(Landy and Dicken, 1942) Pfanstiehl H. P. casein, 5 g per L, fortified by cysteine

Transcription

1 STUDIES OF THE EFFECT OF PARA-AMINOBENZOIC ACID, FOLIC ACID, AND SULFANILAMIDE ON DEXTRAN SYNTHESIS BY LEUCONOSTOC1 VIRGINIA WHITESIDE-CARLSON AND WARNER W. CARLSON DepartmeWnt of Biochemistry, Medical College of Alabama, Birmingham, Alabam,a Received for publication May 9, 1949 The factor stimulating dextran synthesis by Leucono8toc has been claimed by Stacey (1947) to be p-aminobenzoic acid. In studies on the vitamin requirements of Leucono8toc (Whiteside-Carlson and Carlson, 1949), it was reported that omission of p-aminobenzoic acid from the media did not adversely affect acid production or polysaccharide synthesis by the organisms. Since, however, the only identification of the substance in raw cane sugar, molasses, and the like that stimulates dextran synthesis by Leucono8toc has been that given by Stacey, this point was investigated further. The effect of sulfanilamide on polysaccharide synthesis by Leuconostoc was also studied, since Stacey suggested that the identity of the dextran synthesis factor as p-aminobenzoic acid was of interest in relation to the mode of action of the sulfonamides. EXPERIMENTAL METHODS Cultures. Two strains of Leuconostoc mesenteroides, 683 and 535, and two strains of Leuconostoc dextranicum, 8086 and elai, were employed in this investigation. The method of mainaining the cultures and preparing the inocula was the same as that described in the preceding paper. Media. Two basal media were utilized. The first of these, an amino acid, purine, pyrimidine medium, had the composition given in the preceding paper. In the second type the amino acid mixture was replaced by acid-hydrolyzed (Landy and Dicken, 1942) Pfanstiehl H. P. casein, 5 g per L, fortified by cysteine 100 mg. per L, and tryptophan 100 mg per L. The purines, adenine and xanthine, were added in a concentration of 20 mg per L. As indicated, guaine, thymine, and uracil when used were present in a concentration of 10 mg per L. Vitamin concentrations in both basal media were the same except when noted otherwise. The sugar component used was present in a concentration of 50 g per L. Acid production and dextran yields were measured by the methods given in the preceding publication. RESULTS AND DISCUSSION Stacey (1947) did not specify whether p-aminobenzoic acid functioned directly in dextran synthesis or indirectly as a precursor of folic acid; hence we used both folic-acid-dependent and folic-acid-independent strains of Leuconstoc. In table 1 are presented data on the effect of a great variation in p-aminobenzoic acid concentration on acid formation and dextran synthesis by two folic-acid-inde- I Presented before the Biological Division, the Society, St. Louis, Missouri, September 7, th meeting, American Chemical

2 144 VIRGINIA WHITESIDE-CARLSON AND WARNER W. CARLSON [VOL. 58 pendent strains, L. dextranicum elai and L. mesenteroides 535. The results show that a thousandfold variation in p-aminobenzoic acid concentration affected dextran synthesis and acid production only slightly, and then in the direction of inhibition. Sulfanilamide, in a range of concentrations, sharply decreased acid production but affected dextran synthesis to a lesser extent. Both results minimize the effect of p-aminobenzoic acid on polysaccharide synthesis by these strains of Leuonostoc. Table 2 presents the results of added folic acid and sulfanilamide in the cases of two strains of Leuconostoc one of which, L. dextranicum 8086, requires folic acid. As is seen from the table, growth of the other strain, L. mesenteroides 535, is stimulated by the addition of folic acid, although the polysaccharide yield is TABLE 1 Effect of added PAB and sulfanilamide on dextran yield and acid production in cp 8ucro8e casein hydrolyzate medium ACID PRODUUCTON* ADDED PAB ADDED Tam_ SULFANILA TIM E DEXTRAN YELD L. mes. 535 L. dcx. elai L. mes. 535 L. dcx. elai pg/lm og/mi hr ml ml % % , , t t o 2,000 ~~ , t t 0 1,000 ~~ ~~ t The basal medium contained no added PAB or folic acid. * In this and subsequent tables, as ml 0.01 N NaOH used per ml medium. t Less than 5 per cent. approximately equivalent with or without added vitamin. Sulfanilamide in a relatively low concentration inhibited acid production and slowed dextran synthesis without affecting ultimate polysaccharide yield. In the presence of added folic acid both organisms were insensitive to the effects of high levels of the sulfonamide, as has been noted for other lactic acid organisms (Lampen and Jones, 1946). It was thought that if p-aminobenzoic acid functions directly in dextran synthesis, rather than as a precursor of folic acid, it might be possible to block this action by high concentrations of sulfanilamide in the presence of added folic acid. The failure to influence polysaccharide yield by this means again suggests that p-aminobenzoic acid is not involved in dextran synthesis, at least, by these strains. The data so far presented were obtained with strains of Leuconostoc usually giving only low or moderate yields of dextran even after prolonged incubation. In table 3 are recorded data on the effect of folic acid, p-aminobenzoic acid, and

3 1949] FACTORS AFFECTING DEXTRAN SYNTHESIS 145 TABLE 2 Effect of added folic acid and sulfanilamide on dextran yield and acid production in cp sucrose casein hydrolyzate medium FOLIC ACID SULPANILAME TIME ACID PRODUCTION DEXTRAN YIELD L. mes. 535 L. dcx L. mes. 535 L. dcx pg/mml g/mi hr ml ml % % ** O.001 O ~~~ ~~ * , * { ~ * Less than 5 per cent. TABLE 3 Effect of folic acid, PAB, and raw sugar on dextran yield and acid production in casein hydrolyzate medium by L. dextranicum elai SUCROSE ADDED POLIC ACID ADDED PAB TIME ACID PRODUCTION DEXTILAN YIELD C g/mi pg/mi hr ml % Cp) * cp * cp * cp * cp Raw Rawt * Less than 5 per cent. t Adsorbed at ph 3 with norit A.

4 146 VIRGU WMTE5IDE-CARL5ON AND WARNER W. CARLSON [VOL. 58 raw sugar on acid and dextran production by a strain, L. dextranicum elai, that consistently gives yields of dextran approaching theoretical conversion of the glucose half of the sucrose molecule into the polysaccharide. This strain does not require the addition of folic acid for growth, although it was found to be stimulated by the vitamin. Added p-aminobenzoic acid did not affect the rate of dextran synthesis. In contrast, substitution of raw cane sugar for cp sucrose resulted in marked stimulation of both acid and dextran production. When the raw sugar was treated in aqueous solution with norit A at ph 3 for 30 minutes prior to being added to the medium, the rates of acid and dextran synthesis decreased, although they still exceeded those obtained with added folic acid or p-aminobenzoic acid. The fractionation of raw sugar by this and other methods will be reported at a later date. TABLE 4 Effect of folic acid, PAB, and raw sugar on dextran yield and acid production in casein hydrolyzate medium by L. mesenteroides 688 SUCtOSE ADDED JOLIC ACID ADDED PAB TIME ACID NODUCTION DEXTIAN YIELD pg/m g/m hi im% l Ce) * cp * cp ~ cpop ,000 1; p Raw 0 0 ~~~~~ Raw * Less than 5 per cent. Table 4 presents corresponding results in the case of L. mesenteroi4eo 683, a strain that requires folic acid. p-aminobenzoic acid was supplied in a range covering a 101 variation in concentration, the resulting effect on dextran synthesis being one of slight inhibition at the higher levels. Substitution of raw cane sugar for cp sucrose in the absence of added vitamins permitted only a low level of growth and dextran formation. This result indicates that raw sugar is a poor source of folic acid and suggests, when taken in conjunction with the data in table 3, that the stimulating effect of raw sugar is not due to its content of this vitamin. The results so far discussed were obtained in casein hydrolyzate media containing, in addition to various salts and vitamins, the purines, adenine and xanthine. Such media satisfactorily supported growth and dextran formation with all the strains of LeuconostoC utilized, the substitution of raw cane sugar for cp sucrose generally resulting in increased rates of acid and dextran production. A chemically defined medium containing 19 amino acids, vitamins, salts,

5 1949] FACTORS AFFECTING DEXTRAN SYNTHESIS adenine, xanthine, guanine, uracil, and thymine in the concentrations described in the preceding paper was found incapable of supporting the growth of all the various strains. In table 5 are presented dlata for the effects of folic acid, p-aminobenzoic acid, and sulfanilamide on a strain, L. mesenteroides 683, which grew in this chemically defined medium. Growth and dextran synthesis in the absence of folic acid, though better than that observed in casein hydrolyzate, still were low. Thus, in contrast to results reported for some other lactic acid bacteria (Stokes, 1944), thymine apparently has only a limited ability to replace the folic acid requirement of this organism. The addition of folic acid in concentrations of and 0.01,ug per ml strongly stimulated acid production and polysaccharide synthesis, titratable acidity being favored by the higher level of the vitamin and dextran formation by the lower. Supplementation of the medium with both folic acid and p-aminobenzoic acid gave results similar to those noted in casein hydrolyzate, TABLE 5 Effect of folic acid, PAB, and sulfanilamide on dextran yield and acid production by L. mesenterosdes 688 in a synthetic medium (48 hours' incubation time) SUCROSE ADDED POLIC ACI ADDED PAB ADDED ACID PRODUCTION DEXTRAN YrELD SULFANILAMIDE pg/ml pg/ml g/mi m % cp cp cp cp cp cp , i.e., inhibition at a high level of p-aminobenzoic acid. The effect on L. mesenteroides 683 of a high level of sulfanilamide in the presence of folic acid was similar to that previously recorded for strain 535 in table 3. The failure to decrease dextran synthesis by this means again indicates the noninvolvement of p-aminobenzoic acid in polysaccharide formation by these organisms. To investigate further the effect of raw sugar in various media, acid production and dextran synthesis by three organisms were compared in a chemically defined medium and in one containing Difco peptone, as summarized in table 6. When added to the complete chemically defined medium, raw sugar caused a relatively greater stimulation in titratable acidity than in dextran synthesis. In the cp sucrose peptone (Difco) medium without added vitains, the growth of all strains was retarded. In contrast, moderate growth with high yields of dextran was observed with two of the orgnisms when raw sugar was substituted for the cp sucrose. Results of this kind probably have been the basis of the reported stimulation of dextran synthesis by materials such as raw sugar (Carruthers and Cooper, 1936; Stacey and Youd, 1938). From table 6 it is seen that the differ- 147

6 148 VIRGINIA WHITESIDE-CARLSON AND WARNER W. CARLSON [VOL. 58 ence between cp sucrose and raw sugar can be partially eliminated simply by supplying a complete mixture of vitamins. There is, nevertheless, a very definite stimulation by raw sugar which cannot be duplicated by the vitamin mixture and which is especially manifested in enhancement of growth and polysaccharide synthesis early in the incubation period, as shown, for example, in table 3. Also to be noted in table 6 is the tendency, with high dextran-forming strains such as L. mesznteroides 683, for an inverse relationship to eist between titratable acidity and polysaccharide yield. TABLE 6 Effect of added vitamins and raw sugar on dextran yields and acid production in synthetic and peptone media (48 hours' incubation) SUCROSE ACID PRODUCTION ADDED DEXTRN YELD L. ms. 683 L. mes. 535 j L. dex L. es. 683 L. mes. 535 L. dex.886 Amino acid, purine, pyrimidine medium* ml ml ml % % % cp 9t Raw 9t Difco peptone medium: cp Raw cp 9t Raw 9t * Nineteen amino acids, adenine, guanine, xanthine, thymine, and uracil. t Folic acid, PAB, pyridoxal, pyridoxamine, riboflavin, thiamine, pantothenic acid, nicotinic acid, and biotin per cent concentration. Leass than 5 per cent. Recently Stacey and Swift (1948) described dextran synthesis by Leuconostoc in a medium containing inorganic salts and a low concentration of peptone with p-aminobenzoic acid (500 jug per ml) as the only added vita. In similar vitamin-deficient media we have observed occasional stimulation of growth and dextran synthesis by the addition of p-aminobenzoic acid up to the level of 100 ug per ml. However, in view of the results obtained in more complete media, it is believed that this is a nonspecific stimulation not related to the possible presence of this vitan in raw sugar. ACKNOWLEDGMENTS We wish to thank Mr. Leon Godchaux, II, of Godchaux Sugars, Inc., New Orleans, and Dr. Robert C. Hockett of the Sugar Research Foundation, Inc., New York, for samples of raw cane sugar.

7 19491 FACTORS AFFECTING DEXTRAN SYNTHESIS 149 SUMMARY Low levels of p-aminobenzoic acid failed to stimulate dextran synthesis, and higher concentrations retarded polysaccharide formation by the strains of Leuconostoc employed. In the case of folic-acid-independent strains, sulfanilamide produced a greater inhibition of acid production than of dextran synthesis. In the presence of added folic acid, sulfanilamide in high concentrations did not affect polysaccharide formation by Leuconostoc strains that were dependent on this vitamin. These results indicate that p-aminobenzoic acid is not involved in dextran synthesis by the strains of Leuconostoc studied. The stimulating effect of raw cane sugar on growth and dextran synthesis could not be duplicated by p-aminobenzoic acid, folic acid, or a mixture of nine B vitamins. REFERENCES CARRUTHERS, ALBERT, AND COOPER, EVELYN A Enzyme formation and polysac. charide synthesis by bacteria. II. Biochem. J., 30, LAMPEN, J. O., AND JONES, M. J Antagonism of sulfonamides by pteroylglutamic acid and related compounds. J. Biol. Chem., 164, LANDY, MAURICE, AND DICKEN, DOROTHY M A microbiological assay method for six B vitamins using Lactobacillus casei and a medium of essentially known composition. J. Lab. Clin. Med., 27, STACEY, M Macromolecules synthesized by micro-organisms. J. Chem. Soc., STACEY, M., AND SWIFT, G Structure of the dextran synthesized from sucrose by,a new strain of Betacoccus arabinosaceous. J. Chem. Soc., STACEY, MAURICE, AND YouD, FREDERICK R CCL. A note on the dextran produced from sucrose by Betacoccus arabinosaceou8 haenwolyticus. Biochem. J., 32, STOKES, J. L Substitution of thymine for "folic acid" in the nutrition of lactic acid bacteria. J. Bact., 48, WMITESIDE-CARLSON, VIRGINIA, AND CARLSON, WARNER W The vitamin requirements of Leuconostoc for dextran synthesis. J. Bact., 58,