Amino Acid Utilization by Alcaligenes viscolactis

JOURNAL OF BACrERIOLOGY, June, 1965 Copyright a 1965 American Society for Microbiology Vol. 89, No. 6 Printed in U.S.A. Amino Acid Utilization by Alcaligenes viscolactis for Growth and Slime Production1 J. D. PUNCH, J. C. OLSON, JR., AND J. V. SCALETTI2 Departments of Dairy Industries, Animal Husbandry, and Microbiology, University of Minnesota, St. Paul and Minneapolis, Minnesota Received for publication 3 March 1965 ABSTRACT PUNCH, J. D. (University of Minnesota, St. Paul), J. C. OLSON, JR., AND J. V. SCA- LETTI. Amino acid utilization by Alcaligenes viscolactis for growth and slime production. J. Bacteriol. 89:1521-1525. 1965.-The ability of Alcaligenes viscolactis to utilize amino acids in a basal salts solution (K2HPO4, KH 2PO4, MgSO4, MnSO4, FeSO4, NaCl) was studied. Of 27 amino acids, only L-asparagine, L-glutamic acid, L-aspartic acid, L-glutamine, L-proline, and L-tyrosine supported growth sufficient to give a viscous supernatant solution. L-Proline and L-tyrosine, singly or combined, fulfilled the carbon, nitrogen, and energy requirements for growth and slime production. None of eight inorganic nitrogenous compounds supported growth with lactose as the carbon source. The addition of L-asparagine, L-aspartic acid, or L-glutamine to L-tyrosine or L-proline, singly or combined, did not increase growth or slime production, indicating no nutritional interactions among these amino acids. Neither lactose nor glucose was found to be required or utilized by A. viscolactis in a medium containing basal salts, L-proline, L-tyrosine, and lactose or glucose. This was established by the fact that total carbohydrate and total reducing activity remained constant during growth and slime production. The chemically defined nutritional requirements for growth and slime production by Alcaligenes viscolactis have not been determined. Long and Hammer (1936) used a complex agar medium containing milk in their characterization of A. viscosus (A. viscolactis). Nutrient agar plus 1% glucose was used by Gainor and Wegemer (1954a, b). Jones (1954b) reported that A. vtiscolactis would grow in a basal salts medium containing 0.2 g of asparagine, 0.2 g of sodium lactate, and 2.0 g of glucose per liter. He stated, however, that albumin was required for vigorous growth and slime production. Stamer and VanDemark (1961) used an 8%O whey agar for growth and slime production by this bacterium, although they indicated that lactose, sodium lactate, peptone, and calcium phosphate were sufficient for slime production. Evidence is presented in this communication that A. viscolactis may be cultivated in a chemically defined medium and that neither lactose or glucose, nor complex nitrogen-containing substances such as albumin, peptone, or whey are required for growth and slime production. 1 Scientific Journal Series Paper No. 5399, Minnesota Agricultural Experiment Station, St. Paul. 2 Present address: Department of Microbiology, Medical School, University of New Mexico, Albuquerque. In connection with studies on the physical and chemical nature of the slime produced by A. viscolactis, we became interested in the nutritional requirements of this species. It is apparent that the availability of a chemically defined medium would obviate some of the problems inherent in the purification of the slime material and, in addition, facilitate studies related to its biosynthesis. With a complex medium, we were always faced with the problem of constituents of such a medium being occluded and tenaciously held in the slime, thus complicating purification. Accordingly, a study is reported on the utilization of various inorganic and organic nitrogenous compounds by three strains of A. viscolactis and the establishment of a chemically defined medium adequate for growth and slime production. MATERIAL AND METHODS Cultures. Three cultures of A. viscolactis were studied: 73, from the Dairy Bacteriology culture collection of the University of Minnesota; a culture obtained from P. J. V'anDemark of Cornell University, New York, in 1963; and 9036, from the American Type Culture Collection. All three cultures conformed to the characteristics of A. viscolactis, as given in Bergey's Manual, except that No. 73 grew and produced ropy milk at 5 C but not at 37 C, and did not hsdrolyze milk fat. Cultures were maintained on nutrient agar slants at 4 C. 1521

1522 PUNCH, OLSON, AND SCALETTI J. BACTERIOL. Vitamin Free Casamino Acids medium. This medium contained (per liter) Vitamin Free Casamino Acids, 10 g; DL-tryptophan, 0.2 g; and cysteine, 0.2 g. The ph was adjusted to 7.0 prior to sterilization. Basal salts. The basal salts solution contained (per liter) K2HPO4, 5 g; KH2PO4, 3 g; Mg++, 3 X 10-3 M as MgSO4.7H20, 0.74 g; Mn++, 2 X 10-4 M as MnSO4-H20, 0.34 g; Fe++, 2 X 104 M as FeSO4-7H20, 0.056 g; and NaCl, 2 X 10-4 M, 0.012 g. When media were prepared containing basal salts solution, the MgSO4*7H20 was sterilized separately and added aseptically after sterilization to the rest of the particular medium. Supplements to basal salts. Lactose, glucose, peptone (Difco), yeast extract (Difco), Vitamin Free Casamino Acids (Difco), and various inorganic and organic nitrogenous compounds were used to supplement the basal salts solution. Concentrations and combinations of these additives are given in the Results section. All nitrogenous compounds were sterilized separately and added aseptically. Inoculum. The inoculum was prepared from a 24-hr culture grown at 20 C in the Casamino Acids medium. A 1:10 dilution of this culture was made in phosphate buffer (3.1 X 104 M, ph 7.0); 0.1 ml of this dilution was used as the initial inoculum for the various test media. After incubation for 48 hr, 0.1 ml of culture from each respective medium was used to inoculate fresh test media. Subsequently, all measurements were made at the end of 48 hr of incubation. Incubation. All media were dispensed in 20-ml amounts to 150-ml Erleuimeyer flasks. This provided adequate aeration for A. viscolactis during stationary incubation. Preliminary experiments indicated that slime was not produced when the culture was agitated even at a moderate rate. Incubation was at 20 C for 48 hr. Agitation, when used, was accomplished with an Eberbach reciprocating shaker (100 strokes per min). Measurements. Growth was measured with a Klett-Summerson colorimeter with a 42-filter (420 m,). Vliscosity measurements were determined at 30 C with an Ostwald viscometer. Relative viscosities are reported as the ratio, time for sample-time for control medium. Total carbohydrate was determined by the method of Dubois et al. (1956). The procedure of Nelson (1944) and Somogyi (1952) was used to measure total reducing activity. RESULTS A series of media containing basal salts, with and without lactose, and various inorganic and organic nitrogen sources was prepared (Table 1). All amino acids and inorganic nitrogen compounds were added at the rate of 2 X 10-2 M, except for DL-amino acids, in which case the concentration was doubled (4 X 10-2 M) to give a 2 X 10-2 M concentration of the L-form. The concentration each of yeast extract, peptone, and Vitamin Free Casamino Acids was 0.2%. Results obtained after inoculation of each medium with A. viscolactis are given in Table 1. Of the 27 TABLE 1. Growth and slime production by Alcaligenes viscolactis 73 in various synthetic media* Nitrogenous compound 0.5% lactose No lactose Klett value ph Relaostivy Relative Klett lt value vle ph viscosity Relative L-Asparagine... 83 7.1 1.40 64 7.1 1.32 L-Aspartic acid... 73 7.1 1.25 69 7.1 1.24 L-Citrulline... 37 7.0 1.00 12 7.0 1.00 L-Glutamic acid... 94 7.2 1.44 121 7.2 1.47 L-Glutamine..... 110 7.2 1.50 130 7.2 1.57 L-Proline... 198 7.6 2.49 192 7.5 2.38 L-Tyrosine...19.194 7.5 2.58 195 7.6 2.44 L-Lysinet...0... 7.0 1.00 0 7.0 1.00 (NH4)2SO4t... 0 7.0 1.00 Yeast extract... 143 7.4 2.06 149 7.6 2.14 Peptone... 158 7.4 2.17 162 7.6 2.20 Casamino Acids... 164 7.5 2.18 157 7.5 2.09 Control (basal salts)... 0 7.0 1.00 0 7.0 1.00 * Basal salts plus additives indicated. t Identical results were obtained for cysteine HCl, glycine, L-histidine-HCl, DL-isoleucine, L-leucine, DL-methionine, DL-norvaline, DL-norleucine, DL-serine, DL-threonine, and DL-valine. Klett readings ranging from 1 to 8 with no change in ph or viscosity, with or without lactose, were obtained with: DL-alanine, L-arginine, L-cystine, glutathione, L-hydroxyproline, L-ornithine, DL-phenylalanine, and DL-tryptophane. t Identical results were obtained for: ammonium chloride, NH20H-HCI, thiourea, sodium nitrate, sodium nitrite, ammonium nitrate, and sodium ammonium phosphate.

VOL. VAMINO 89, 1965 ACID UTILIZATION BY A. VISCOLACTIS 1523 amino acids tested, only L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, L-proline, and L-tyrOsine supported growth sufficient to give any appreciable turbidity, alkaline reaction, and a viscous supernatant solution. All three complex additives, peptone, yeast extract, and Casamino Acids, supported good growth and slime formation. An alkaline reaction was produced as growth and slime formation progressed. L-Citrulline gave very slight growth with no increase in ph or viscosity. Greatest turbidity and viscosity were observed in the flasks containing L-tyrosine and L-proline. The inclusion of lactose did not increase growth or viscosity. None of the inorganic nitrogenous compounds tested was able to support growth with lactose as the carbon and energy source. In all cases, with the exception of L- citrulline, growth was accompanied by slime production. In a subsequent experiment, it was observed that somewhat better growth and slime production was obtained with L-proline at 4 X 10-2 M concentration than with 2 X 10-2 M concentration; however, for L-tyrosine, L-asparagine, and L-glutamine, growth and slime production were not enhanced by increasing the concentration above 2 X 102 M. Furthermore, no significant increase or decrease in growth and slime formation occurred throughout six serial transfers at 48-hr intervals in media containing basal salts plus either L-proline or L-tyrosine. Thus, the ability of these amino acids to support sustained growth was established. Since L-proline and L-tyrosine yielded significantly more growth and viscosity than did yeast extract, peptone, or Vitamin Free Casamino Acids (Table 1) at comparable levels (weight basis, 2.3, 3.6, 2.0, 2.0, and 2.0 g per liter, respectively), one might conclude that only specific nitrogenous compounds contained in the complex additives were utilized; furthermore, preformed growth factors probably are not necessary. The latter was verified by an experiment in which various growth factors (0.002 g per liter each of adenine sulfate, p-aminobenzoic acid, calcium pantothenate, folic acid, guanine. HCI, nicotinic acid, pyridoxine -HCl, pyridoxal, pyridoxalamine, riboflavine, thiamine, uracil, xanthine, biotin, choline, inositol, lipoic acid, niacin, and ascorbic acid) were added singly and combined to a basal salts-iproline medium. No detectable increase in growth or viscosity over that which occurred in the absence of growth factors was obtained. As a check on possible nutritional interactions among the amino acids that resulted in the most abundant growth and slime production, a study was made of growth and viscosity resulting from TABLE 2. Growth and slime production by three cultures of Alcaligenes viscolactis in a medium containing basal salts + L-proline and L-tyrosine with and without lactose No lactose 0.5% lactose Culture Cult ett ph Relative Klett H Relative value P viscosity value P viscosity 73 240 7.6 3.00 237 7.6 3.00 9036 232 7.6 2.82 236 7.6 2.83 Cornell 223 7.5 3.04 214 7.6 2.90 various combinations of L-proline, L-tyrosine, L-asparagine, L-aspartic acid, and L-glutamine added to basal salts solution. It was observed that 4 X 10-2 M L-proline with 10-2 M L-tyrosine supported growth and viscosity equivalent to that obtained with 2 X 10-2 M each of the four amino acids combined. Additions of L-asparagine, L-aspartic acid, and L-glutamine to L-proline or L-tyrosine, singly or combined, did not stimulate or increase growth or viscosity. The data in Table 1 and the above observations suggested that lactose was not required for either growth or slime production. Further evidence in support of this is shown by the data in Table 2. As indicated, all three cultures of A. viscolactis gave equivalent growth and viscosity readings in media containing basal salts, L-proline (4 X 10-2 M), and L-tyrosine (10-2 M) with or without added lactose. To determine whether lactose or glucose was being metabolized or utilized in some way by A. viscolactis, the following experiment was conducted. Four flasks, two containing basal salts, i-proline (4 X 10-2 M), L-tyrosine (10-2 M), and 0.5% lactose, and two containing basal salts, L-proline (4 X 10-2 M), L-tyrosine (10-2 M), and 0.5% glucose were inoculated with culture 73. One flask containing lactose and one containing glucose were incubated under stationary conditions. The other set was agitated during incubation. Preliminary studies indicated that good growth occurred but no slime was synthesized when cultures were agitated during growth. The total carbohydrate and total reducing activity were determined at zero-time and after 24 and 48 hr of incubation by use of a sample from which the cells had been removed by centrifugation at 15,000 X g for 20 min. As shown in Table 3, the total carbohydrate and total reducing activity remained constant except for a possible slight increase in the 48-hr samples taken from the stationary flasks. This slight increase may be attributed either to the incomplete removal of the cells or to some carbo-

1524 PUNCH, OLSON, AND SCALETTI J. BACTERIOL. TABLE 3. Total carbohydrate and reducing activity in synthetic media* supernatant liquids from stationary and agitated cultures of Alcaligenes viscolactis Medium containing glucose Medium containing lactose Incubation Stationary Agitated Stationary Agitated RAt TCt Kiett ph RA TC Klet ph RA TC Klett ph RA TC Kiett ph hr._ 0 5,200 5,300 0 7.0 5,100 5,200 0 7.0 3,100 6,100 0 7.0 3,100 6,200 0 7.0 24 5,000 5,100 93 7.1 5,000 5,300 210 7.2 3,000 6,100 97 7.0 2,900 6,000 197 7.1 48 5,400 5,400 232 7.7 5,200 5,100 337 7.3 3,300 6,400 227 7.6 3,000U6,100 323 7.3 * Basal salts + L-proline and L-tyrosine + glucose or lactose. t Total reducing activity expressed as micrograms of glucose equivalents. t Total carbohydrate expressed as micrograms of glucose equivalents. hydrate material present in the slime which formed during stationary incubation. These possibilities are indicated since, with agitation, good growth but no increase in viscosity (no slime) was observed, and in these flasks no detectable change occurred either in total carbohydrate or total reducing activity. From these data, one can conclude that A. viscolactis can utilize L-proline or L-tyrosine as the sole sotirce of nitrogen, carbon, and energy, and that neither lactose nor glucose is required. DISCUSSION These studies show that certain individual amino acids satisfy the carbon, nitrogen, and energy requirements of A. viscolactis for growth and slime production. This makes it possible to proyvide a simple, chemically defined dialyzable medium useful for study of the physiological processes of slime formation by this organism, and in obtaining relatively pure slime, uncomplicated by occluded medium constituents, for study of its chemical composition. Among the amino acids utilized, proline and tyrosine supported the most abundant growth and slime formation. It is interesting to note that milk, a common habitat for A. viscolactis, is rich in both proline and tyrosine, as are the complex additives used by previous investigators in studying this species. The specificity of A. viscolactis for certain amino acids is reflected by the fact that only 6 of the 27 tested were actively metabolized. Furthermore, L-hydroxyproline and L-phenylalanine, structurally related to L-proline and L-tyrosine, respectively, were not utilized. The inability of.4. viscolactis to utilize lactose and glucose either oxidatively or fermentatively under the conditions described in this study is apparent. It has been reported by other investigators that A. viscolactis uses common carbohydrates oxidatively. For example, Long and Hammer (1936) stated that lactose was utilized in the synthesis of a polysaccharide slime, although they presented no evidence that lactose was utilized or that the slime was a polysaccharide. Gainor and Wegemer (1954a, b), Jones (1954a, b, c), and Stamer and VanDemark (1961) used complex media containing lactose and organic nitrogenous compounds in their studies of growth and slime production by this organism. As shown in the present study, lactose or glucose was not required for growth and slime production. Further studies are in progress to determine the physical and chemical characteristics of the slime produced by A. viscolactis. ACKNOWLEDGMENT This investigation was supported by the Minneapolis-St. Paul Quality Control Committee. LITERATURE CITED DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS, AND F. SMITH. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28:350-356. GAINOR, C., AND D. E. WEGEMER. 1954a. Studies of a psychrophilic bacterium causing ropiness in milk. I. Morphological and physiological considerations. Appl. Microbiol. 2:95-97. GAINOR, C., AND D. E. WEGEMER. 1954b. Studies of a psychrophilic bacterium causing ropiness in milk. II. Chemical nature of the capsular material. Appl. Microbiol. 2:97-99. JONES, D. 1954a. Ropy milk. I. The biology of rope production. Food Res. 19:246-249.

VOL. 89, 1965 AMINO ACII) UTILIZATION BY A. VISCOLACTIS 1525 JONES, D. 19546. Ropy milk. II. Influence of physiological environment on the formation of ropy substance. Food Res. 19:250-253. JONES, D. 1954c. Ropy milk. III. Chemical studies of ropy substances. Food Res. 19:254-256. LONG, H. F., AND B. W. HAMMER. 1936. Studies on Alcaligenes viscosuis. Iowa State J. Sci. 10:261-265. NELSON, N. 1944. A photometric adaption of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380. SOMOGYr, M. 1952. Notes on sugar determinations. J. Biol. Chem. 195:19-23. STAMER, J. R., AND P. J. VANDEMARK. 1961. Studies on slime formation by Alcaligenes viscolactis. Bacteriol. Proc., p. 67.