composition (Snell, Tatum, and Peterson, 1937):

Transcription

1 THE NUTRITIONAL REQUIREMENTS OF THE LACTIC ACID BACTERIA AND THEIR APPLICATION TO BIOCHEMICAL RESEARCH' ESMOND E. SNELL The University of Texas, Biochemical Institute, and the Clayton Foundation for Research, Austin Received for publication June 22, 1945 Early studies by a number of workers, particularly those of Orla-Jensen and coworker (1919, 1930), established the fact that lactic acid bacteria require complex media for rapid growth. Among the constituents of such media which were found necessary for these organisms were riboflavin and certain amino acids (Orla-Jensen et al., 1936a, 1936b). A detailed investigation of the organic factors required by these bacteria was begun by the author at the University of Wisconsin in 1936, under the direction of Professor W. H. Peterson, and has been continued under varied circumstances, and with many interruptions, to the present time. A brief, and therefore incomplete, summary of our present knowledge of this subject, of its development, and certain of its applications to biochemical research will be attempted. The medium chosen as a starting point for our investigation had the following composition (Snell, Tatum, and Peterson, 1937): Medium I Bacto peptone Glucose Mineral salts (K+, Na+, Mg++, Fe++, Mn++, Cl-, PO4-) The mineral mixture used was that of Speakman (1923) and was used in the concentration recommended by him. No detailed investigation of the mineral nutrition of these organisms has been made. Speakman's salt mixture was chosen because it proved particularly suitable for the organisms used, probably because of its content of manganese, which is required in comparatively large amounts by certain organisms of this group (Moeller, 1939; Woolley, 1941). On this medium Lactobacillus delbrueckii grew poorly, even when calcium carbonate was added as buffer, unless tissue extracts were also added. Fractionation experiments were begun to elucidate the nature of the active substances in tissue extracts. It was soon found that fractions which contained acetate greatly stimulated growth and acid production of the test organism, so that the medium, when supplemented with adequate amounts of sodium acetate, supported rather good growth of a variety of lactic acid bacteria.2 1 Adapted from an address delivered to the Texas Branch of the Society of American Bacteriologists on the occasion of the presentation of the Eli Lilly Award in Bacteriology and Immunology for U 2 At present, acetate is widely used in media for these organisms. Although the assumption is often made that it serves only as a buffer, this is not true, since in purified media 373

2 374 ESMOND E. SNELL The peptone of such a medium now contained all the substances of unknown nature required for these organisms. When the peptone was treated with sodium hydroxide, however, it no longer permitted the growth of any of several organisms tested. Orla-Jensen et al. (1936a) had shown that mild treatment with sodium hydroxide destroyed riboflavin, which they found stimulated growth of all cultures of lactic acid bacteria which they investigated. Riboflavin was therefore added to the new medium, but even in its presence, the medium failed to support growth of any of our test organisms unless small amounts of tissue extracts were also added. A new basal medium which would not support growth was thus developed with the following cqmposition (Snell, Strong, and Peterson, 1937): Medium II Amount per 100 ml Sodium-hydroxide-treated peptone... 5 g Glucose g Sodium acetate... Cystine... Riboflavin... Mineral salts 0.6 g 0.01 g 0.01 mg C3ystine, although not essential for growth on such supplemented media, appeared to improve the response, and was therefore included. An extensive investigation of the substance present in tissue extracts which permitted growth was then undertaken (Snell, Strong, and Peterson, 1937). After considerable purification fractions were obtained the growth-promoting properties of which were evident at concentrations as low as ug per ml of medium. At this point similarity in properties of the active substance to those described for concentrates of pantothenic acid (Williams et al., 1938) led us to test concentrates of the latter substance obtained from Dr. R. J. Williams. These concentrates proved highly active, and it was concluded that the active substance present in these extracts was that named pantothenic acid by Williams (Snell, Strong, and Peterson, 1938, 1939). The procedures developed during this work for concentrating pantothenic acid and testing for its presence supplemented those developed by Williams and coworkers, and were later used extensively at the Merck Laboratories for the preparation of large-scale concentrates of pantothenic acid, preliminary to the isolation and identification of the lactone moiety of this vitamin (Stiller, Keresztesy, and Finkelstein, 1940; cf. Williams, 1943). The addition of small amounts (0.05,ig per ml suffice) of pure pantothenic acid, or of concentrates of this substance, to the medium permitted excellent growth of a number of test organisms, including Lactobacillus delbrueckii, Lactobacillus casei, or Lactobacillus arabinosus. If riboflavin was now omitted, the other buffers do not duplicate its action. Unpublished data support the view that one function of acetate is as a starting material for synthesis of lipoidal and steroidal materials, 4ice a variety of such materials, when added to media adequately buffered with phosphate, partially duplicate the action of acetate. Tissue extracts also contain water-soluble substances of unknown nature, which surpass acetate in activity (Guirard, Snell, and Williams, to be published).

3 NUTRITIONAL REQUIREMENTS OF LACTIC ACID BACTERIA 375 medium became specifically deficient in this substance. Opportunity was thus afforded to test a number of these organisms for their riboflavin requirement (Snell and Strong, 1939a). Of eleven organisms tested, three proved unableto grow in the absence of riboflavin. Those which grew in the absence of riboflavin synthesized it. The response of L. casei to a number of synthetic flavin analogues paralleled closely the effect of these same flavins on the growth of rats. These findings laid the groundwork for subsequent use of L. casei in slightly modified media for the microbiological determination of riboflavin (Snell and Strong, 1939b) and pantothenic acid (Pennington, Snell, and Williams, 1940; Neal and Strong, 1943). For the demonstration of additional nutritional requirements of these organisms, further purification of the nitrogen source in the medium was necessary. For this purpose the sodium-hydroxide-treated peptone was replaced by an acid hydrolyzate of purified casein. Acid hydrolysis destroys tryptophane, which was found necessary for the test organisms and was added back to the medium. The composition of the medium was now as follows (Snell, Strong, and Peterson, 1939): Medium III "Vitamin-free" casein, acid hydrolyzed % Glucose % Sodium acetate % Cystine % Tryptophane % Riboflavin ppm Pantothenic acid (concentrate, 5% pure) ppm When this medium was inoculated heavily with washed cells of L. casei or L. arabinosus only very scanty growth occurred. Rather good growth occurred if nicotinic acid was added to the medium, however. This demonstrated the requirement of these organisms for nicotinic acid, but it was evident that other substances were also required, since growth failed upon subculture in the same medium. Apparently the small amount of these additional growth essentials carried over with the inoculum (which had been grown in medium II plus pantothenic acid) sufficed to permit considerable growth in the first culture when nicotinic acid was added. The nature of these additional requirements was next investigated, L.xasei being used as the test organism. The basal medium was medium III, supplemented with nicotinic acid (0.2 ppm). It was shown (Snell and Peterson, 1940) that at least three distinct substances were involved. One of these was vitamin B6, which Moeller (1938) had previously shown was required by certain lactic acid bacteria. In addition, two substances were required which were present in yeast and liver extracts, and which could be partially separated from each other by differential adsorption on activated charcoal. One of these was adsorbed readily by small amounts of charcoal and was eluted from the charcoal with difficulty. This substance had both acidic and basic properties and was concentrated about 100-fold over a potent source material. This substance

4 376 ESMOND E. SNELL was called the "norite eluate factor" (Snell and Peterson, 1939, 1940). Subsequent findings permitted development of more suitable media for its determination (Mitchell and Snell, 1941; Tepley and Elvehjem, 1945). It is now known to be both a growth factor for various microorganisms and a vitamin for animals and is variously known as the "eluate factor," "L. casei factor," "folic acid," "vitamin B.," and "vitamin M." It has been isolated in apparently pure form in three different industrial laboratories, and elucidation of its chemical nature represents one of the spearheads of vitamin research at the present time.' The factor necessary for L. caoei on the described medium which was less readily adsorbed on charcoal is now kiown to have been a mixture of substances, chief among which was biotin. The pattern used for establishing the nutritional requirements of these organismns is now evident. As successive essential nutrients were identified, the pure substances or highly purified concentrates of. them were added to the basal medium. A more highly purified nitrogen source was then used in the medium, and the additional compounds which then proved necessary for growth were identified. As a result of contributions from several sources, all of the organic accessory factors necessary for growth of a large number of lactic acid bacteria are now known. These are listed in table 1. The composition of a semisynthetic medium which will support good growth of most species of lactic acid bacteria tested by the author is given in table 2. The casein hydrolyzate of this medium can be replaced by a mixture of the amino acids which compose it without decreasing the growth-supporting properties of the medium. Thus, a complete medium for most of these organisms, in which all necessary constituents are pure, chemically defined materials, awaits only the synthesis of folic acid. It was early evident that rather marked differences existed in the nutritional requirements of various species of the lactic group. An idea of the extent of such variation can be gained from table 3, in which the vitamin requirements of several species of organisms which have proved useful to the biochemist are listed. Such variation may also be evident in different "strains" or cultures of a single species, as was shown by Niven (1944) for Streptococcus lacti8. It is noteworthy that all of the cultures require pantothenic acid, biotin, and nicotinic acid; variation is evident in requirements for thiamine, riboflavin, vitamin B6, and folic acid. A similar variation is met when one investigates requirements of various cultures for the purine bases, and for the individual amino acids. The amino acid requirements of a variety of cultures have been determined (for literature references see Snell, 1945b). At the present time, cultures are known which require seventeen of the twenty-one or so amino acids which occur normally in proteins. Indeed, a single culture, Leuconostoc mesenteroides, has been reported to require all seventeen (Duinn et al., 1944). ' The synthesis of this substance has been announced recently (Angier et al., Science, 102, , 1945),

5 NUTRITIONAL REQUIREMENTS OF LACTIC ACID BACTERIA As the growth factors required by these organisms became known, it was natural that the growth tests originally used in identifying them should be applied TABLE 1 Vitamins and growth factors essential for or stimulatory to growth of various lactic acid bacteria GROWTH FACTOR TYPICALL ORGANISM RESPONDING R1EFERENCE 377 Riboflavin Streptococcus lactis Orla-Jensen et al. (1936) Lactobacillus casei Snell, Strong, and Peterson (1937) Pantothenic acid Lactobacillus casei Snell, Strong and Peterson (1938) Nicotinic acid Lactobacillus casei Snell, Strong, and Peterson (1938) Lactobacillus arabinosus Biotin Lactobacillus plantarum Moeller (1939) Lactobacillus arabinosus Snell and Wright (1941) Vitamin B6t Lactobacitlus plantarum Moeller (1938) Thiamine Streptococcus lactis Niven (1944) Lactobacillus fermentum Sarett and Cheldelin (1944) p-aminobenzoic Lactobacillus arabinosus Isbell (1942) acid Folic acid (eluate Lactobacillus casei Snell and Peterson (1939, 1940) factor, vitamin B,) Streptococcus faecalis Mitchell, Snell, and Williams (1941) Adenine$ Lactobacillus plantarum Moeller (1939) Guaninel Leuconostoc mesenteroides Snell and Mitchell (1941) Uracil Lactobacillus arabinosus Snell and Mitchell (1941) Thymine Streptococcus faecalis Snell and Mitchell (1941) Lactobacillus casei Stokstad (1941) * Classification of substances as "growth factors" is entirely arbitrary. The table does not include the variety of amino acids required by these organisms, or the related substances, asparagine, and glutamine, which are essential or stimulatory for some species (cf. Niven, 1944). Similarly acetate and various fatty substances which greatly stimulate growth under some conditions are not included. t The growth-promoting effect of vitamin B,, originally discovered with pyridoxine, is much more strikingly demonstrated with pyridoxamine or pyridoxal (see text). For some organisms, d(-)-alanine replaces vitamin B. under some conditions (Snell, 1945a). $ For certain organisms, the four purine bases, adenine, guanine, xanthine, and hypoxanthine, are interchangeable, but various organisms differ in the ease with which they utilize the individual compounds. For Leuconostoc mesenteroides, adenine alone is almost inactive (Snell and Mitchell, 1941, 1942); guanine or xanthine is readily available. For organisms such as L. arabinosus, purine bases are not required if adequate amounts of p-aminobenzoic acid are present in the medium. Similarly, organisms such as S. faecalis or L. casei do not require the purine bases if adequate amounts of folic acid are present in the medium. Thymine was originally found to permit growth of S. faecalis (S. lactis R) in a medium which contained no folic acid (Snell and Mitchell, 1941). The same medium with thymine omitted was later used for the assay of folic acid (Mitchell and Snell, 1941). The effect of thymine upon L. casei is also observed only in the absence of folic acid (Stokstad, 1941), which it replaces more or less completely when purine bases are also present in the medium. to their quantitative detection in natural materials. One of the first tests to be adapted to such quantitative use was that using L. casei for the determination of riboflavin (Snell and Strong, 1939b). This method has been widely tested

6 378 ESMOND E. SNELL and adopted, and is now recognized as an official method by the American Association of Agricultural Chemists and the the U. S. Pharmacopoeia. The reasons for its success are readily apparent. The test organism used is nonpathogenic and can be handled with impunity. Its response to riboflavin can be accurately and conveniently followed by titrating with standard alkali the lactic acid which it produces dug growth; or, if special equipment is available, TABLE 2 A complete medium for lactic acid bacteria* SUBSTANCE ADDED AOUNT PER LrTERl Acid hydrolyzed casein, charcoal treated g Sodium acetate g Glucose g Cystine g Tryptophane g Asparagine g Glutaminet g Riboflavin...; pg Calcium pantothenate g Nicotinicacid.500 pg Thiamine hydrochloride.200 pg p-aminobenzoicacid.200 pg Biotin pug Folic acid (eluate factor, vitamin B.) pg Pyridoxinehydrochloride.1,000 pg Adenine sulfate.10 mg Guanine hydrochloride.10 mg Xanthine.10 mg Uracil.10 mg Mineral salts * Adapted from Snell and Rannefeld (1945). t Filtered and added aseptically. Growth of most commonly used organisms of this group occurs without this addition. Both glutamine and asparagine can be dispensed with if small quantities (0.5 g) of a charcoal-treated tryptic digest of vitamin-free casein are added. $ Much smaller amounts of pyridoxal or pyridoxamine may be used with as good or better result (Snell and Rannefeld, 1945). Five-tenths grams K2HPO4, 0.5 g KH2PO4, 0.2g MgSO4*7H20,0.01 g NaCl,0.01 g FeSO4-7H20, and 0.01 g MnSO4.4H20 per liter. Other trace elements may be required; if so, they are present in sufficient quantity as contaminants in the other ingredients of the medium, or are furnished by the culture vessel. turbidimetric determinations of growth yield accurate results. The method is of unparalleled sensitivity as compared with either animal assay or chemical procedures; only small samples are necessary to furnish the minute amounts of riboflavin required by the organism. When necessary, the method canbe made even more sensitive by cutting the scale upon which it is carried out, without impairing its accuracy (Lowry and Bessey, 1944). As comparedwith animal assay, the method is incomparably faster, more reliable, and more economical. It is well

7 NUTRITIONAL REQUIREMENTS OF LACTIC ACID BACTERIA adapted to routine use. Finally, when adequate precautions in the preparation of samples are taken, the method has proved reliable by every test applied. The advantages listed are shared to a greater or lesser degree by other procedures which utilize these organisms for determining the vitamins. Such procedures were rapidly developed and studied as knowledge of the nutritional requirements of the organisms permitted formulation of media free from specific vitamins but adequate in other respects. Nicotinic acid (Snell and Wright, 1941), pantothenic acid (Pennington et al., 1940), folic acid (Mitchell and Snell, 1941), p-aminobenzoic acid (Lewis, 1942), biotin (Wright and Skeggs, 1944b) and thiamine (Sarett and Cheldelin, 1944) may at present all be determined with organisms of this group. Reference to table 3 shows that one is not limited to any specific organism in developing such methods, but at present L. casei and L. arabinosus are the most widely used. For the determination of biotin, TABLE 3 Vitamin requirements of various species of lactic acid bacteria* LACTOBACIL- LACTOBACIL- LACTOBACILk STREPTO- LEUCONOS- LACTOBAC I- LUS DEL- LUS EER- LUS ARABI- COCCUS TOC MESEN- LUS CASEI BRIuECKI 3 ENTum 36 NOSUS 17-5 FAECALS R TEROPES P.60 Riboflavin Pantothenic acid Nicotinic acid Biotin Vitamin B _ + Thiamine.- _ + Folic acid * Determined in parallel on media similar to that of table 2 but lacking in one appropriate vitamin. The p-aminobenzoic acid requirements of these organisms were not determined. A "+" sign indicates that no growth is obtained in the absence of the vitamin; a "-)) sign indicates that the substance is not required for growth. Frequently, a nonessential substance stimulates rate of growth, although the final amount of growth obtained is not affected. folic acid, and pantothenic acid, microbiological methods are the only rapid procedures available at the present time. With the development of chemically defined media for these organisms they have been applied to the determination of the various amino acids which they require (see above). Here the general lack of sensitive chemical procedures has made the microbiological methods of special interest. In instances investigated so far, results of great promise have been obtained, and if preliminary expectations are realized, significant advances in our knowledge of the amino acid composition of rare proteins and biological materials of all kinds will become possible (cf. Snell, 1945b). Occasionally, microbiological assays of this type yield apparently anomalous results, the explanation of which involves fundamental findings of a biochemical nature. Vitamin B6 is a case in point. Since the isolation and synthesis of

8 380 30ESMOND E. SNELL pyridoxine in 1939, it has been assumed that this substance fully accounted for the vitamin Be activity of biological materials. When the "pyridoxine" content of such materials was determined by a procedure involving comparative growth response of certain lactic acid bacteria to pyridoxine and the material in question, unreasonably large figures were obtained (Snell, Guirard, and Williams, 1942). Elucidation of the reasons for this behavior led to the eventual recognition and synthesis of two new substances with vtamin B1 activity, which were named pyridoxamine and pyridozal (Snell, 1944a, 1944b). Both of these compounds occur naturally and possess from several hundred to several thousand times the activity of pyridoxine for lactic acid bacteria (Snell and Rannefeld, 1945; Snell, 1945c). Their recognition has led, in turn, to a partial elucidation of the catalytic role which vitamin B6 plays in living organisms (Gunsalus and Bellamy, 1944; Schlenk and Snell, 1945). In summary, study of the nutrition of lactic acid bacteria has led to the following results: (a) the discovery of three substances which may function as vitamins for both these organisms and higher animals, i. e., folic acid (L. casei factor), pyridoxal, and pyridoxamine, and contributions to the isolation of a fourth vitamin, pantothenic acid; (b) the development of methods of unsurpassed ensitivity for the quantitative determination of seven of the watersoluble vitamins; and (c) the development of sensitive and accurate methods for the quantitative determination of several amino acids, a development which shows great promise of future results. It is now possible to grow many organisms of this group in chemically defined media, which should permit more intimate study of the details of their metabolism. However, substances which are stimulatory or essential for growth of certain of these organisms on media similar to that of table 2 remain to be identified (Wright and Skeggs, 1944a; Snell, 1945a; Cheldelin et al., 1945). It may be expected that further study of such substances will lead to additional findings of interest to both the biochemist and the bacteriologist. REFERENCES CHELDELIN, V. H., RIGGS, T., AND SARzTT, H. P A growth factor for Lactobacillu gayonii. Federation Proc., 4, 85. DUNN, M. S., SHANKMAN, S., CAMIEN, M. N., FRANKL, W., AND ROCKLAND, L. B Investigations of amino acids, peptides, and proteins. XVIII. The amino acid requirements of Leuconostoc mesenteroides P-60. J. Biol. Chem., 156, GuNsAlus, J. C., AND BELLAMY, W. D A function of pyridoxal. J. Biol. Chem., 155, IsBEELJ, H Effect of p-aminobenzoic acid on the microbiological assay for nicotinic acid. J. Biol. Chem., 144, LEWIS, J. C A lactobacillus assay method for p-aminobenzoic acid. J. Biol. Chem., 148, Loway, 0. H., AND BESSEY, 0. A A method for the determination of five-tenths to two miulimicrograms of riboflavin. J. Biol. Chem., 155, MITCHELL, H. K., AND SNELL, E. E Assay method for "folic acid." Univ. Texas, Pub. 4137, MICHELL, H. K., SNELL, E. E., AND WILLIAMS, R. J The concentration of "folic acid." J. Am. Chem. Soc., 63, 2284.

9 NUTRITIONAL REQUIREMENTS OF LACTIC ACID BACTERIA MOELLER, E. F Vitamin B. (Adermin)als Wuchstoff ffir physiol. Chem., 254, M ilchsfiurebakterien. Z. MOELLER, E. F Das Wuchstoffsystem der Milchsiiurebakterien. Z. phyoiol. Chem., 260, NEAL, A. T., AND STRONG, F. M. 1943Microbiological determination of pantothenic acid. Further studies.ind. Eng. Chem., Anal. Ed., 15, NIVEN, C. F., JR Nutrition of Streptococcus lactis. J. Bact., 47, ORLA-JENSEN,S The lactic acid bacteria. Mem. acad. roy. sci. Danemark (Sec. Sci.), VIII, 5, ORLA-JENSEN, S., AND JACOBSEN, J Neue Untersuchungen ueber die bakteriziden Eigenschaften dermilch. Zentr. Bakt. Parasitenk.,II, 80, ORLA-JENSEN, S.,OTTE, N. C., AND SNOG-KJAER, A. 1936a Der Vitaminbedarf der Milchsaurebakterien. Zentr. Bakt. Parasitenk., II, 94, ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAER, A. 1936b DieStickstoffnahrung der Milchsiurebakterien. Zentr. Bakt. Parasitenk., II, 94, PENNINGTON, D., SNELL, E. E., AND WILLIAMS, R. J An assay method for pantothenic acid. J. Biol. Chem., 135, SARETT, H. P., AND CHELDELIN, V. H The use of Lactobacillus fermentum 36 for thiamine assay. J. Biol. Chem., 155, SCHLENK, F., AND SNELL, E. E Vitamin B. and transamination. J. Biol. Chem., 157, SNELL, E. E. 1944a The vitamin activities of "pyridoxal" and "pyridoxamine." J. Biol. Chem., 154, SNELL, E. E. 1944b The vitamin Be group. I. Formation of additional members from pyridoxine and evidence concerning their structure. J. Am. Chem. Soc., 66, SNELL, E. E. 1945a The vitamin Be Group. VII. Replacement of vitamin Be for some microorganisms by d(-)-alanine and an unidentified factor from casein. J. Biol. Chem., 158, SNELL, E. E. 1945b The microbiological assay of amino acids. Advances in Protein Chem., 2, Academic Press, New York. SNELL, E. E. 1945c The vitamin B. group. IV. Evidence for the occurrence of pyridoxamine and pyridoxal in natural products. J. Biol. Chem., 157, SNELL, E. E., AND MITCHELL, H. K Purine and pyrimidine bases as growth substances for lactic acid bacteria. Proc. Nat. Acad. Sci. U.S., 27, 1-7. SNELL, E. E., AND MITCHELL, H. K Some sulfanilamide antagonists as growth factors for lactic acid bacteria. Arch. Biochem., 1, SNELL, E. E., AND PETERSON, W. H Properties of a new growth factor for lactic acid bacteria. J. Biol. Chem., 128, xciv-xcv. SNELL, E. E., AND PETERSON, W. H Growth factors for bacteria. X. Additional factors required by certain lactic acid bacteria. J. Bact., 39, SNELL, E. E., AND RANNEFELD, A. N The vitamin B, group. III. The vitamin activity of pyridoxal and pyridoxamine for various organisms. J. Biol. Chem., 157, SNELL, E. E., AND STRONG, F. M. 1939a The effect of riboflavin and of certain synthetic flavins on the growth of lactic acid bacteria. Enzymologia, 6, SNELL, E. E., AND STRONG, F. M. 1939b A microbiological assay for riboflavin. Ind. Eng. Chem., Anal. Ed., 11, SNELL, E. E., AND WRIGHT, L. D A microbiological method for the determination of nicotinic acid. J. Biol. Chem., 139, SNELL, E. E., GUIRARD, B. M., AND WILLIAMS, R. J Occurrence in natural products of a physiologically active metabolite of pyridoxine. J. Biol. Chem., 143, SNELL, E. E., STRONG, F. M., AND PETERSON, W. H Growth factors for bacteria. VI. Fractionation and properties of an accessory factor for lactic acid bacteria. Biochem. J., 31,

10 382 32SMOND E., SNELL SNELL, E. E., STRONG, F. M., AND PETERSON, W. H Pantothenic and nicotinic acids as growth factors for lactic acid bacteria. J. Am. Chem. Soc., 60, SNELL, E. E., STRONG, F. M., AND PETERSON, W. H Growth factors for bacteria. VIII. Pantothenic and nicotinic acids as essential growth factors for lactic and propionic acid bacteria. J. Bact., 38, SNELL, E. E., TATum, E. L., AND PETERSON, W. H Growth factors for bacteria. III. Some nutritive requirements of Lactobacillus delbrueckii. J. Bact., 33, SPEAxMN, H. B Molecular configuration in the sugars and acid production by Bacillu granulobacter pectinovorum. J. Biol. Chem., 58, STILLER, E. T., KEREszTTEY, J. C., AND FIxzELsTEIN, J Pantothenic *cid. VI. The isolation and structure of the lactone moiety. J. Am. Chem. Soc., 62, TEPLEY, L. J., AND ELVEHJEM, C. A The titrimetric determination of "Lactobacilluw casei factor" and "folic acid." J. Biol. Chem., 157, 303,309. W Iis, R. J The chemistry and biochemistry of pantothenic acid. Advances in Enzymol., 3, Interscience Publishers, Inc., New York. WILLIs, R. J., TRuESDAIL, J. H., WEINSTOCK, H. H., JR., ROHRANN, E., LyiAN, C. M., A.ND MCBumquy, C. H Pantothenic acid. II. Its concentration and purification from liver. J. Am. Chem. Soc., 60, WOOLLEY, D. W Manganese and the growth of lactic acid bacteria. J. Biol. Chem., 140, WmGHT, L. D., ArND SEOGGs, H. R. 1944a The growth factor requirements of certain streptococci. J. Bact., 48, WRIGHT, L. D., AND S1zu:s, H. R. 1944b Determination of biotin with Lactobacillus arabinosue. Proc. Soc. Exptl. Biol. Med., 56,