revtersed by methionine, they postulate that 2-Cl-PAB inhibits only the

INHIBITION OF METHIONINE SYNTHESIS IN ESCHERICHIA COLI BY 2-CHLORO-4-AMINOBENZOIC ACID AND SULFANILAMIDE FREDE B. STRANDSKOV The Research Department of Wallace and Tiernan Products, Inc., Belleville, New Jersey Received for publication January 29, 1947 The bacteriostatic action of 2-chloro-4-aminobenzoic acid (2-Cl-PAB) was found by Wyss, Rubin, and Strandskov (1943) to be completely reversed by p-aminobenzoic acid. This, according to the theory of Woods (1940) that p-aminobenzoic acid is a specific antagonist for sulfanilamide, classified the bacteriostatic action of 2-Cl-PAB as sulfonamide activity. The bacteriostatic action of this compound did, however, differ from that of the sulfonamides in that it could be completely reversed by methionine. The action of the sulfonamides is only partially reversed by this amino acid. Shive and Roberts (1946) hav-e suggested that the sulfonamides interfere wvith a number of enzyme systems in which p-aminobenzoic acid functions and that the enzyme system which is most sensitive to the sulfonamides is the one responsible for the synthesis of methionine. Because the bacteriostatic action of 2-Cl-PAB is completely revtersed by methionine, they postulate that 2-Cl-PAB inhibits only the enzyme system involved in the synthesis of this amino acid. The work reported here is a study of the inhibition of methionine synthesis bv 2-Cl-PAB and sulfanilamide. Strains of Escherichia coli made resistant to 2-Cl-PAB and a strain made resistant to sulfanilamide were employed. The mechanism of the development of resistance to 2-Cl-PAB was also investigated. METHODS Stock solutions of the compounds to be tested were prepared in sterile distilled water and sterilized by minimal heating. The desired quantities were added to each test tube and the volume was made up to 2 ml with sterile distilled water. A glucose, inorganic salts, asparagine medium, adjusted to ph 7.0, was inoculated with the strain of E. coli to be tested, and 8 ml of the inoculated medium were added to each of the tubes containing 2 ml of the drug solution, as well as to controls containing 2 ml of sterile distilled water. The inoculum was prepared by growing the test organism in the synthetic medium at 37 C for 24 hours. The cells were washed twice with saline and diluted so that the inoculated medium contained approximately 1,000 cells per ml. The test solutions were incubated at 37 C, and turbidity readings were made at selected time intervals. EXPERIMENTAL We intended to develop resistance to 2-Cl-PAB in E. coli by the technique employed in developing resistance to sulfanilamide. This involves continuous 555

556 FREDE B. STRANDSKOV transfers in growth-limiting concentrations. We found, however, that complete resistance to 2-Ci-PAB was developed in a single transfer of the organisms in a medium containing.inhibitory..concentrations of the drug. This is shown by the data in tables 1 and 2. The data in table 1 show that after the 24-hour incu- TABLE 1 The bacteriostatic action of 2-Cl-PAB on E. coli [VOL. 53 TUBE NO. 2-Cl-PAB TURBIDITY AFTER 24 hours 72 hours mg % 1 0 0.342 0.342 2 0.2 0.337 0.377 3 0.5 0.2924 0.372 4 1.0 0 0.382 5 2.0 0 0.310 6 5.0 0 0.305 7 10 0 0.387 8 25 0 0.259 TABLE 2 The resistance to 2-CI-PAB of strains of E. coli previously grown in 0, 5, and 2' mg per cent of the drug CULTURE NO. 2-Cl-PAB 24-HOUR TURBIDITY mg % 1 0 0.328 1 0.2 0.319 1 0.5 0.036 1 1.0 0 6 0 0.276 6 2.0 0.276 6 5.0 0.102 6 10 0.082 6 25 0.046 6 50 0 8 0 0.310 8 5.0 0.305 8 10 0.276 8 25 0.260 8 50 0.194 bation period no turbidity had developed in the tubes containing 1.0 mg per cent or more of 2-Cl-PAB. After 72 hours, however, the growth in the tubes containing as high as 25 mg per cent of the drug approximated that in the control. The resistance of these cultures to 2-Cl-PAB was tested by using cultures prepared from tubes 1, 6, and 8 (table 1) as inocula for the experiment reported in table 2. The data show that the organisms had developed a resistance to the

19471 METHIONINE SYNTHESIS IN E. COLI 557 drug and that the degree of resistance of the culture in its first 24 hours of growth is governed by the concentration of the drug in which it was originally grown. The relationship of the degree of resistance to the concentration of the drug used in developing resistance has previously been observed for the sulfonamides by Kirby and Rantz (1943). Organisms isolated from the other concentrations of 2-Cl-PAB, reported in table 1, were also tested, and the same relationship was observed. This resistance to the drug was retained through one year of culturing on nutrient agar. The relationship of 2-Cl-PAB resistance to sulfanilamide resistance in E. coli is shown in table 3. The sulfanilamide-resistant organism is a strain of the TABLE 3 The resistance of a parent strain (1), a sulfonamide-resistant strain (138), and a 2-CI-PABresistant strain (8) of E. coli to sulfanilamide and -Cl-PAB CULTUIR NO. 2-Cl-PAB SULJANILAEMIE 24-HouR TRBIDITY mg% mg% 1 0 0.328 1 0.2 0.336 1 0.5 0 8 0 0.305 8 25 0.310 8 50 0 133 0 0.301 133 0.5 0.292 133 1.0 0.102 133 2.0 0.082 133 5.0 0 1 2.0 0.337 1 5.0 0.201 1 10 0 8 2.0 0.319 8 5.0 0.027 8 10 0 133 50 0.347 133 100 0 E. coli culture used in these experiments that was made resistant to sulfanilamide several years ago in this laboratory by continuous transfer in growth-limiting concentrations of the drug. The 24-hour turbidity readings show that the ratio of the lowest bacteriostatic concentrations of 2-Cl-PAB to sulfanilamide is the same for the sulfanilamide-resistant strain as it is for the parent strain. The 2-Cl-PAB-resistant organism was, however, found to be slightly but consistently more sensitive to sulfanilamide than was the parent strain. This shows that resistance to 2-Cl-PAB is not a true sulfonamide resistance, as it has been previously demonstrated by Wyss, Strandskov, and Schlmelkes (1942) that organisms made resistant to bacteriostatic concentrations of sulfanilamide are equally resistant to similar bacteriostatic concentrations of the other sulfonamides that are antagonized by p-aminobenzoic acid.

558 FREDU B. STRAND13KOV If the inhibition of methionine synthesis is the only function of 2-Ci-PAB and if this inhibition is identical with sulfonamide inhibition of this synthesis, one would expect a 2-Cl-PAB-resistant organism to behave in the presence of sulfanilamide as though methionine had been added to the medium. The data in table 4 show, however, that the concentration of sulfanilamide required to reduce the rate of growth to half-maximum was increased by the addition of methionine for the 2-Cl-PAB-resistant strain as well as for the parent strain. This shows that although the organism has been trained to grow in the presence of this drug, which is completely antagonized by methionine, it cannot synthesize methionine in the presence of sulfanilamide. Sulfanilamide must, therefore, inhibit a process in the synthesis of methionine not affected by 2-C1-PAB. TABLE 4 The effect of methionine on the bacterio8tatic action of sulfanilamide on a parent and 2-CI-PABresistant strain of E. coli ORGANISM MdETHIONINE. CONCENTRATION OF SULFAN1LMIDE GIVING mg % mg % 1 0 3.8 1 1.0 5.8 8 0 1.9 8 1.0 3.7 1 = parent strain. 8 = 2-Cl-PAB-resistant strain. [VOL. 53 MAXIMUM RATE OF GROWTH DISCUSSION That the bacteriostatic action of 2-Cl-PAB is closely related to true sulfonamide bacteriostasis is emphasized by the relative resistance of the parent and sulfanilamide-resistant organisms to the drug. This is also added evidence that the inhibition of methionine synthesis is a fundamental function of the sulfonamides. The lack of resistance to sulfanilamide in the 2-Cl-PAB-resistant strain, as well as the increased resistance to sulfanilamide observed in the presence of methionine, shows, however, that sulfanilamide, in addition to inhibiting methionine synthesis in the same manner as does 2-CI-PAB, exerts an additional inhibitory effect on the synthesis of this amino acid. The ease with which resistance to 2-Cl-PAB is developed in E. coli is unusual. Resistance to the sulfonamides in this organism is developed only after a series of transfers in growth-limiting concentrations. The fact that 2-Ci-PAB inhibits only the synthesis of methionine, whereas sulfanilamide interferes with the synthesis of additional products, could account for this difference. The rapid development of resistance to 2-Cl-PAB indicates that either a very small percentage of the inoculum was originally resistant to the drug, and that the delayed growth as compared with that in the control was due to the extremely small inoculum, or that all the cells are able slowly to adapt themselves, through some alteration in their growth mechanism, to growing in concentrations of the

19471 METHIONINE SYNTHESIS IN E. COLI 559 drug that were originally inhibitory. To determine the mechanism involved, the parent strain was plated on a series of synthetic medium agar plates containing increasing concentrations of 2-Cl-PAB. We found that after 24 hours' incubation the plates containing as high as 0.5 mg per cent of the drug supported growth of a number of colonies equal to the control. No colonies were visible on the plates containing higher drug concentrations. After 96 hours, the plate containing 1.0 mg per cent 2-Cl-PAB contained 25 per cent as many colonies as the control, and each of those having higher concentrations, up to 25 mg per cent, supported growth of one-tenth the number of colonies which grew on the control. Continued incubation for 2 weeks gave no increase in the number of colonies on any of the plates. Organisms isolated from these plates were resistant to the drug, and the degree of resistance was again dependent on the concentration to which they had been exposed. This shows that approximately one-tenth of the cells in the parent culture can develop a resistance to inhibitory concentrations of 2-Cl-PAB and that apparently none of the cells were originally resistant to this drug. ACKNOWLEDGMENT The author is indebted to Dr. L. Reiner and Dr. H. C. Marks for helpful suggestions and criticisms. CONCLUSIONS Permanent resistance to 2-Cl-PAB in Escherichia coli is developed during the first 72-hour growth period in the presence of bacteriostatic concentrations of the drug. The degree of resistance obtained depends on the concentration of drug in which the organisms are grown. Resistance to 2-Cl-PAB is not a property of a small percentage of the original inoculum but is developed in approximately 10 per cent of the cells. This is not a general sulfonamide resistance. Sulfonamideresistant E. coli cells do, however, show a parallel increase in resistance to 2-Cl-PAB. Sulfanilamide appears to inhibit a process in the synthesis of methionine additional to that inhibited by 2-Cl-PAB. REFERENCES KIRBY, W. M. M., AND RANTZ, L. A. 1943 Quantitative studies of sulfonamide resistance. J. Exptl. Med., 77, 29-39. SEIVE, W., AND ROBERTS, E. C. 1946 Biochemical transformations as determined by competitive analogue-metabolite growth inhibitions. J. Biol. Chem., 162, 463-471. WOODS, D. D. 1940 The relation of p-aminobenzoic acid to the mechanism of the action of sulphanilamide. Brit. J. Exptl. Path., 21, 74-90. WYSS, O., RUBIN, M., AND STRANDSKOV, F. B. 1943 Mechanism of sulfonamide action. III. Biological action of substituted p-aminobenzoic acids. Proc. Soc. Exptl. Biol. Med., 52, 155-158. WYSS, 0., STRANDSKOV, F. B., AND SCHMELKES, F. C. 1942 Mechanism of sulfonamide action. II. Inhibition of bacterial respiration by sulfanilamide and by its inactive isomers. Science, 96, 236-237.