Amino Acid Requirements of Acetobacter Suboxydans J

Home , Alanine (data page), Isoleucine, Isoleucine (data page), Norleucine

AMINO ACID REQUIREMENTS OF ACETOBACTER SUBOXYDANS J. L. STOKES AND ALMA LARSEN Research Laboratories, Merck & Co., Inc., Rahway, N. J. Received for publication January 16, 1945 Acetobacter suboxydans is of considerable interest because of its remarkable inability to dissimilate organic carbon compounds beyond the intial stages of oxidation (Visser't Hooft, 1925). This property of A. suboxydans and the fact that large amounts of substrate are transformed have suggested use of the organism for the large-scale production of various organic compounds. The oxidation of sorbitol to sorbose, which is used in the synthesis of vitamin C, is a well-established commercial process. It has been necessary to add yeast extract or other similar organic nitrogenous complexes to media for A. suboxydans, as a source of unknown growth factors and nitrogen. Recently Underkofler, Bantz, and Peterson (1943) established that the required growth factors are pantothenic, nicotinic, and p-aminobenzoic acids. This has made it possible to determine for the first time the specific nitrogen requirements of A. suboxydans. METHODS The strain of A. suboxydans was obtained from the University of Wisconsin. Stock cultures were maintained on yeast extract glycerol agar slants. The inoculum was prepared as described by Underkofler et al. (1943) except that the cells were washed once with water. One drop (ca. 0.05 ml) was used to inoculate 10 ml of medium contained in a 50-ml Erlenmeyer flask. Cultures were in- cubated at 30 C for 48 hours unless otherwise indicated. Growth was measured in an Evelyn photoelectric colorimeter. The basal medium consisting of glycerol, hydrolyzed casein as nitrogen source, inorganic salts, and growth factors was that described by Underkofler et al. (1943) except that when amino acids, (NH4)2SO4, or yeast extract was used as a source of nitrogen in place of hydrolyzed casein, tryptophane and cystine were omitted. All media were adjusted to pH 6.0. The 20 amino acids (Merck) employed included synthetic leucine, isoleucine, valine, methionine, phenylalanine, glutamic acid, threonine, alanine, aspartic acid, lysine, serine, norleucine, and glycine. Those isolated from natural sources were cystine, tryptophane, tyrosine, arginine, histidine, proline, and hydroxyproline. Each 10 ml of medium received 2 mg of the natural amino acids and 4 mg of the synthetic amino acids. A larger amount of the latter was used since it was considered probable that only one of the two isomers would be available to A. suboxydans. EXPERIMENTS In agreement with the results of Underkofler et al. (1943) a mixture of the 20 amino acids listed above could replace hydrolyzed casein for growth of A. 495 496 J. L. STOKES AND ALMA LARSEN suboxydans. To determine which amino acids of the 20 are required, each one was omitted in turn from the medium and the effect on growth noted. Valine is essential since no growth occurred in its absence (table 1). Growth was slight without isoleucine or alanine. The need for isoleucine may be absolute since the small amount of growth obtained in its absence may be due to a small amount of isoleucine present as an impurity in the synthetic leucine of the medium (Hegsted and Wardell, 1944). Somewhat less than maximum development occurred when histidine, cystine, glutamic acid, aspartic acid, proline, or hydroxyproline was omitted. These amino acids can be classified as stimulatory. The remaining amino acids are not required by A. suboxydans since maximum growth occurred without them. On repeating this experiment, similar results were obtained except that cystine and aspartic acid, in contrast to their previous slight stimulatory action, had no effect on growth.

TABLE 1 Effect of omission of individual amino acidefrom the "20 amino acid medium" upon the growth of Acetobacter suboxydan8

ESSENTIL PROBABLY ESSENTIAL STIMULATORY NONESSENTIfAL

Leucine ...... 31 Valine...... 98* Isoleucine...... 68 Histidine ...... 45 Methionine ...... 32 Alanine...... 72 Cystine.37 Tryptophane . 30 Glutamic acid..... 41 Tyrosine. 33 Aspartic acid..... 38 Phenylalanine. 28 Proline...... 39 Threonine 30 Hydroxyproline... 38 Lysine. 33 Arginine 32 Serine 28 Norleucine 29 Glycine 33 * Per cent transmissible light of cultures grown without the amino acid indicated; un- inoculated medium = 100. Cultures grown with all 20 amino acids gave a reading of 32. Although valine, isoleucine, and alanine are essential components of the nitrogen requirements of A. suboxydans, growth did not take place in a medium containing only those three amino acids. This was not due to an insufficiency of nitrogen since a threefold increase of each amino acid did not alter the results. The further addition of histidine permitted slight growth, which increased on continued incubation as described below. The same effect was obtained also with proline but not with any of the other amino acids. Glutamic acid, aspartic acid, or hydroxyproline, when added to the four amino acids, did not materially increase growth. However, on the addition of either of the sulphur-containing amino acids, cystine and methionine, to the medium containing valine, isoleucine, alanine, and histidine, A. suboxydans developed fairly well although less than with the 20 amino acids. This marked effect of cystine and methionine is in sharp contrast to their inactivity in the previous experiment in which each amino acid, in turn, was omitted from the 20 amino acid mixture (table 1). In that AMINO ACID REQUIREMENTS OF ACETOBACTER SUBOXYDANS 497 experiment, methionine was present in the medium when cystine was omitted and vice versa, so that the effect of each was masked by the presence of the other. The apparent nonessentiality of a particular amino acid in this type of experiment may, therefore, simply mean that some other remaining amino acid may serve the same but, nevertheless, necessary physiological function. On the con- trary, absence of growth when one amino acid of the mixture is omitted is fairly conclusive evidence that the omitted amino acid is required for growth. Growth with the 20 mino acids was about equal to that obtained with hydrolyzed casein.I The cultures in the medium containing valine, isoleucine, alanine, and histidine read only 94 after 48 hours of incubation, and this decreased to 78 on incubation for 3 additional days. Although growth was slow, sparse, and occurred in smnall clumps, all indicative of an unfavorable environment, A. suboxydan8 was successfully subcultured through six serial transfers in the same medium. TABLE 2 Growth of Acetobacter suboxydane with various combinations of amino acids

% TRANSMISSIBLE LIGHT AMINO ACIDS IN BASAL MEDIUM_ Exp. 1 Exp. 2 Valine + isoleucine + alanine + histidine + cystine (A)... 52 50 (A) + proline...... 29 34 (A) + hydroxyproline ...... 38 37 (A) + serine...... 32 40 (A) + phenylalanine ...... 41 37 20 Amino acids...... 29 29 Hydrolyzed casein...... 17 27

The combination of valine, isoleucine, alanine, and histidine represents, therefore, the smallest number of amino acids which can consistently support some growth of A. suboxydans. Growth was raised to the same level as with 20 amino acids by the addition of proline to the medium containing valine, isoleucine, alanine, histidine, and cystine (table 2). Hydroxyproline, serine, and phenylalanine were only slightly less effective than proline. The somewhat limited specificity of the latter suggests that it may be utilized by A. suboxydans for the synthesis of the 15 or more other amino acids normally present in cellular proteins rather than directly as a structural building block. The remainiTng amino acids were substantially inactive when substituted for proline. It is thus established that the nitrogen requirements of A. suboxydans are satisfied by a mixture of six amino acids, namely, valine, isoleucine, alanine, histidine, cystine, and proline. The medium containing these six amino acids, which supported, on repeated serial subculture, as much growth as the medium containing 20 amino acids, was used in the following experiments. ' Prepared from S. M. A. Corp. "vitamin-free" casein by hydrolysis with H2S04. 498 J. L. STOKES AND AMA LAMRSEN Effect of amino acid concentration and ammontum sufate. Increasing the concentration of the six amino acids in the basal medium from the customary 2 mg of the naturally occurring isomer per 10 ml of medium to 2,3, or 4 times this amount did not appreciably increase the amount of cell substance. However, use of less than 2 mg caused a reduction in growth (table 3). With 0.01 to 0.1 mg of each amino acid, little or no growth occurred. The decrease may be due to a critical reduction of one or more of the essential amino acids, but probably not all. Nitrogen supply was a limiting factor under these conditions since addition of (NH4)S04 led to better growth. The effect of (NH4)2S04 Was decisive at the lowest amino acid concentrations, which alone failed to support development of A. suboxydans. At these low concentrations it was necessary to extend the incubation period to 4 days to obtain maximlm effect of (NIL)2SO4. There is no doubt, therefore, that A. suboxydans can utilize NH4-nitrogen for growth provided it is supplied with the 6 amino acids which it is unable to syn- thesize or which are formed too slowly for normal development.

TABLE 3 Influence of amino acid concentration and (NH4)2SO4 on the development of Acetobacter suboxydan8 in the "6 amino acid medium"

MOUNT 07 EACH GROwT AlTER 2 DAYS GRowTH AlTER 4 DAYS AMINO ACID PER .___ .- 10XL OF ~ D Without (NE)42S04 With (NHQ)2SO4 Without NH)2SO4 With (NH)2OS4 mg* Per ceng trexsmissible light 2.0 33 24 25 21 1.0 43 30 36 27 0.5 51 40 47 36 0.1 91 81 87 65 0.05 94 90 93 62 0.01 94 93 96 60 * In terms of the l-isomer. (NH4)2S04 could not be substituted for any of the 6 o acids required by A. suboxydans. Influence of nitrogen source 'on rate and type of growth. When bactoyeast extract (0.4 per cent) was used as a source of nitrogen, A. suboxydans multiplied more rapidly and somewhat more extensively than with the six amino acids (figure 1). The lag phase with the latter lasted for about 12 hours compared to 6 hours with yeast extract. It was not reduced by the addition of asparagine, glutamine, glucose, or (NH4)2S04, although a combination of 0.1 per cent of the latter two substances increased total growth slightly. Also, substitution of mannitol or sorbitol for glycerol, addition of reducing agents (sodium thiogly- collate and ascorbic acid), or use of inoculum grown in the amino acid medium did not affect either the rate or amount of growth in the amino acid medium. Delayed growth was noted both in stationary and agitated cultures. During the period of rapid increase, however, the rates in both media were approximately equal. AMINO ACID REQUIREMENTS OF ACETOBACTER SUBOXYDANS 499 Growth with yeast extract occurred uniformly throughout the medium, and within 48 hours usually a delicate, fragile, surface membrane was evident. In contrast, initial growth in the synthetic amino acid mediumn adhered to the bottom and sides of. the flask, leaving the body of the medium almost entirely clear. Later, a well-defined, surface pellicle formed, which could be dispersed only by vigorous shaking. The pellicle- contained most of the cells of the culture. This type of surface growth was almost always obtained and appears to be characteristic for the synthetic amino acid medium. Deamination of amino acids by resting cell suspensions. Cells were obtained from approximately 20-hour cultures grown on yeast extract glycerol agar medium. They were washed and suspended in M/15 phosphate buffer at pH

S0 J1o aj YEAST EXTtACT VW 30 40. AMN AC-IDS

z - w 70

'U, lop HOUORS OF INCUBATION FIG. 1. RATE OF GROWTH OF A. suboxydanfs IN SYNTHETIC AMINo ACID AND YEAST ExTRACT MEDIA 6.7 to give a reading of 3 on the photometer. Five ml of cell suspension (equivalent to 32 mg of dry cells) and 5 ml of buffer containing an amount of amino acid equivalent to 2 mg of nitrogen of the natural isomer were mixed in a large tube fitted for aeration. One-tenth ml of tributyl citrate was added to reduce foaming, and a slow stream of either air or nitrogen was bubbled through the mixture (placed at 37 C) for 16 hours. The ammonia liberated was determined by distillation into N/70 HCl and titration of excess acid with N/70 NaOH. Under aerobic conditions, 10 of the amino acids were deaminated either com- pletely or more than 50 per cent (table 4). More than 100 per cent of the nitrogen in alanine, aspartic acid, and serine, calculated in terms of the 1-isomer, was obtained as NH-N, indicating that both enantiomorphs of those amino acids were attacked. Proteus and Pseudomonas aeruginosa, under similar conditions, also decompose both isomers of alanine and serine (Bernheim, Bernheim, and .500 J. L. STOKES AND ALMA LARSEN Webster, 1935; Wpbster and he, 1936). Seven of the amino acids were dearninated to the extent of 50 per cent or les. Little or no NH was obtained from. leucine, isoleucine, or vaine. Since -the latter two amino acids.also are required,. preformed, for growth of A. sboydans. it is probable.that they.are utilized forsynthesis. of protein without extewsive mo ficftiQn. Anaerobically, little or no d tion ocourred with any of the io acids withf the exception ofasrine both isomers f which were attacked. According to these results, A. suboxydan decoiposes amino acids primarily by a process of.oxidative d nation. This is in contrast to the data of Miyaji (1925) which indicate. that other acetic acid bacteria reductively deaminate glycine and tyrosine, but is perhaps more in.accord with the highly aerobic character of these organism.

TABLE 4 Aerobic deamination of amino acids by resting cell suspensions of Acetobacter 8uboxydans

PM CENT NITROGUN W RD AS I1 100-50 S0-10 10-0 Glutamic acid Cystine Leucine Aspartic acid Methionine Isoleucine Alanine Tyrosine Valine Lysine Phenylalanine Arginine Threonine Tryptophane Norleucine Histidine Glycine Serine Proline Hydroxyproline

SUMMARY The amino acids required by Acetobacter uboxydans for growth were determined. The organism multiplies to a limited extent in a medium consisting of glycerol, salts, essential growth factors, and a combination-of valine, isoleucine, alanine, and histidine as nitrogen source. Growth is considerably improved by the addition of either cystine or methiorLine. The further addition of proline increases growth to the level obtainable with a mixture of 20 amino acids or hydrolyzed casein. However, development is not so rapid and somewhat less extensive than with yeast extract. At suboptimum concentrations of the six required amino acids, (NH4)2S04 stimulated growth. Resting cell suspensions of A. suboxydans deaminate most amino acids under aerobic conditions. Both optical isomers of alanine, serine, and aspartic acid are attacked. Under anaerobic conditions only serine is deaminated to any appreciable extent. AMINO ACID REQUIREMENTS OF ACETOBACTER SUBOXYDANS 501

REFERENCES BERNHEIM, F., BERNHEIM, M. L. C., AND WEBSTER, M. D. 1935 Oxidation of certain amino acids by "resting" Bacillus proteus. J. Biol. Chem., 110, 165-172. HEGSTED, D. M., AND WARDELL, E. D. 1944 On the purity of dl-leucine. J. Biol. Chem., 153, 167-170. MIYAJI, K. 1925 Products of decomposition of amino acids by acetic bacteria. J. Chem. Soc. Japan, 45, 391-450. Cited by Bernhauer, K. 1938 Biochemie der Essigbak- terien. Ergeb. Enzymforsch., 7, 246-280. UNDERKOFLER, L. A., BANTZ, A. C., AND PETERSON, W. H. 1943 Growth factors for bacteria. XIV. Growth requirements of Acetobacter suboxydans. J. Bact., 45, 183- 190. VISSER'T HOOFT, F. 1925 Biochemische onderzoekingen over het geslacht Acetobacter. Thesis, Technische Hoogeschool, Delft. WEBSTER, M. D., AND BERNHEIM, F. 1936 Oxidation of amino acids by Bacillus pyo- cyaneus (Pseudomonas aeruginosa). J. Biol. Chem., 114, 265-271.