 rpm in a Beckman SW 39 L rotor. Fractions of 0.2 ml were collected and radioactivity determined by liquid scintillation counting. Enzyme assay. Determination of dihydrofolate reductase activity was carried out according to Warner and Lewis (1966). The assay was performed at 30°C in a thermostated Zeiss PM 6 spectrophotometer equipped with a Servogor 5 recorder. Enzyme activity was followed for at least 5 min. Unspecific oxidation of NADPH, i.e., decrease in absorbance at 340 nm in the absence of dihydrofolate, was subtracted. Enzyme preparation. Bacteria were grown in 700 ml of mineral salts medium containing 40 pg/ml of methionine, 0.05% casamino acids, and 15 pg/ml of trimethoprim. At about 5 x lo8 per ml, cells were harvested by centrifugation, washed twice with buffer A, and frozen. In order to culture larger quantities of
bacteria, a 3 1 fermentor (FL-103, Biotec, Stockholm, Sweden) was used in a procedure described by Holme et al. (1970). The cultivation was performed at 37°C for 5-6 h at pH 7.2, which value was kept constant by the automatic addition of 1 M NaOH. Bacteria were harvested by centrifugation, washed in buffer A, and finally frozen. Partial purification of dihydrofolate reductase was performed as described by Burchall and Hitchings (1965) as modified by Skold and Widh (1974). The frozen cells were disintegrated in a pressure cell (X-press, Biotec, Stockholm, Sweden). The crude extract (in buffer A) was treated with 0.1 volume of 5% w/v streptomycin sulfate solution and the supernatant was fractionated by ammonium sulfate precipitation. The fraction at 40-90% saturation was dialyzed for 17 h against buffer A and chromatographed in the same buffer on a Sephadex G-100 column (3 x 90 cm). Active fractions were pooled and concentrated to 2-6 mg of protein/ml by ultrafiltration (Amicon, Diaflo filter UM-2). Protein determinations. Protein was measured by the method of Lowry et al. (1951) using trypsin as standard. Polyacrylamide gel electrophoresis, The method of Davis (1964) was followed. The samples were mixed with sucrose solution and were applied directly to the separation gels. The gel concentration was 11.1% and the ratio of acrylamide to methylene bisacrylamide was 37: 1 (w/w). Electrophoresis was conducted at 6°C and pH 8.3 under the conditions described by Davis (1964) and until the marker of bromophenol blue migrated to the bottom of the gel. Zymography. Bands of enzyme protein were located on the gels by staining for dihydrofolate reductase activity. For this purpose a tetrazolium salt (MTT) was used (Altman, 1972). A water-insoluble formazan deposit was formed by the tetrahydrofolate reduction of tetrazolium at the site of reduction of dihydrofolic acid to tetrahydrofolic acid. Immediately after the electro-
PLASMID-BORNE
DIHYDROFOLATE
phoretic run, the gels were transferred into glass tubes containing 0.2 M potassium phosphate buffer, pH 6.9, 1.2 mM NADPH, and 0.8 mM dihydrofolic acid, prepared according to Blakley (1960). After 5 min of incubation at 37°C 0.6 mM tetrazolium salt was added. The gels were incubated in the dark for another 30-45 min at 37°C and finally washed in 0.2 M potassium phosphate buffer, pH 6.9. Unspecific reduction of the tetrazolium salt was identified on con100000
10000
r a 0 1000
\i
I
I
I
1
5
10
15
20
Fraction
1
25
number
Fig. 1. Analysis of plasmid content by alkaline sucrose gradient centrifugation. Bacterial cells were radioactively labeled and lysates were subjected to centrifugation as described under Materials and Methods. Radioactivity values in cpm per 0.2 ml fraction are given along the ordinate on a logarithmic scale; fraction numbers are given along the abscissa. EC1005, a plasmid-free E. coli K-12 strain used as recipient in subsequent transfer experiments (x-x); EC1005(R6275), trimethoprim resistance transferred to EC1005 from clinical isolate (LO).
REDUCTASES
337
trol gels, stained as described but in the absence of dihydrofolic acid. RESULTS
Characteristics of Bacterial Strains Highly Resistant to Trimethoprim A survey of the strains carrying highlevel resistance to trimethoprim and studied in this report is given in Table 1. It can be seen that the clinically isolated E. coli strains, or laboratory strains with R factors from clinical isolates, were all insensitive to 1000 E.Lg/mlor more of trimethoprim. By sucrose gradient centrifugation analysis all were found to harbor plasmids. It was also observed that when the trimethoprim resistance trait was transferred to a sensitive recipient strain, the latter always acquired plasmid CCC-DNA. An example of this is shown in Fig. 1, where the plasmid-free K- 12 strain EC 1005in a gradient centrifugation analysis was seen to have obtained a plasmid concomitantly with the trimethoprim resistance from the clinical isolate 6275. Table 1 lists strains carrying four different R factors obtained from the laboratory of Dr. N. Datta in London. These are well established as trimethoprim resistancemediating plasmids (cf. references given in Table 1) and they all confer high-level resistance. It has seemed that resistance to 1000 pg/ml or more of trimethoprim was always plasmid-borne. Of the clinically isolated high-level resistant strains, however, two, 3048 and 1810, could not transfer their trimethoprim resistance traits to a sensitive recipient. Several attempts at direct transfer to different recipients and at mobilization with other plasmids were all unsuccessful. Both strains contained plasmids, however, and 1810 was observed to be able to transfer sulfonamide resistance to EC1005. Table 1 gives the general resistance patterns of the studied strains and plasmid classification according to pilus characteristics and incompatibility properties. The cellular levels of the trimethoprim
338
TENNHAMMAR-EKMAN
target enzyme, dihydrofolate reductase , in the different resistant strains are also shown in Table 1. It can be seen that these levels can be grouped into three classes. In the first, represented by strains EC1005 (R388) and EC1005(R751), the enzyme content is similar to that of the drug sensitive host strain EC1005. In the second, represented by strain 1810, the enzyme level is about 200 times higher than in the K-12 strain, and in the third, consisting of EC1005(R6275), EC1005(R22259), EC1005(R4891), 3048, EC1005(R483), and EC1005(R721), the enzyme content is IO-20 times larger than in K-12.
AND SKGLD
.._ EC1005
IO
20
30 Fraction
Analysis for Plasmid-Borne Dihydrofolate Reductases In earlier work, it was shown that the trimethoprim resistance mediated by the plasmid R483 was due to a drug-resistant variant of dihydrofolate reductase, the gene of which was R factor-borne (Skold and Widh, 1974). In order to find out if plasmids of different origins carrying trimethoprim resistance also directed synthesis of dihydrofolate reductases, extracts from different resistant strains were analyzed by gel chromatography. The variable contents of dihydrofolate reductase shown to occur (Table 1) in different plasmidcarrying strains imply the existence of different enzymes on different plasmids, or at least differences in the regulation of enzyme formation. In Fig. 2 a gel chromatogram of an extract from EC1005(R751) is shown. Two peaks of enzyme activity are clearly discernible. The peak of activity emerging second behaved chromatographically like that obtained with an extract of the plasmidfi-ee host strain EC1005 (data not shown), and was shown to be completely inhibited by the presence of 10F6M trimethoprim (Fig. 2). The first peak of activity, however, was insensitive to this concentration of the drug. The R751 plasmid thus seemed to carry the gene for a trimethoprim-resistant
LO
50
IF!7511
60
70
80
number
FIG. 2. Gel chromatography of dihydrofolate reductase from EClOOS(R751). A 40-90% ammonium sulfate precipitate was prepared from EC1005(R751) grown in rich medium in a fermentor as described, and 100 mg was applied to a 3 x 90 cm Sephadex G-100 column and eluted with buffer A at a flow rate of 15 mVh at 4°C. Fractions of 5 ml were collected and assayed for dihydrofolate reductase in the absence (X-X) and in the presence (O-O) of trimethoprim 1OWM. Enzyme activity is shown as decrease in absorption at 340 nm/5 mitt/O.1 ml eluate. Protein is represented as absorption at 280 nm (O--O). Fraction numbers are along the abscissa.
enzyme with a molecular weight larger than that of the chromosomal enzyme. The relative amounts of the two enzymes were about 35% for the plasmid enzyme and 65% for the chromosomal enzyme. A very similar chromatogram was obtained with an extract from EC1005(R388), where the druginsensitive enzyme peak comprised 20-25% of total activity (data not shown). With the plasmid R483 the resistant peak was much more dominant. This is illustrated in Fig. 3, where an experiment is described with EC1005 (R483) grown in rich medium in a fermentor as described under Materials and Methods. It can be seen that the resistant enzyme activity was 94% of total during rapid growth (Fig. 3a) but decreased to about 78% later in the growth period (Fig. 3b). With strains EC1005(R6275), EC1005(R22259), EC1005(R4891), EC1005(R721), and 3048 (cf. Table 1) chromato-
PLASMID-BORNE
3
R
DIHYDROFOLATE
REDUCTASES I3
EC1005 iRL831
EC1005 IRL831
0250
0.200 x C ; 0150 0 E I=z- 0100
0050
a
15
55 65 Fraction number
b
L5
55 Fraction
65 number
FIG. 3. Gel chromatography of dihydrofolate reductase from fermentor-grown EClOW(R483). Cells were grown in an automatic fermentor as described under Materials and Methods. Enzyme preparation, gel chromatography, and enzyme assays were as described in the legend to Fig. 2. (a) Rapid growth; (b) stationary phase, judged from the cessation of the automatic addition of alkali. (X x), no trimethoprim; (0 O), trimethoprim 1Om6M.
graphic patterns very similar to that described for EC1005(R483) were obtained. It was noted that 3048 showed two enzyme peaks, one of which was drug resistant. This suggests plasmid localization of trimethoprim resistance, in spite of the nontransferability of the resistance trait in this case. With strain 1810,however, only one enzyme peak was observed and the dihydrofolate reductase in this peak was only slightly less sensitive to trimethoprim than the chromosomal enzyme (data not shown). The peak was much larger (about loo-fold) than with any other strain, reflecting the high enzyme content of the cells of this strain (cf. Table 1). Methotrexate titration showed this high enzyme content to be due to an increased number of enzyme molecules (to be published). Electrophoresis of Dihydrofolate Reductases from Trimethoprim-Resistant Strains
The existence of different drug-resistant, plasmid-borne dihydrofolate reductases is
implied by the results of the gel chromatography experiments described above. In order to characterize these enzymes further, electrophoresis on polyacrylamide gels combined with a zymographic detection technique was employed on enzyme preparations purified through the gel chromatography step. Electrophoresis was performed as described under Materials and Methods. The localization of dihydrofolate reductase bands on the gels was done by incubating the gels with NADPH and dihydrofolate. The enzymatically formed tetrahydrofolate was then detected by addition of the tetrazolium salt MTT (cf. Materials and Methods section), which was reduced by tetrahydrofolate to give a blue, insoluble, formazan deposit. Unspecific bands of MTT reduction, not related to tetrahydrofolate, were identified by control incubations without added dihydrofolate. Bands of drug-resistant, plasmid enzyme could easily be distinguished from chromosomal enzyme bands, which were absent at staining in the presence of 10m3M trimethoprim. In Fig: 4 results from several
340
TENNHAMMAR-EKMAN
(I)
+TP
(2)
(31
(4)
-DMF +Tp
(5)
AND SKGLD
(6)
(7)
(8)
(9)
-DHF +Tp
FIG. 4. Gel electrophoresis and zymographic identification of plasmid-borne, trimethoprim-resistant dihydrofolate reductases. Gel electrophoresis was carried out in 11.1% polyacrylamide gel columns 6 x 70 mm as described under Materials and Methods. To each column 40 pg of gel-chromatographed chromosomal enzyme and the amounts mentioned below of plasmid enzyme were added in a mixture. Electrophoresis was for 60 min at 6°C. The procedure for zymographic staining was described. Gel columns 1-3 contained partially purified enzyme from EC1005(R751) (200 pg), columns 4-6 enzyme from EC1005(R388)(300 pg), and columns 7-9 enzyme from EClOOS(R483)(50 pg). In columns 3.6, and 9 the zymographic incubation took place in the absence of dihydrofolate (- DHF), and in columns 1,4, and 7 in the presence of trimethoprim (+Tp) 10m3M.
electrophoresis runs are shown. In column 2 a mixture of enzymes from the drugsensitive and drug-insensitive peak, respectively, of EClOOS(R751) was subjected to electrophoresis. Two bands disappear at staining without added dihydrofolate (column 3, but only one in the presence of trimethoprim (column 1). The latter band is interpreted to represent the chromo-
somal, drug-sensitive enzyme. This interpretation is supported by the appearance of this band at the same location in electrophoresis of similar mixtures from EC1005 (R388) (column 5) and EClOOS(R483) (column 8), respectively. The trimethoprimresistant enzymes on the other hand did show different electrophoretic migration distances. If the migration distance of
PLASMID-BORNE
DIHYDROFOLATE
chromosomal enzyme was set at 1.0, the resistant, plasmid-determined enzyme from R751 migrated 0.35 of that distance, the enzyme from R388 0.53 and that from R483 0.45. Drug-resistant enzymes from strains EC 1005(R6275), EC 1005(R22259), EC 1005(R4891), and 3048, respectively, all behaved like that from EC1005(R483). With strain 1810, however, using enzyme from the single gel chromatography peak (see above) a band was seen that migrated
(1)
(21
(31
REDUCTASES
341
slightly slower (relative migration = 0.92) than chromosomal enzyme from EC1005 (Fig. 5). Its zymographic staining was completely inhibited by the presence of trimethoprim (data not shown). The unspecific band (Fig. 5) seen to occur in the absence of dihydrofolate was observed to disappear if the starting material was chosen from only a narrow portion of the peak activity fractions of the dihydrofolate reductase gel chromatogram. In conclusion the electrophoresis data are interpreted to demonstrate the existence of at least three different drug-resistant dihydrofolate reductases, borne by plasmids mediating resistance to trimethoprim. Sensitivity of Dihydrofolate Reductases from Different Strains to Trimethoprim and Other Folic Acid Analogs
FIG. 5. Gel electrophoresis of dihydrofolate reductase from trimethoprim-resistant strain 1810. Procedures were as described in the legend to Fig. 4, but trimethoprim inhibition was not performed. In gel column 1, 40 pg of partially purified (chromosomal) enzyme from EC1005 was added, and in column 2 a mixture of 40 wg of EC 1005enzyme and 1fig of partially purified 1810 enzyme was added. With column 3 the staining incubation took place in the absence of dihydrofolate (- DHF).
A quantitative determination of the trimethoprim sensitivity of partially purified dihydrofolate reductases from different resistant strains is shown in Fig. 6. It can be seen that the drug-sensitive chromosomal enzyme is 50% inhibited at a trimethoprim concentration of about lo-’ M. The overproduced enzyme from 1810 was slightly less drug sensitive. The inhibition curves for resistant, plasmid enzymes, on the other hand, could be grouped into three classes. One, consisting of activities from EClOOS(R6275) and EC1005(R483), showed inhibition to 50% at about low4 M trimethoprim, whereas dihydrofolate reductase from EC 1005(R751) was similarly inhibited only at lo-‘- lo-* M trimethoprim. Enzyme from EC1005(R388) showed a slowly decreasing inhibition curve reaching 50% inhibition at lo-*- 1O-3M trimethoprim. Enzyme preparations from strains EC1005(R22259), EC1005(R4891),3048, and EC1005 (R721) all showed behavior very similar to those of EC1005(R483) and EC1005(R6275), demonstrating 50% inhibition at about 10e4 M concentration of trimethoprim. More detailed measurements of enzyme activities at varying dihydrofolate concen-
342
TENNHAMMAR-EKMAN
AND SKijLD
TABLE 2 trations, and with and without trimethoprim, yielded values of inhibitor constants (Ki) INHIBITION CONSTANTS OF DIHYDROFOLATE REDUCTASES FOR TRIMETHOPRIM, METHOby reciprocal plotting. The inhibition was TREXATE, AND AMINOPTERIN, of a competitive type in all cases. In Table RESPECTIVELY 2, Ki values are given for enzymes from different strains; in all cases (except for L (MS’ Trimethoprim EC1005 and 1810, but including 3048) an enStrain Methotrexate Aminopterin K CM) zyme from the trimethoprim-resistant peak 0.3 x 10-e 1.1 x 10-a 3.0 x 10.” in the gel chromatogram was used. A pattern EC1005 EC1005 (R6275) 0.7 x 10-s 3.2 x 10” 3.4 x losimilar to that of Fig. 7 can be observed EC1005 (R22259) 1.0 x 10-s 17 x 10” 23 x IO” 17 x 10” 1.0 x 10-S 7.9 x 104 here, with three classes of plasmid enzymes EC1005 (R4891) 16 x 10’ 3048 0.8 x 10-S 7.2 x IO4 discernible. It should be noted that dihydro- 1810 0.5 x 10-R 0.1 x 10-7 0.6 x IO-” 0.6 x 10m5 5.6 x IO” 6.8 x 10m6 folate reductase from strain 1810, which EC1005 (R483) 1.0 x lo-” EC1005 (R751) 0.4 x 10-a 1.5 x 10-J dramatically overproduced the enzyme (see EClOO5 (R388) 0.2 x lo-,’ 1.4 x 10-a I 2 x 10-S above), had a Ki value which was about n Enzyme assays performed as described. At inhibition, ttimethoprim three times larger than that of the chromo- was added 2 min before the reaction was started by the addition of somal enzyme from EC 1005. The K, values dihydrofolate. Values of K, were calculated from reciprocal plots obtained at two different inhibitor concentrations. for dihydrofolate varied only between h Values are given as inhibitor concentrations at 50% inhibition, as 0.5 x lop5 and 1.0 x 10V5M for the dif- judged from curves similar to those described in Fig. 6. ferent enzymes. Dihydrofolate reductases from the dif- tern of three different sensitivity classes ferent strains were also tested for sensitivity for plasmid enzymes was again observed. to two other folic acid analogs, methotrexate and aminopterin. The results are Differences among Plasmid-Borne shown in Table 2. The same general patDihydrofolate Reductases in Heat Stability, pH-Optimum, and Sensitivity to p-Chloromercuribenzoate
c
10-10
10-e I Trl:lbhopr!m
7
IM/
1o-2
FIG. 6. Relative sensitivity to trimethoprim of dihydrofolate reductases borne by different plasmids. Enzymes were purified through the gel chromatography step and concentrated as described. Assays were performed as described; trimethoprim was added 2 min before the reaction was started by the addition of dihydrofolate. Values along the ordinate denote relative enzyme activities. Activities obtained in the absence of inhibitor (100%) were in the range of 0.0900.150 (decrease in A&5 mitt/O.1 ml eluate). (X-X), ECl005; (O-O), EClOOXR6275); (m--W), EClOO5(R751); (A-A), EClOO5(R483); (O-O), EClOOS(R388);(O-O), 1810.
In Fig. 7 temperature inactivation curves are shown for chromosomal and plasmid enzymes of the different classes distinguished above. It is shown that drug-resistant dihydrofolate reductases from EClOOXR6275) and EC1005(R483), respectively, are quite heat sensitive, in that they are inactivated to 50% in less than 1 min at 45°C. Similar behavior was observed for the corresponding enzymes from EC1005(R22259),EC1005(R4891), 3048, and EC1005(R721) (data not shown). Enzymes from EClOOXR388) and EC1005(R751) on the other hand were as heat stable as the chromosomal enzyme, i.e., only a few percent decrease in enzyme activity was observed after lo-15 min of treatment at 45°C. The latter behavior was also seen for the enzyme from strain 1810 (data not shown). In the experiments described in Fig. 8, the optimal pH was sought for the different di-
PLASMID-BORNE
DIHYDROFOLATE
343
REDUCTASES
mercuribenzoate) was used according to Stanley er al. (1971). However, not only did this inhibitor interfere with NADPH diaphorase but also with some of the dihydrofolate reductases, possibly reflect-
1 EC1005
0
FIG. 7. Heat sensitivity of dihydrofolate reductases from different plasmids. Partially purified (gel chromatography) enzymes were diluted to a protein concentration of l-3 mg/ml in assay buffer and treated at 45°C on a water bath. At the indicated times, 0. l-ml samples were withdrawn and assayed for activity as described. Values along the ordinate denote relative enzyme activities on a logarithmic scale. Initial values (100%) were in the range 0.090-o. 130 decrease in A,,45 min. (X-X), EClOO5; (A-A), EC1005 (R483); (O-O), EC1005’(R6275); (U-U), EClOOS(R388);(m-m), EClOOS(R751).
hydrofolate reductase activities. It can be seen that the pH curve for the K-12 chromosomal enzyme was without any clear maximum (Fig. 8a). With the drug-resistant enzyme from EC1005(R751), however, a distinct optimum at about pH 6.5 was observed (Fig. 8d). The drug-resistant enzyme from EClOOS(R6275)also showed a maximum at pH 6.5, but in this case the curve was broader and the maximum less distinct (Fig. 8b). With the enzyme from EC1005(R22259), finally, the activity increased monotonously with decreasing pH (Fig. 8~). The enzyme from EC1005(R388) behaved very similarly to that of EC1005(R751), whereas resistant enzymes from the other strains of Table 1 showed curves similar to that for EC1005(R6275) (data not shown). The experiments of Fig. 8 were performed in Tris-chloride or Tris-succinate buffer, but very similar results were obtained with 0.02 M potassium phosphate buffer. In order to minimize the unspecific reduction of NADPH in our dihydrofolate reductase assays the addition of 1.5-2.0 mM p-chloromercuribenzoate (p-hydroxy-
0 OLO x T:
z 0020 B E
0
:,"
EC10051R62751 0160
c
:2. z
EC1005lR222591
1
0 EC1005
IF37511
0160
I 6
7
8
PH
FIG. 8. Optimal pH for dihydrofolate reductases coded for by different plasmids. Enzyme preparations were purified through the gel chromatography step. Assays were performed as described, but in 0.1 M Tris-chloride (pH 7.0-8.0) or Tris-succinate (pH 5.5-7.0) buffers. Enzyme activities are given as decreases in A,d5 mitt/O.1 ml of eluate. Amount of protein added in each eluate ranged between 0.05 and 0.20 mg.
344
TENNHAMMAR-EKMAN
AND SKiiLD
ing differences in relative dependence on intact SH-groups. From the inhibition curves shown in Fig. 9 it can be seen that the plasmid enzymes borne by R6275, R483, and R751 were relatively insensitive to p-chloromercuribenzoate, whereas the chromosomal enzyme from EC1005, that of 1810, and the plasmid enzyme of R388 were clearly more sensitive. DISCUSSION
Bacterial resistance to folic acid analogs is most easily thought of as a mutational change in the target enzyme, dihydrofolate reductase. Such changes, however, would not be expected to result in a pronounced resistance to high inhibitor concentrations without changes also in substrate affinity. The molecular mechanism of transferable trimethoprim resistance carried by the R factor R483 was shown to be explained by the R factor-induced formation of a highly drug-resistant dihydrofolate reductase, with a substrate affinity comparable to that of the chromosomal enzyme (Skiild and Widh, 1974). It has been demonstrated that the R483-induced enzyme has a molecular weight about twice as large as that of the chromosomal E. coli K-12 enzyme (Pattishall et al., 1977). Plasmid-borne druginsensitive dihydrofolate reductases seem to represent a general mechanism of transferable trimethoprim resistance (Amyes and Smith, 1976; Pattishall et al., 1977). Insensitivity to very high concentrations of trimethoprim, 1000pg/ml or more, is a characteristic of R factor-mediated resistance to this drug. Furthermore, Pattishall et al. (1977) used enzyme inhibition data to classify dihydrofolate reductases into two groups, one with highly resistant enzymes and one with reductases which were practically insensitive to folic acid analogs. The same pattern is seen in this report with the extremely resistant R388 and R751 enzymes, and several others with varying degrees of drug sensitivity. A general correlation between drug resis-
FIG. 9. Inhibition by p-chloromercuribenzoate of chromosomal and plasmid-borne dihydrofolate reductases. Enzyme preparations were purified through the gel chromatography step. Enzyme assays were performed as described, but enzyme was incubated at 30°C with p-chloromercuribenzoate at the indicated concentrations for 10 min before the reaction was started by the addition of NADPH and dihydrofolate. Values along the ordinate denote relative enzyme activities on a logarithmic scale. Uninhibited values (100%) were in the range of 0.050-0.170 (decrease in A&S min/O.l ml eluate). Concentrations of added inhibitor are given along the abscissa. (X-X), EC1005; (O-O), EClOOS(R6275); (t&---Cl), ECIOOS(R388); (Lm), EC1005(R751); (&----A), EC1005(R483); (O-O), 1810.
tance and cellular enzyme content is also discernible with strain 1810 as an extreme example, where enzyme is overproduced some 200-fold and shows only a slight increase in trimethoprim resistance over the chromosomal enzyme. The very marked overproduction of dihydrofolate reductase in this strain could be explained by either a change in transcriptional regulation or in a gene dosage effect. Preliminary results (to be published) indicate that the latter explanation is the valid one. Barth et al. (1976) and Shapiro and Sporn (1977) have shown that the trimethoprim resistance traits from R483 and R751 were localized on transposable elements, Tn7 and Tn402, respectively. From the enzyme inhibition data, pH profiles, heat lability curves, and electrophoresis characteristics reported here it could be concluded that the dihydrofolate reductase genes in these two transposons must be distinct from
PLASMID-BORNE
DIHYDROFOLATE
each other. The differences are so pronounced that they indicate different origins for the two genes. It should be noted that Tn402 is much smaller than Tn7 and lacks the streptomycin resistance trait (Shapiro and Spom, 1977). The enzyme characteristics presented here allow a clear distinction between the dihydrofolate reductases carried by the previously described (cf. Table 1) R factors, R483, R751, and R388, respectively. The overproduced dihydrofolate reductase from strain 1810 is also separable from the chromosomal E. coli K-12 enzyme. The newly isolated bacterial strains with plasmid-borne trimethoprim resistance contained drug-insensitive enzymes very similar to. that coded for by R483, which in turn seemed to be identical to that of R721. One exception was the enzyme carried by the plasmid of EC1005(R22259). This strain lacked streptomycin resistance which was present in the other strains (cf. Table 1). The plasmid-borne enzyme from EC1005(R22259), furthermore, showed a slightly lower sensitivity to methotrexate and aminopterin and also gave a different pH optimum curve. That most of the newly isolated plasmids mediating trimethoprim resistance carried dihydrofolate reductases similar to that of R483 might ,indicate an epidemiological dominance of Tn7 in the spread of resistance toward this drug. In conclusion, three and possibly four different plasmid-borne, drug-resistant dihydrofolate reductases can be clearly discerned as responsible for transferable trimethoprim resistance. ACKNOWLEDGMENTS This work was supported by a grant to O.S. from the Swedish Medical Research Council, B.T-E. gratefully acknowledges fellowships from The Swedish Academy of Pharmaceutical Sciences and from The I. F. Foundation for Pharmaceutical Research.
REFERENCES ALTMAN, F. P. (1972). An introduction to the use of tetrazolium salts in quantitative enzyme cyto-
REDUCTASES
345
chemistry, Koch-Light Laboratories Ltd. Colnbrook, Bucks., England. AHYES, S. G. B., AND SMITH, J. T. (1974). R-Factor trimethoprim resistance mechanism: An insusceptible target site. Biochem. Biophys. Res. Commun. 58, 412-418. AMYES, S. G. B., AND SMITH, J. T. (1976). The purification and properties of the trimethoprimresistant dihydrofolate reductase mediated by the R-factor, R388. Eur. J. Biochem. 61: 597-603. BARTH, P. T., DATTA, N., HEDGES, R. W., AND GRINTER, N. J. (1976). Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bacterial. 125, 800-810. BLAKLEY, R. L. (1960). Crystalline dihydropteroylglutamic acid. Narure (London) 188, 231-232. BURCHALL, J. J., AND HITCHINGS, G. H. (1965). Inhibitor binding analysis of dihydrofolate reductases from various species. Mol. Pharmacol. 1, 126-136. DATTA, N., AND HEDGES, R. W. (1972). Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae. J. Gen. Microbial. 12, 349-3.55. DAVIS, B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. GRINSTED, J., SAUNDERS, J. R., INGRAM, L. C., SYKES, R. B., and RICHMOND, M. H. (1972). Properties of an R-factor which originated in Pseudomonas aeruginosa 1822. J. Bacterial. 110: 529-537. HEDGES, R. W., DATTA, N., AND FLEMING, M. P. (1972). R-factors conferring resistance to trimethoprim but not sulfonamides. J. Gen. Microbial. 73, 573-575. HOLME, T., ARVIDSON, S., LINDHOLM, B., and PAVLU, B. (1970). Enzymes-laboratory-scale production. Process Biochem. 5, 62-66. JOBANPUTRA, R. S., AND DATTA, N. (1974). Trimethoprim resistance factors in enterobacteria from clinical specimens. J. Med. Microbial. 7, 169- 177. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MEYNELL, E., MEYNELL, G. G., AND DATTA, N. (1968). Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacterial. Rev. 32: 55-83. PATTISHALL, K. H., ACAR, J., BURCHALL, J. J., GOLDSTEIN, F. W., AND HARVEY, R. J. (1977). Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J. Biol. Chem. 252,2319-2323. SHAPIRO, J. A., AND SPORN. P. (1977). Tn402: A new transposable element in determining trimethoprim resistance that inserts in bacteriophage lambda. J. Bacterial. 129, 1632- 1635.
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Skold, O., AND WIDH, A. (1974). A new dihydrofolate reductase with low trimethoprim sensitivity induced by an R factor mediating high resistance to trimethoprim. J. Biol. Chem. 249, 4324-4325. SK~LD, 0. (1976). R-factor-mediated resistance to sulfonamides by a plasmid-borne, drug-resistant dihydropteroate synthase. Antimicrob. Agents Chemother. 9, 49-54.
STANLEY, B. G., NEAL, G. E., AND WILLIAMS, D. C.
AND SKGLD
(1971). Dihydrofolic reductase (5,6,7,%tetrahydrofolate: NADP oxidoreductase, EC 1.5.1.3). In “Methods in Enzymology,” (D. B. McCormick and L. D. Wright, eds.), Vol. XVIII, pp. 775-779. Academic Press, New York. WARNER,H. R., AND LEWIS, N. (1966). The synthesis of deoxycytidylate deaminase and dihydrofolate reductase and its control in Escherichia coli infected with Bacteriophage T4 and T4 amber mutants. Virology 29, 172- 175.
2,334-346
Trimethoprim
(1979)
Resistance Plasmids of Different Origin Encode Different Drug-Resistant Dihydrofolate Reductases BIRGITTATENNHAMMAR-EKMAN
Department
of Microbiology,
Faculty
of Pharmacy,
AND OLA SK~LD University
of Uppsala,
Uppsala,
Sweden
Received June 27, 1978 Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofoiate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R7.51 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a lO- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.
Plasmids mediating resistance to trimethoprim have been known for several years (Hedges et al., 1972) and the molecular mechanism of plasmid-borne trimethoprim resistance has been investigated. This was done with one of the R factors isolated first, R483 (Hedges, et al., 1972). It was found that this plasmid possessed the ability to induce the formation of a drugresistant target enzyme, dihydrofolate reductase (SkGld and Widh, 1974). This extrachromosomally coded enzyme was shown to be distinct from the chromosomal enzyme of E. cofi by several chemical criteria. Several different R factors carrying trimethoprim resistance have been shown to code for aberrant dihydrofolate reductases (Amyes and Smith, 1974; Amyes and Smith, 1976; Pattishall et al., 1977). These drugresistant enzymes were shown to have 0147-619X179/030334-13$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
molecular weights about twice as large as that of the chromosomal enzyme (PattishalI et al., 1977). The latter authors also clearly demonstrated the existence of two classes of these large plasmid-borne enzymes, which showed remarkable differences in their folic acid analog sensitivity. The aim of the work presented here was to compare plasmid enzymes from several, earlier characterized R factors and from plasmids newly isolated from clinical material. Clear characteristics of different plasmid-coded enzymes could give a clue to their origin and to their relationship to different transposons mediating trimethoprim resistance (Barth et al., 1976; Shapiro and Spom, 1977). By several techniques, including gel electrophoresis, at least three different plasmid-borne dihydrofolate reductases could be discerned.
334
PLASMID-BORNE
DIHYDROFOLATE
MATERIALS AND METHODS Bacterial
strains. The E. coli K-12 strain
EC1005 met-, nal’ was supplied by K. Nordstrom, Odense, Denmark (cf. Table 1). The R factors R388, R483, R721, and R751 were supplied by N. Datta, London (cf. Table 1). Bacterial strains 6275, 22259, 4891, and 1810 were trimethoprim-resistant isolates from clinical material obtained from hospitals in the area of Stockholm, Sweden. They were characterized as E. cofi by routine tests. Materials. Dihydrofolic acid was purchased from Sigma Chemical Co. (St. Louis, MO.) or prepared by the method of Blakley (l%O). NADPH, 3-(4,5-dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide (MTT), ribonuclease-A (type I-A), lysozyme (grade I), and trypsin were bought from Sigma Chemical Co. Methotrexate was obtained from Nutritional Biochemicals Corporation (Cleveland, Ohio) and aminopterin from KochLight and Co. Ltd. (Colnbrook, England). Trimethoprim lactate was a gift from the Wellcome Research laboratories (England) and nalidixic acid was donated by Winthrop AB (Sweden). [methyl-3H]Thymidine was purchased from the Radiochemical Centre (Amersham, England). Sephadex G-100 was from Pharmacia (Uppsala, Sweden).
335
REDUCTASES
Media. The mineral salts medium used contained per liter: Tris(hydroxymethyl)aminomethane (Tris) 12.0 g, KCl, 2.0 g, NH&l 2.0 g, MgCl,*6H,O 0.5 g, NqHPO,. 2H20 0.178 g, Na$O, 0.05 g, and casamino acids (Difco, certified) 0.5 g; the pH was adjusted to 7.2 with HCl and, after autoclaving, 5 g of glucose and FeCl, to 1O-5M was added. Required methionine was added to 40 pg/ml. Solid media were obtained by the addition of 2% agar. The rich medium TY-2 used in the fermentor contained per liter: tryptone 30.0 g, yeast extract 10.0 g, NH&l 2.5 g, N%HPO,. 2H,O 7.5 g, KH,POI 3.0 g, Na.#O,* 10HzO 2.5 g, glucose 40.0 g, MgS04.7H,0 0.2 g, and trace elements (Holme et al., 1970). Buffer A was 0.05 M in Tris-HCl, pH 7.2, 0.05 M in KCl, 0.01 M in mercaptoethanol, and 0.001 M in EDTA. Drug resistance transfer. Analysis for R factors was performed by mixing the presumptive donor with a nalidixic acid resistant recipient strain (EC1005 met-, naf’) as described earlier (Skold, 1976). Bacteria were grown in mineral salts medium supplemented with casamino acids (0.05%) and mixed at 2 x 1OBcells/ml of donor and 2 x lo9 cells/ml of recipient. After 2 h of incubation, recombinants were sought by plating the mixture on mineral salts medium
TABLE
1
CHARACTERISTICSOFBACTERIALSTRAINSHIGHLY RESISTANTTOTRIMETHOPRIM MIC, trimethoprim strain EC1005 EC1005 EC1005 EClOO5 3048 1810 EClOO5 EC1005 EC1005 EC1005
Resistance
markew
ham
(R627J) (R22259) (R4891)
Nd Nd,Tp,Sm Nal,Tp,Su.Tc, Cap Nd,Tp,Su, Sm
(R483) (R721) (R751) (R388)
Tp, Tp, Nal, NaI, N=L Nal,
Su, Su, Tp, Tp, Tp Tp,
Tc, Nitro, Sm Sm Sm Su
Sm
I >looo >lCKlO >locm ZlcoO >lcKw >lOCWl ZlooO >looo >lOOil
Dihydrofobate reductase contentb 0.3-0.6 6.8 6.1 8.3 5.8 77.3 8.2 13.9 0.9 0.3
Plasmid classification’
I F I I I ILZ 16 P W
Reference Grinsted et 01. (1972) This report This report This report This report This report Hedges er al. (1972) Jobanputra and Datta ( 1974) Jobanputra and Datta (1974) Data and Hedges ( 1972)
a Antibiotic sensitivity testing was perfomxd by the paper disc diffusion method. Abbreviations: Sm. streptomycin; Nal, nalidixic acid; Tp, trimethoprim; Su, sulfonamide; Tc, tetracyline; Cap, chloramphenicol: Nitro, nitrofurantoin. * Activity expressed as nmol of dibydrofolate reduced per min per 10s cells. c PiIus type of clinical isolates was determined by phage sensitivity as referred to by Meynell et a/. (196@. The phages used were ,.~2 and iFi. Compatibility groups are as described in the given references.
336
TENNHAMMAR-EKMANANDSKijLD
containing methionine (40 pg/ml), nalidixic acid (50 pg/ml) , and trimethoprim ( 15 pgrnl) . Gradient centrifugation analysis. Alkaline sucrose gradient centrifugation was performed as described by Grinstedet al. (1972). Bacterial cells growing logarithmically in mineral salts medium supplemented with deoxyadenosine, 250 pg/ml, were labeled for two generations with [3H]thymidine, 5 &i/ml (specific activity 15.8 Ci/mmol). The labeled culture was chilled on ice, centrifuged, and washed twice with ice-cold TES buffer (0.05 M Tris-HCl, pH 8.0, 0.005 M EDTA, 0.05 M NaCl). The pellet (about 8 x log cells) was resuspended in TES buffer containing lysozyme (1 mg/ml) and RNase A (500 I.Lg/ml). After incubation at 37°C for 15 min, sodium dodecyl sarcosinate (1% w/w) was added to give a clear, viscous lysate, which was sheared by passage through a 50-mm-long 21-gauge hypodermic needle five to six times at about 0.1 ml/s. Of the sheared lysate 0.1-0.2 ml was layered onto a 5-ml, 5-20% (w/w) linear sucrose gradient, in 0.3 M NaOH and 0.01 M EDTA. Centrifugation was for 60 min at 4°C and at 35,000 rpm in a Beckman SW 39 L rotor. Fractions of 0.2 ml were collected and radioactivity determined by liquid scintillation counting. Enzyme assay. Determination of dihydrofolate reductase activity was carried out according to Warner and Lewis (1966). The assay was performed at 30°C in a thermostated Zeiss PM 6 spectrophotometer equipped with a Servogor 5 recorder. Enzyme activity was followed for at least 5 min. Unspecific oxidation of NADPH, i.e., decrease in absorbance at 340 nm in the absence of dihydrofolate, was subtracted. Enzyme preparation. Bacteria were grown in 700 ml of mineral salts medium containing 40 pg/ml of methionine, 0.05% casamino acids, and 15 pg/ml of trimethoprim. At about 5 x lo8 per ml, cells were harvested by centrifugation, washed twice with buffer A, and frozen. In order to culture larger quantities of
bacteria, a 3 1 fermentor (FL-103, Biotec, Stockholm, Sweden) was used in a procedure described by Holme et al. (1970). The cultivation was performed at 37°C for 5-6 h at pH 7.2, which value was kept constant by the automatic addition of 1 M NaOH. Bacteria were harvested by centrifugation, washed in buffer A, and finally frozen. Partial purification of dihydrofolate reductase was performed as described by Burchall and Hitchings (1965) as modified by Skold and Widh (1974). The frozen cells were disintegrated in a pressure cell (X-press, Biotec, Stockholm, Sweden). The crude extract (in buffer A) was treated with 0.1 volume of 5% w/v streptomycin sulfate solution and the supernatant was fractionated by ammonium sulfate precipitation. The fraction at 40-90% saturation was dialyzed for 17 h against buffer A and chromatographed in the same buffer on a Sephadex G-100 column (3 x 90 cm). Active fractions were pooled and concentrated to 2-6 mg of protein/ml by ultrafiltration (Amicon, Diaflo filter UM-2). Protein determinations. Protein was measured by the method of Lowry et al. (1951) using trypsin as standard. Polyacrylamide gel electrophoresis, The method of Davis (1964) was followed. The samples were mixed with sucrose solution and were applied directly to the separation gels. The gel concentration was 11.1% and the ratio of acrylamide to methylene bisacrylamide was 37: 1 (w/w). Electrophoresis was conducted at 6°C and pH 8.3 under the conditions described by Davis (1964) and until the marker of bromophenol blue migrated to the bottom of the gel. Zymography. Bands of enzyme protein were located on the gels by staining for dihydrofolate reductase activity. For this purpose a tetrazolium salt (MTT) was used (Altman, 1972). A water-insoluble formazan deposit was formed by the tetrahydrofolate reduction of tetrazolium at the site of reduction of dihydrofolic acid to tetrahydrofolic acid. Immediately after the electro-
PLASMID-BORNE
DIHYDROFOLATE
phoretic run, the gels were transferred into glass tubes containing 0.2 M potassium phosphate buffer, pH 6.9, 1.2 mM NADPH, and 0.8 mM dihydrofolic acid, prepared according to Blakley (1960). After 5 min of incubation at 37°C 0.6 mM tetrazolium salt was added. The gels were incubated in the dark for another 30-45 min at 37°C and finally washed in 0.2 M potassium phosphate buffer, pH 6.9. Unspecific reduction of the tetrazolium salt was identified on con100000
10000
r a 0 1000
\i
I
I
I
1
5
10
15
20
Fraction
1
25
number
Fig. 1. Analysis of plasmid content by alkaline sucrose gradient centrifugation. Bacterial cells were radioactively labeled and lysates were subjected to centrifugation as described under Materials and Methods. Radioactivity values in cpm per 0.2 ml fraction are given along the ordinate on a logarithmic scale; fraction numbers are given along the abscissa. EC1005, a plasmid-free E. coli K-12 strain used as recipient in subsequent transfer experiments (x-x); EC1005(R6275), trimethoprim resistance transferred to EC1005 from clinical isolate (LO).
REDUCTASES
337
trol gels, stained as described but in the absence of dihydrofolic acid. RESULTS
Characteristics of Bacterial Strains Highly Resistant to Trimethoprim A survey of the strains carrying highlevel resistance to trimethoprim and studied in this report is given in Table 1. It can be seen that the clinically isolated E. coli strains, or laboratory strains with R factors from clinical isolates, were all insensitive to 1000 E.Lg/mlor more of trimethoprim. By sucrose gradient centrifugation analysis all were found to harbor plasmids. It was also observed that when the trimethoprim resistance trait was transferred to a sensitive recipient strain, the latter always acquired plasmid CCC-DNA. An example of this is shown in Fig. 1, where the plasmid-free K- 12 strain EC 1005in a gradient centrifugation analysis was seen to have obtained a plasmid concomitantly with the trimethoprim resistance from the clinical isolate 6275. Table 1 lists strains carrying four different R factors obtained from the laboratory of Dr. N. Datta in London. These are well established as trimethoprim resistancemediating plasmids (cf. references given in Table 1) and they all confer high-level resistance. It has seemed that resistance to 1000 pg/ml or more of trimethoprim was always plasmid-borne. Of the clinically isolated high-level resistant strains, however, two, 3048 and 1810, could not transfer their trimethoprim resistance traits to a sensitive recipient. Several attempts at direct transfer to different recipients and at mobilization with other plasmids were all unsuccessful. Both strains contained plasmids, however, and 1810 was observed to be able to transfer sulfonamide resistance to EC1005. Table 1 gives the general resistance patterns of the studied strains and plasmid classification according to pilus characteristics and incompatibility properties. The cellular levels of the trimethoprim
338
TENNHAMMAR-EKMAN
target enzyme, dihydrofolate reductase , in the different resistant strains are also shown in Table 1. It can be seen that these levels can be grouped into three classes. In the first, represented by strains EC1005 (R388) and EC1005(R751), the enzyme content is similar to that of the drug sensitive host strain EC1005. In the second, represented by strain 1810, the enzyme level is about 200 times higher than in the K-12 strain, and in the third, consisting of EC1005(R6275), EC1005(R22259), EC1005(R4891), 3048, EC1005(R483), and EC1005(R721), the enzyme content is IO-20 times larger than in K-12.
AND SKGLD
.._ EC1005
IO
20
30 Fraction
Analysis for Plasmid-Borne Dihydrofolate Reductases In earlier work, it was shown that the trimethoprim resistance mediated by the plasmid R483 was due to a drug-resistant variant of dihydrofolate reductase, the gene of which was R factor-borne (Skold and Widh, 1974). In order to find out if plasmids of different origins carrying trimethoprim resistance also directed synthesis of dihydrofolate reductases, extracts from different resistant strains were analyzed by gel chromatography. The variable contents of dihydrofolate reductase shown to occur (Table 1) in different plasmidcarrying strains imply the existence of different enzymes on different plasmids, or at least differences in the regulation of enzyme formation. In Fig. 2 a gel chromatogram of an extract from EC1005(R751) is shown. Two peaks of enzyme activity are clearly discernible. The peak of activity emerging second behaved chromatographically like that obtained with an extract of the plasmidfi-ee host strain EC1005 (data not shown), and was shown to be completely inhibited by the presence of 10F6M trimethoprim (Fig. 2). The first peak of activity, however, was insensitive to this concentration of the drug. The R751 plasmid thus seemed to carry the gene for a trimethoprim-resistant
LO
50
IF!7511
60
70
80
number
FIG. 2. Gel chromatography of dihydrofolate reductase from EClOOS(R751). A 40-90% ammonium sulfate precipitate was prepared from EC1005(R751) grown in rich medium in a fermentor as described, and 100 mg was applied to a 3 x 90 cm Sephadex G-100 column and eluted with buffer A at a flow rate of 15 mVh at 4°C. Fractions of 5 ml were collected and assayed for dihydrofolate reductase in the absence (X-X) and in the presence (O-O) of trimethoprim 1OWM. Enzyme activity is shown as decrease in absorption at 340 nm/5 mitt/O.1 ml eluate. Protein is represented as absorption at 280 nm (O--O). Fraction numbers are along the abscissa.
enzyme with a molecular weight larger than that of the chromosomal enzyme. The relative amounts of the two enzymes were about 35% for the plasmid enzyme and 65% for the chromosomal enzyme. A very similar chromatogram was obtained with an extract from EC1005(R388), where the druginsensitive enzyme peak comprised 20-25% of total activity (data not shown). With the plasmid R483 the resistant peak was much more dominant. This is illustrated in Fig. 3, where an experiment is described with EC1005 (R483) grown in rich medium in a fermentor as described under Materials and Methods. It can be seen that the resistant enzyme activity was 94% of total during rapid growth (Fig. 3a) but decreased to about 78% later in the growth period (Fig. 3b). With strains EC1005(R6275), EC1005(R22259), EC1005(R4891), EC1005(R721), and 3048 (cf. Table 1) chromato-
PLASMID-BORNE
3
R
DIHYDROFOLATE
REDUCTASES I3
EC1005 iRL831
EC1005 IRL831
0250
0.200 x C ; 0150 0 E I=z- 0100
0050
a
15
55 65 Fraction number
b
L5
55 Fraction
65 number
FIG. 3. Gel chromatography of dihydrofolate reductase from fermentor-grown EClOW(R483). Cells were grown in an automatic fermentor as described under Materials and Methods. Enzyme preparation, gel chromatography, and enzyme assays were as described in the legend to Fig. 2. (a) Rapid growth; (b) stationary phase, judged from the cessation of the automatic addition of alkali. (X x), no trimethoprim; (0 O), trimethoprim 1Om6M.
graphic patterns very similar to that described for EC1005(R483) were obtained. It was noted that 3048 showed two enzyme peaks, one of which was drug resistant. This suggests plasmid localization of trimethoprim resistance, in spite of the nontransferability of the resistance trait in this case. With strain 1810,however, only one enzyme peak was observed and the dihydrofolate reductase in this peak was only slightly less sensitive to trimethoprim than the chromosomal enzyme (data not shown). The peak was much larger (about loo-fold) than with any other strain, reflecting the high enzyme content of the cells of this strain (cf. Table 1). Methotrexate titration showed this high enzyme content to be due to an increased number of enzyme molecules (to be published). Electrophoresis of Dihydrofolate Reductases from Trimethoprim-Resistant Strains
The existence of different drug-resistant, plasmid-borne dihydrofolate reductases is
implied by the results of the gel chromatography experiments described above. In order to characterize these enzymes further, electrophoresis on polyacrylamide gels combined with a zymographic detection technique was employed on enzyme preparations purified through the gel chromatography step. Electrophoresis was performed as described under Materials and Methods. The localization of dihydrofolate reductase bands on the gels was done by incubating the gels with NADPH and dihydrofolate. The enzymatically formed tetrahydrofolate was then detected by addition of the tetrazolium salt MTT (cf. Materials and Methods section), which was reduced by tetrahydrofolate to give a blue, insoluble, formazan deposit. Unspecific bands of MTT reduction, not related to tetrahydrofolate, were identified by control incubations without added dihydrofolate. Bands of drug-resistant, plasmid enzyme could easily be distinguished from chromosomal enzyme bands, which were absent at staining in the presence of 10m3M trimethoprim. In Fig: 4 results from several
340
TENNHAMMAR-EKMAN
(I)
+TP
(2)
(31
(4)
-DMF +Tp
(5)
AND SKGLD
(6)
(7)
(8)
(9)
-DHF +Tp
FIG. 4. Gel electrophoresis and zymographic identification of plasmid-borne, trimethoprim-resistant dihydrofolate reductases. Gel electrophoresis was carried out in 11.1% polyacrylamide gel columns 6 x 70 mm as described under Materials and Methods. To each column 40 pg of gel-chromatographed chromosomal enzyme and the amounts mentioned below of plasmid enzyme were added in a mixture. Electrophoresis was for 60 min at 6°C. The procedure for zymographic staining was described. Gel columns 1-3 contained partially purified enzyme from EC1005(R751) (200 pg), columns 4-6 enzyme from EC1005(R388)(300 pg), and columns 7-9 enzyme from EClOOS(R483)(50 pg). In columns 3.6, and 9 the zymographic incubation took place in the absence of dihydrofolate (- DHF), and in columns 1,4, and 7 in the presence of trimethoprim (+Tp) 10m3M.
electrophoresis runs are shown. In column 2 a mixture of enzymes from the drugsensitive and drug-insensitive peak, respectively, of EClOOS(R751) was subjected to electrophoresis. Two bands disappear at staining without added dihydrofolate (column 3, but only one in the presence of trimethoprim (column 1). The latter band is interpreted to represent the chromo-
somal, drug-sensitive enzyme. This interpretation is supported by the appearance of this band at the same location in electrophoresis of similar mixtures from EC1005 (R388) (column 5) and EClOOS(R483) (column 8), respectively. The trimethoprimresistant enzymes on the other hand did show different electrophoretic migration distances. If the migration distance of
PLASMID-BORNE
DIHYDROFOLATE
chromosomal enzyme was set at 1.0, the resistant, plasmid-determined enzyme from R751 migrated 0.35 of that distance, the enzyme from R388 0.53 and that from R483 0.45. Drug-resistant enzymes from strains EC 1005(R6275), EC 1005(R22259), EC 1005(R4891), and 3048, respectively, all behaved like that from EC1005(R483). With strain 1810, however, using enzyme from the single gel chromatography peak (see above) a band was seen that migrated
(1)
(21
(31
REDUCTASES
341
slightly slower (relative migration = 0.92) than chromosomal enzyme from EC1005 (Fig. 5). Its zymographic staining was completely inhibited by the presence of trimethoprim (data not shown). The unspecific band (Fig. 5) seen to occur in the absence of dihydrofolate was observed to disappear if the starting material was chosen from only a narrow portion of the peak activity fractions of the dihydrofolate reductase gel chromatogram. In conclusion the electrophoresis data are interpreted to demonstrate the existence of at least three different drug-resistant dihydrofolate reductases, borne by plasmids mediating resistance to trimethoprim. Sensitivity of Dihydrofolate Reductases from Different Strains to Trimethoprim and Other Folic Acid Analogs
FIG. 5. Gel electrophoresis of dihydrofolate reductase from trimethoprim-resistant strain 1810. Procedures were as described in the legend to Fig. 4, but trimethoprim inhibition was not performed. In gel column 1, 40 pg of partially purified (chromosomal) enzyme from EC1005 was added, and in column 2 a mixture of 40 wg of EC 1005enzyme and 1fig of partially purified 1810 enzyme was added. With column 3 the staining incubation took place in the absence of dihydrofolate (- DHF).
A quantitative determination of the trimethoprim sensitivity of partially purified dihydrofolate reductases from different resistant strains is shown in Fig. 6. It can be seen that the drug-sensitive chromosomal enzyme is 50% inhibited at a trimethoprim concentration of about lo-’ M. The overproduced enzyme from 1810 was slightly less drug sensitive. The inhibition curves for resistant, plasmid enzymes, on the other hand, could be grouped into three classes. One, consisting of activities from EClOOS(R6275) and EC1005(R483), showed inhibition to 50% at about low4 M trimethoprim, whereas dihydrofolate reductase from EC 1005(R751) was similarly inhibited only at lo-‘- lo-* M trimethoprim. Enzyme from EC1005(R388) showed a slowly decreasing inhibition curve reaching 50% inhibition at lo-*- 1O-3M trimethoprim. Enzyme preparations from strains EC1005(R22259), EC1005(R4891),3048, and EC1005 (R721) all showed behavior very similar to those of EC1005(R483) and EC1005(R6275), demonstrating 50% inhibition at about 10e4 M concentration of trimethoprim. More detailed measurements of enzyme activities at varying dihydrofolate concen-
342
TENNHAMMAR-EKMAN
AND SKijLD
TABLE 2 trations, and with and without trimethoprim, yielded values of inhibitor constants (Ki) INHIBITION CONSTANTS OF DIHYDROFOLATE REDUCTASES FOR TRIMETHOPRIM, METHOby reciprocal plotting. The inhibition was TREXATE, AND AMINOPTERIN, of a competitive type in all cases. In Table RESPECTIVELY 2, Ki values are given for enzymes from different strains; in all cases (except for L (MS’ Trimethoprim EC1005 and 1810, but including 3048) an enStrain Methotrexate Aminopterin K CM) zyme from the trimethoprim-resistant peak 0.3 x 10-e 1.1 x 10-a 3.0 x 10.” in the gel chromatogram was used. A pattern EC1005 EC1005 (R6275) 0.7 x 10-s 3.2 x 10” 3.4 x losimilar to that of Fig. 7 can be observed EC1005 (R22259) 1.0 x 10-s 17 x 10” 23 x IO” 17 x 10” 1.0 x 10-S 7.9 x 104 here, with three classes of plasmid enzymes EC1005 (R4891) 16 x 10’ 3048 0.8 x 10-S 7.2 x IO4 discernible. It should be noted that dihydro- 1810 0.5 x 10-R 0.1 x 10-7 0.6 x IO-” 0.6 x 10m5 5.6 x IO” 6.8 x 10m6 folate reductase from strain 1810, which EC1005 (R483) 1.0 x lo-” EC1005 (R751) 0.4 x 10-a 1.5 x 10-J dramatically overproduced the enzyme (see EClOO5 (R388) 0.2 x lo-,’ 1.4 x 10-a I 2 x 10-S above), had a Ki value which was about n Enzyme assays performed as described. At inhibition, ttimethoprim three times larger than that of the chromo- was added 2 min before the reaction was started by the addition of somal enzyme from EC 1005. The K, values dihydrofolate. Values of K, were calculated from reciprocal plots obtained at two different inhibitor concentrations. for dihydrofolate varied only between h Values are given as inhibitor concentrations at 50% inhibition, as 0.5 x lop5 and 1.0 x 10V5M for the dif- judged from curves similar to those described in Fig. 6. ferent enzymes. Dihydrofolate reductases from the dif- tern of three different sensitivity classes ferent strains were also tested for sensitivity for plasmid enzymes was again observed. to two other folic acid analogs, methotrexate and aminopterin. The results are Differences among Plasmid-Borne shown in Table 2. The same general patDihydrofolate Reductases in Heat Stability, pH-Optimum, and Sensitivity to p-Chloromercuribenzoate
c
10-10
10-e I Trl:lbhopr!m
7
IM/
1o-2
FIG. 6. Relative sensitivity to trimethoprim of dihydrofolate reductases borne by different plasmids. Enzymes were purified through the gel chromatography step and concentrated as described. Assays were performed as described; trimethoprim was added 2 min before the reaction was started by the addition of dihydrofolate. Values along the ordinate denote relative enzyme activities. Activities obtained in the absence of inhibitor (100%) were in the range of 0.0900.150 (decrease in A&5 mitt/O.1 ml eluate). (X-X), ECl005; (O-O), EClOOXR6275); (m--W), EClOO5(R751); (A-A), EClOO5(R483); (O-O), EClOOS(R388);(O-O), 1810.
In Fig. 7 temperature inactivation curves are shown for chromosomal and plasmid enzymes of the different classes distinguished above. It is shown that drug-resistant dihydrofolate reductases from EClOOXR6275) and EC1005(R483), respectively, are quite heat sensitive, in that they are inactivated to 50% in less than 1 min at 45°C. Similar behavior was observed for the corresponding enzymes from EC1005(R22259),EC1005(R4891), 3048, and EC1005(R721) (data not shown). Enzymes from EClOOXR388) and EC1005(R751) on the other hand were as heat stable as the chromosomal enzyme, i.e., only a few percent decrease in enzyme activity was observed after lo-15 min of treatment at 45°C. The latter behavior was also seen for the enzyme from strain 1810 (data not shown). In the experiments described in Fig. 8, the optimal pH was sought for the different di-
PLASMID-BORNE
DIHYDROFOLATE
343
REDUCTASES
mercuribenzoate) was used according to Stanley er al. (1971). However, not only did this inhibitor interfere with NADPH diaphorase but also with some of the dihydrofolate reductases, possibly reflect-
1 EC1005
0
FIG. 7. Heat sensitivity of dihydrofolate reductases from different plasmids. Partially purified (gel chromatography) enzymes were diluted to a protein concentration of l-3 mg/ml in assay buffer and treated at 45°C on a water bath. At the indicated times, 0. l-ml samples were withdrawn and assayed for activity as described. Values along the ordinate denote relative enzyme activities on a logarithmic scale. Initial values (100%) were in the range 0.090-o. 130 decrease in A,,45 min. (X-X), EClOO5; (A-A), EC1005 (R483); (O-O), EC1005’(R6275); (U-U), EClOOS(R388);(m-m), EClOOS(R751).
hydrofolate reductase activities. It can be seen that the pH curve for the K-12 chromosomal enzyme was without any clear maximum (Fig. 8a). With the drug-resistant enzyme from EC1005(R751), however, a distinct optimum at about pH 6.5 was observed (Fig. 8d). The drug-resistant enzyme from EClOOS(R6275)also showed a maximum at pH 6.5, but in this case the curve was broader and the maximum less distinct (Fig. 8b). With the enzyme from EC1005(R22259), finally, the activity increased monotonously with decreasing pH (Fig. 8~). The enzyme from EC1005(R388) behaved very similarly to that of EC1005(R751), whereas resistant enzymes from the other strains of Table 1 showed curves similar to that for EC1005(R6275) (data not shown). The experiments of Fig. 8 were performed in Tris-chloride or Tris-succinate buffer, but very similar results were obtained with 0.02 M potassium phosphate buffer. In order to minimize the unspecific reduction of NADPH in our dihydrofolate reductase assays the addition of 1.5-2.0 mM p-chloromercuribenzoate (p-hydroxy-
0 OLO x T:
z 0020 B E
0
:,"
EC10051R62751 0160
c
:2. z
EC1005lR222591
1
0 EC1005
IF37511
0160
I 6
7
8
PH
FIG. 8. Optimal pH for dihydrofolate reductases coded for by different plasmids. Enzyme preparations were purified through the gel chromatography step. Assays were performed as described, but in 0.1 M Tris-chloride (pH 7.0-8.0) or Tris-succinate (pH 5.5-7.0) buffers. Enzyme activities are given as decreases in A,d5 mitt/O.1 ml of eluate. Amount of protein added in each eluate ranged between 0.05 and 0.20 mg.
344
TENNHAMMAR-EKMAN
AND SKiiLD
ing differences in relative dependence on intact SH-groups. From the inhibition curves shown in Fig. 9 it can be seen that the plasmid enzymes borne by R6275, R483, and R751 were relatively insensitive to p-chloromercuribenzoate, whereas the chromosomal enzyme from EC1005, that of 1810, and the plasmid enzyme of R388 were clearly more sensitive. DISCUSSION
Bacterial resistance to folic acid analogs is most easily thought of as a mutational change in the target enzyme, dihydrofolate reductase. Such changes, however, would not be expected to result in a pronounced resistance to high inhibitor concentrations without changes also in substrate affinity. The molecular mechanism of transferable trimethoprim resistance carried by the R factor R483 was shown to be explained by the R factor-induced formation of a highly drug-resistant dihydrofolate reductase, with a substrate affinity comparable to that of the chromosomal enzyme (Skiild and Widh, 1974). It has been demonstrated that the R483-induced enzyme has a molecular weight about twice as large as that of the chromosomal E. coli K-12 enzyme (Pattishall et al., 1977). Plasmid-borne druginsensitive dihydrofolate reductases seem to represent a general mechanism of transferable trimethoprim resistance (Amyes and Smith, 1976; Pattishall et al., 1977). Insensitivity to very high concentrations of trimethoprim, 1000pg/ml or more, is a characteristic of R factor-mediated resistance to this drug. Furthermore, Pattishall et al. (1977) used enzyme inhibition data to classify dihydrofolate reductases into two groups, one with highly resistant enzymes and one with reductases which were practically insensitive to folic acid analogs. The same pattern is seen in this report with the extremely resistant R388 and R751 enzymes, and several others with varying degrees of drug sensitivity. A general correlation between drug resis-
FIG. 9. Inhibition by p-chloromercuribenzoate of chromosomal and plasmid-borne dihydrofolate reductases. Enzyme preparations were purified through the gel chromatography step. Enzyme assays were performed as described, but enzyme was incubated at 30°C with p-chloromercuribenzoate at the indicated concentrations for 10 min before the reaction was started by the addition of NADPH and dihydrofolate. Values along the ordinate denote relative enzyme activities on a logarithmic scale. Uninhibited values (100%) were in the range of 0.050-0.170 (decrease in A&S min/O.l ml eluate). Concentrations of added inhibitor are given along the abscissa. (X-X), EC1005; (O-O), EClOOS(R6275); (t&---Cl), ECIOOS(R388); (Lm), EC1005(R751); (&----A), EC1005(R483); (O-O), 1810.
tance and cellular enzyme content is also discernible with strain 1810 as an extreme example, where enzyme is overproduced some 200-fold and shows only a slight increase in trimethoprim resistance over the chromosomal enzyme. The very marked overproduction of dihydrofolate reductase in this strain could be explained by either a change in transcriptional regulation or in a gene dosage effect. Preliminary results (to be published) indicate that the latter explanation is the valid one. Barth et al. (1976) and Shapiro and Sporn (1977) have shown that the trimethoprim resistance traits from R483 and R751 were localized on transposable elements, Tn7 and Tn402, respectively. From the enzyme inhibition data, pH profiles, heat lability curves, and electrophoresis characteristics reported here it could be concluded that the dihydrofolate reductase genes in these two transposons must be distinct from
PLASMID-BORNE
DIHYDROFOLATE
each other. The differences are so pronounced that they indicate different origins for the two genes. It should be noted that Tn402 is much smaller than Tn7 and lacks the streptomycin resistance trait (Shapiro and Spom, 1977). The enzyme characteristics presented here allow a clear distinction between the dihydrofolate reductases carried by the previously described (cf. Table 1) R factors, R483, R751, and R388, respectively. The overproduced dihydrofolate reductase from strain 1810 is also separable from the chromosomal E. coli K-12 enzyme. The newly isolated bacterial strains with plasmid-borne trimethoprim resistance contained drug-insensitive enzymes very similar to. that coded for by R483, which in turn seemed to be identical to that of R721. One exception was the enzyme carried by the plasmid of EC1005(R22259). This strain lacked streptomycin resistance which was present in the other strains (cf. Table 1). The plasmid-borne enzyme from EC1005(R22259), furthermore, showed a slightly lower sensitivity to methotrexate and aminopterin and also gave a different pH optimum curve. That most of the newly isolated plasmids mediating trimethoprim resistance carried dihydrofolate reductases similar to that of R483 might ,indicate an epidemiological dominance of Tn7 in the spread of resistance toward this drug. In conclusion, three and possibly four different plasmid-borne, drug-resistant dihydrofolate reductases can be clearly discerned as responsible for transferable trimethoprim resistance. ACKNOWLEDGMENTS This work was supported by a grant to O.S. from the Swedish Medical Research Council, B.T-E. gratefully acknowledges fellowships from The Swedish Academy of Pharmaceutical Sciences and from The I. F. Foundation for Pharmaceutical Research.
REFERENCES ALTMAN, F. P. (1972). An introduction to the use of tetrazolium salts in quantitative enzyme cyto-
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chemistry, Koch-Light Laboratories Ltd. Colnbrook, Bucks., England. AHYES, S. G. B., AND SMITH, J. T. (1974). R-Factor trimethoprim resistance mechanism: An insusceptible target site. Biochem. Biophys. Res. Commun. 58, 412-418. AMYES, S. G. B., AND SMITH, J. T. (1976). The purification and properties of the trimethoprimresistant dihydrofolate reductase mediated by the R-factor, R388. Eur. J. Biochem. 61: 597-603. BARTH, P. T., DATTA, N., HEDGES, R. W., AND GRINTER, N. J. (1976). Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bacterial. 125, 800-810. BLAKLEY, R. L. (1960). Crystalline dihydropteroylglutamic acid. Narure (London) 188, 231-232. BURCHALL, J. J., AND HITCHINGS, G. H. (1965). Inhibitor binding analysis of dihydrofolate reductases from various species. Mol. Pharmacol. 1, 126-136. DATTA, N., AND HEDGES, R. W. (1972). Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae. J. Gen. Microbial. 12, 349-3.55. DAVIS, B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. GRINSTED, J., SAUNDERS, J. R., INGRAM, L. C., SYKES, R. B., and RICHMOND, M. H. (1972). Properties of an R-factor which originated in Pseudomonas aeruginosa 1822. J. Bacterial. 110: 529-537. HEDGES, R. W., DATTA, N., AND FLEMING, M. P. (1972). R-factors conferring resistance to trimethoprim but not sulfonamides. J. Gen. Microbial. 73, 573-575. HOLME, T., ARVIDSON, S., LINDHOLM, B., and PAVLU, B. (1970). Enzymes-laboratory-scale production. Process Biochem. 5, 62-66. JOBANPUTRA, R. S., AND DATTA, N. (1974). Trimethoprim resistance factors in enterobacteria from clinical specimens. J. Med. Microbial. 7, 169- 177. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MEYNELL, E., MEYNELL, G. G., AND DATTA, N. (1968). Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacterial. Rev. 32: 55-83. PATTISHALL, K. H., ACAR, J., BURCHALL, J. J., GOLDSTEIN, F. W., AND HARVEY, R. J. (1977). Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J. Biol. Chem. 252,2319-2323. SHAPIRO, J. A., AND SPORN. P. (1977). Tn402: A new transposable element in determining trimethoprim resistance that inserts in bacteriophage lambda. J. Bacterial. 129, 1632- 1635.
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Skold, O., AND WIDH, A. (1974). A new dihydrofolate reductase with low trimethoprim sensitivity induced by an R factor mediating high resistance to trimethoprim. J. Biol. Chem. 249, 4324-4325. SK~LD, 0. (1976). R-factor-mediated resistance to sulfonamides by a plasmid-borne, drug-resistant dihydropteroate synthase. Antimicrob. Agents Chemother. 9, 49-54.
STANLEY, B. G., NEAL, G. E., AND WILLIAMS, D. C.
AND SKGLD
(1971). Dihydrofolic reductase (5,6,7,%tetrahydrofolate: NADP oxidoreductase, EC 1.5.1.3). In “Methods in Enzymology,” (D. B. McCormick and L. D. Wright, eds.), Vol. XVIII, pp. 775-779. Academic Press, New York. WARNER,H. R., AND LEWIS, N. (1966). The synthesis of deoxycytidylate deaminase and dihydrofolate reductase and its control in Escherichia coli infected with Bacteriophage T4 and T4 amber mutants. Virology 29, 172- 175.