NUTRITIONAL REQUIREMENTS OF LACTOBACILLUS 30a FOR GROWTH AND HISTIDINE DECARBOXYLASE PRODUCTION

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 2, p February, 1964 Copyright 1964 by the American Society for Microbiology Printed in U.S.A. NUTRITIONAL REQUIREMENTS OF LACTOBACILLUS 30a FOR GROWTH AND HISTIDINE DECARBOXYLASE PRODUCTION BEVERLY M. GUIRARD AND ESMOND E. SNELL Departmlent of Biochemistry, University of California, Berkeley, California Received for publication 23 September 1963 ABSTRACT GUIRARD, BEVERLY MI. (University of California, Berkeley), AND ESMOND E. SNELL. Nutritional requirements of Lactobacillus 30a for growth and histidine decarboxylase production. J. Bacteriol. 87: The nutritional requirements of Lactobacillus 30a include each of the naturally occuriing amino acids, several B vitamins, ascorbic acid, glucose, acetate, and oleate. The nutritional requirements for optimal histidine decarboxylase production (up to 900,liters of CO2 per hr per rng of cells) differ to some extent from those for optimal growth. Wholly synthetic and partially defined media are described which produce high enzyme activity, together with rapid and luxuriant growth. The high histidine decarboxylase activity of Lactobacillus 30a, an isolate from horse stomach, was discovered by Rodwell (1953a). He also developed a semidefined medium (vitamins, salts, easein hydrolysate) for growth of this organism (Rodwell, 1953b). In studies of its histidine decarboxylase, we experienced difficulty in consistently obtaining cells of this organism in high yield and with high decarboxylase activity. A more detailed study of its nutritional requirements was, therefore, undertaken. MATERIALS AND METHODS Stock culture. Stock cultures of the organism were originally carried on litmus milk or on Briggs' (1953) medium for lactobacilli. At present, stock cultures are transferred weekly on the semidefined medium C, developed during this study (Table 3), solidified with 2% agar. Inactive stocks have been held for long periods by lyophilization. Assay procedure. Customary procedures employed in nutritional studies with lactic acid bacteria were used (Snell, 1950). Nutritional supplements to be tested were diluted to 3 ml with deionized water, 3 ml of double-strength medium were added, and the culture tubes were capped and autoclaved for 7 min at 121 C. An inoculum culture, previously grown on medium C for 16 to 18 hr at 40 C, was centrifuged, and the cells were washed once with sterile water. The density of the suspension was adjusted to 0.04 mg (dry weight) of cells per ml by reference to a previously constructed reference curve relating dry weight of cells to optical density. One drop of this suspension was added to each experimental culture tube, and the assay was incubated at 40 C for 16 hr. Growth response was measured photometrically at 660 (Evelyn colorimeter) or 650 m, (Bausch & Lomb colorimeter). An uninoculated tube of medium set at 100% transmission served as the reference point. illeasurement of histidine decarboxylase activity. The cells were harvested by centrifugation, washed, and resuspended in water at a cell density of 1 mg/ml. Carbon dioxide was measured manometrically in a Warburg apparatus at 37 C. The main compartment contained 1 ml of the aqueous cell suspension, 1 ml of 0.2 M ammonium acetate buffer (ph 4.8), and 0.5 ml of water. A 5-mg amount of histidine hydrochloride dissolved in 0.5 ml of water was placed in the side arm. Gas evolution was recorded at 5-min intervals for a period of 15 min after the substrate was dumped into the main compartment. The same results were obtained whether the gas phase was air or nitrogen; consequently, an air atmosphere was used for routine manometric measurements. Decarboxylase activity is expressed in terms of Qco2 values (microliters of CO2 evolved per hour per milligram of dry cells). RESULTS AND DISCUSSION Growth on defined media. In preliminary experiments, a semidefined medium similar to that used by Rodwell (1953a), but with hypoxanthine and ascorbic acid added, served as the basal medium. By omitting each component of this medium singly and varying its concentration over 370

2 VOL. 87, 1964 NUTRITION OF LACTOBACILLUS 30a 371 TABLE 1. Composition and essential components of synthetic medium A for growth of Lactobacillus 30aa Amt required Amt in final Amt required Amt in final mal growvth mal growth mdu Component for near maxi- medium Component for near maxi- medium mg/6 ml mg/6 ml amt/6 ml amli6 ml Amino acidsb Inorganic salts-cont'd. DL-Alanine... O FeSO 4*7H20...Ọ 01 mg 0.06 mg L-Arginine MnSO4*H20... O. 01 mg mg r,-asparagine Vitamins L-Cysteine * HCl p-aminobenzoic acid ,Ag L-Glutamic acidd Biotin c..... O.0O O.012,ug Glycine Folic acid... O.01,ug 0.1,ug L-Histidine*HCl Nicotinic acid... O.8,ug 10.0 Ag DL-Isoleucine Riboflavine 1... O A,g 2.4 lag DL-Leucine Thiamine 0... O. 1.2,ug L-Lysine * HCl c Calcium pantothen- DL-Methionine ate... 1.O,g 4.8,ug DL-Phenylalanine Pyridoxamine*2HCI ,ug 0. 1,ug L-Proline Ascorbic acide 3.0 mg DL-Serine DL-Threonine DL-Tryptophan Miscellaneous i,-tyrosine Glucose mg 60 mg DL-Valine Tween-oleatef ml 0.06 ml Inorganic salts Hypoxanthine ug 200,g Salts A (0.1 g each of Uracil.80.0 /Ag 100,ug KH2PO4 and K2HPO4 Guanine ,ug per ml) ml 0.10 ml Adenine ,g MgSO4 7H mg 3.0 mg Sodium acetate... O.1 mg 10 mg NaCl mg Potassium acetate - 10 mg ph a The nutrients required for growth can be identified from columns 2 and 5, where the amounts of each required for near maximal growth (80 to 100% of maximum as determined from dose-response curves for that nutrient) at 16 hr are tabulated. Columns 3 and 6 give the amounts included in the final medium. b DL-Amino acids were employed where indicated as a matter of convenience; only the L-amino acids are required for growth. 6 Stimulatory but not essential (see text). d Sample was twice recrystallized. Commercial glutamic acid is often contaminated with methionine; in the present experiments, a requirement for methionine could not be demonstrated until purified glutamic acid was used in the medium. e Required as a reducing agent, not as a vitamin (see text). f A solution containing 2 g of Tween-40, of sodium oleate, and water to 20 ml. g The requirement for these substances displayed by the original culture is no longer found in the culture in current use. a range, an approximation of the nutritional requirements was obtained. The hydrolyzed casein of this semidefined medium was then replaced by a mixture of pure amino acids. Each component of this completely defined medium was then individually varied as before to determine its essentiality and the amount required for optimal growth in the presence of all other components of the medium. Where necessary, concentrations were altered and the process was repeated. The final medium evolved is shown in Table 1. A few compounds that proved neither essential nor stimulatory for growth of this organism, but are frequently required by related organisms, were also added as indicated. On this medium, the optimal ph for growth was 6.5 and the optimal temperature was 40 C, in agreement with the findings of Rodwell

3 '372 GUIRARD ANI) SNELL J. BACTERIOL. (1953a). The cell yield (dry w-eight) obtained in 16 hr oin the complete mediumi-was 0.3 ing/mil. As is evident from Table 1, the organism is unusually demanding in its nutritional needs. Certain of these requirements are discussed briefly below. Amino acid requirements. The organism did not grow on the defined medium if any one of the amino acids, lysine excepted, was omitted. Although not essential, lysine increased the rate of growth during short ( <16 hr) growth periods. Chromatographic and microbiological tests indicated that lysine was not a contaminant of any of the other amino aci(ds of the medium; consequently, Lactobacillus 30a must synthesize lysine slowly. The organism displayed a remarkable tolerance for high concentrations of most amino acidsonly D)L-methionine, DL-serine, DL-isoleucine, DL-phenylalanine, and L-cysteine being consistently toxic at 10 mg/ml. Toxicity due to this concentration of phenylalanine could be prevented completely by increasing the tryptophan content of the medium to 1 mg per 6 ml of culture; none of the other amino acids was effective in preventing the inhibition. Several amino acids, but chiefly alanine and histidine, contributed town-ard preventing DL-serine toxicity. The inhibition produced by high levels of L-cysteine was not prevented by amino acids but was prevented by increasing the concentration of the mixture of vitamins. The most effective single component of the mixture in this respect was pyridoxamine. Vitamin requiirements. Folic acid, nicotinic acid, riboflavine, pantothenate, and vitamin B6 were essential for growth of this organism, and biotin was slightly stimulatory. Pantethine replacedl pantothenate, but about 30 times as much was required to effect the same growth response. The lack of a biotin re(quiremeent for the organism may result from the presence in the medium of asparagine and oleic acid, which reduce or eliminate completely the biotin requiremeat of sev-eral related bacteria (Broquist and Snell, 1951). Both oleate and asparagine were required for grow-th, and biotin, although slightly stimulatorv in the presence of both of these compounds, did not support growth in the absence of either, even at high concentrations. Aspartic acid exerted a slight sparing action on the asparagine requirement in the absence, but not in the presence, of biotin. Oleate may be replaced for this organism by high levels (50 to 100 mg per 6 ml of culture) of an extract of autolyzed yeast, but not by sodium pyruvate or an atmosphere of carbon dioxide. In this respect, its unsaturated fatty acid requirement differs from that of some other species of lactobacilli and the "minute" streptococci (Deibel and Niven, 1955). All forms of vitamin B6 tested supported growth of Lactobacillus 30a, but the concentrations required for optimal growth differed markedly. The most active form, pyridoxamine phosphate, supported optimal growth at a concentration of 2.92 m,um ( m,moles per 6 ml of medium). Pyridoxamine, pyridoxal-5'-phosphate, pyridoxal, and pyridoxine supported similar growth at concentrations 6, 11, 28, and 14,000 times higher, respectively. In contrast to many other lactic acid bacteria (see Holden and Snell, 1949), the requirement for vitamin B6 was not eliminated by addition of D-alanine to a complete assortment of L-amino acids. Folic acid proved slightly more active than folinic acid in supporting growth. Thymidine, but not thymine, partially replaced the folic acid requirement wnhen added to the complete medium. If glucose was sterilized by filtration and added to previously autoclaved medium, little or no growth occurred unless a reducing agent (or compounds giving rise to reducing agents on heating with the medium) was added. The requirement was nonspecific; ascorbic acid and isoascorbic acid were the most effective supplements under these conditions (Table 2). Purine and pyrimiidine requirements. Hypoxanthine was required by the culture studied initially; however, during 6 years of transfer on laboratory media, the requirement for this compound was lost. Uracil stimulated growth, but addition of other purine and pyrimidine bases, of a crude enzymatic digest of deoxyribonucleic acid, or of an alkaline hydrolysate of ribonucleic acid did not further improve growth. Inorganic ion requirenments. No attempt was made to remove contaminating inorganic ions from the medium. The inorganic ion requirements indicated in Table 1 are those demonstrable in the presence of cations and anions supplied as contaminants in the medium. Under the assay conditions, K+, P04-3, Mg+2, and Mn+2 were essential, and Fe+2 was sometimes stimula-

4 VOL. 87, 1964 NUTRITION OF LACTOBACILLUS 30a 373 tory. The requirement for potassium ion was verified by preparing media containing no added K+ or NH4+ (all Na+), or containing no added Na+ or K+ (all NH4+). No growth occurred in either medium unless K+ was added; the potassium requirement on the "all NH4+" medium was higher than that on the "all Na+" medium (see MacLeod and Snell, 1948); excellent growth was obtained also in an "all K+" medium containing neither added Na+ or NH4+..V1iscellaneous requirements. Lactose was not utilized as a carbon source by this organism, but glucose was fermented readily. Its carbohydrate utilization, morphology, and growth characteristics resembled those of L. delbrueckii and L. leichmannii strains. The requirement for oleate was noted. Tween 40 (polyoxyethylene sorbitan palmitate), in the absence of oleate, did not support growth, whereas Tween 80 (polyoxyethylene sorbitan oleate) did permit growth. Tween 40 was retained in the medium because of its effect in detoxifying and promoting availability of unsaturated fatty acids for certain lactic acid bacteria (Williams, Broquist, and Snell, 1947). The growth response to acetate was erratic, varying in different trials from complete dependence to complete independence of its presence in the medium. However, when the potassium phosphate solution (salts A) was autoclaved separately from the other components of the medium, growth was obtained only when acetate was added. Appropriate trials revealed that the interfering material was produced in significant amounts only when glucose was autoclaved with phosphate. Shockman (1956) reported similar findings in studying the acetate requirement of Streptococcus faecalis. The possibility that acetic acid was formed from glucose during the heating process was tested. An autoclaved mixture of glucose and inorganic phosphate was steam-distilled under reduced pressure. A portion of the distillate was oxidized with HgSO4 to quantitatively remove formic acid (Friedemann, 1938), a recognized product of heat degradation of carbohydrates (Auclair and Portmann, 1957; Gould, 1945). The treated distillate, when concentrated to a small volume, supported growth of the test organism in the absence of added acetate, and when chromatographed on paper (Kennedy and Barker, 1951) gave a single acidic zone (RF = 0.27) similar to that given by acetic acid (RF = 0.30). Formate, TABLE 2. Growth response of Lactobacillus 30a to ascorbic acid and related compounds* Amt (mg Per cent Addition per 6 ml transmissiont of culture) (16 hr) Ascorbic acid Isoascorbic acid Dehydroascorbic acid Sodium thioglycolate Sodium pyruvate Glyceraldehyde t * The substances to be tested were appropriately diluted with water in the culture tubes and heated in flowing steam for 10 min. The basal medium without glucose was autoclaved separately and then cooled; filter-sterilized glucose was added aseptically, and the medium was dispensed to the tubes which were inoculated as usual. 100% transmis- t Uninoculated medium reads sion. + Toxic at higher level tested. It-I 300 Acetate alon 40X z -- + Acetate 0 Mevolonic acid - ) 50- (log) LO +Aceae/, < 60 K / r 708_ 0 / 90 I!" "'-~ S30 AC4TAE o Lipoic acid (10 tg) SODIUM ACETATE, 6p z9g per 6m FIG. 1. Growth response of Lactobacillus 3Oa to acetate in the presence and absence of lipoic or mevalonic acids. propionate, and higher homologues of the fatty acid series have no growth-promoting activity under these conditions. The active substance produced by autoclaving inorganic phosphate with the complete medium or with glucose thus appears to be acetic acid. 100

5 374 GUIRARD AND SNELL J. BACTERIOL IIII i" f,0t ai/ ~IenB "~~~~~~~~~1 100 A_ - * * X,/.~~~~~1~00n,// HOURS INCUBATED AT 40 C FIG. 2. Variation of growth (solid lines) and histidine decarboxylase activity (dotted lines) with time on (A) the synthetic mediumn of Table 1, (B) this same medium supplemented with 1 mg of yeast extract per 6 ml, and (C) the simplified medium of Table 3. Lipoic acid was inactive in replacing acetate and inhibited the utilization of acetate (Fig. 1). Mevalonic acid was also inactive and had no sparing action on acetate utilization. The response to acetate appears to be sufficiently specific, sensitive, and reproducible to serve as a microbiological assay for this substance. Although small amounts of acetate satisfied the growth requirement (Fig. 1), substantially larger amounts of sodium and potassium acetate were included in the growth medium for their buffer action. Growth on a medium containing the amounts of each component listed in columns 2 and 5 of Table 1 was substantially improved when all of the components were doubled in concentration. This effect could be duplicated by increasing the concentrations of the amino acids and salts A. The final concentration of each component in the medium is given in Table 1 (columns 3 and 6). All of the components except glucose, ascorbic acid, and the vitamin solution may be combined in one stock solution and preserved under benzene or toluene in the cold. Freshly prepared solutions of these ingredients were added to the stock solution just before use. The organism has been subcultured indefinitely on this medium without evidence of diminished growth. The rate of growth on this medium (A, Fig. 2) was improved slightly by addition of yeast extract (B, Fig. 2) and, during the first few hours, by replacing most of the amino acids with a casein hydrolysate (C, Fig. 2). After 10 to 15 hr of incubation, however, growth on medium C lagged behind that on media A and B (Fig. 2). Effect of composition of the medium on histidine decarboxylase production. Cells grown on each of the media of Fig. 2 showed Qco, values for histidine decarboxylase up to 900 (Fig. 2), approximately three times higher than those reported by Rodwell (1953b). Maximal decarboxylase production was aehieved slightly before or at the time of maximal growth. Because of the relative simplicity of its preparation and its lower cost, medium C (Table 3) was used for growing large quantities of cells as a source of histidine decarboxylase. TABLE 3. Semideftned medium C for culture of Lactobacillus 30a Component Glucose... Commercial acid-hydrolyzed casein (salt free)*... DL-Tryptophan... L-Cysteine HC1... L-Asparagine... L-Histidine HCI... Adenine... Guanine... Uracil... Hypoxanthine... Potassium acetate... Sodium acetate 3H20... Potassium dihydrogen phosphate... Dipotassium hydrogen phosphate... Magnesium sulfate heptahydrate... Sodium chloride... Ferrous sulfate 7H20... Manganese sulfate H20... Tween Sodium oleate... p-aminobenzoic acid... Biotin... Calcium pantothenate... Folic acid... Nicotinic acid... Riboflavine... Thiamine... Pyridoxamine... Ascorbic acid... * Nutritional Biochemicals Corp., Ohio. Amt per liter of strength doublemedium 20 g 16.7 g 33 mg 167 mg 167 mg 500 mg 10 mg 10 mg 33 mg 67 mig 400 mg 2.0 g 400,ug 4.0,g 3.3 mg 33,g 3.3 mg 800 pag 400 pag 33,ug 1.0 g Cleveland,

6 VOL. 87, 1964 NUTRITION OF LACTOBACILLUS 30a 375 TABLE 4. Effect of concentration of individual components of the medium on histidine decarboxylase activity of Lactobacillus 30a Amt per 6 ml Cell yield Amt per Cell yield Compound varied of culture Qco, (mg per Compound varied 6 ml of QCo2 (mg per 6 ml) culture 6 ml) A. Vitamins Pyridoxamine Folic acid Nicotinic acid Calcium pantothenate Sodium ascorbate B. Amino acids Histidine Serine g 0.005,ug 0.1,ug 0.3,g 0.001,ug 0.002,ug 0.03,ug 0.1,ug 1.0,ug 0.05,Ag 0.1,ug 2.4 jug 10. O,ug 0.1,g 0.5 ug 4.8,ug 10.0,g 3.0 mg 0.3 mg 3.0 mg 10.0 mg 0.3 mg 5.0 mg C. Inorganic salts Salts A* Salts Bt MgSO4 7H20 D. Miscellaneous Glucose Acetate Adenine ml 0.01 ml 0.1 ml 0.3 ml ml 0.01 ml 0.03 ml 0.1 ml 0.05 mg 0.5 mg 1.2 mg 3.0 mg 10 mg 30 mg 60 mg 100 mg 10.0 mg 20.0 mg 50.0 mg ug 500,ug 1,000,g * Salts A: 0.1 g of KH2PO4 and 0.1 g of K2HP04 per ml. t Salts B: 40 mg of MgS047H20, and 2 mg each of NaCl, MnS044H20, and FeSO4-7H20 per ml. Production of cells with high specific activity was dependent upon the presence in the growth medium of adequate concentrations of pyridoxamine (see Guirard and Snell, 1954), folic acid, nicotinic acid, calcium pantothenate (or pantethine), ascorbic acid, potassium phosphate, magnesium sulfate, glucose, acetate, adenine, histidine, and serine (Table 4). Indeed, any nutrient so far tested that limits growth appears also to limit production of the enzyme, an indication that growth takes precedence over histidine decarboxylase production. Histidine continued to increase enzyme production even at the highest level tested, where its presence discolored the growth medium. Adenine, though not required for growth (Table 1), was required for high enzyme production. Acetate improved enzyme production at levels much higher than were required for growth. Although maximal cell yields were obtained when the initial ph of the medium was between 5.6 and 7.0, enzyme production reached maximal values only when the initial ph was at the lower end of this range (Table 5). The final ph achieved at 16 hr in each case was near 4.1. Finally, several comparative experiments in which intact cells grown under a variety of conditions were compared with cell extracts prepared by ultra-

7 376 GUIRARD AND SNELL J. BACTERIOL. TABLE 5. Effect of the initial ph of the growth medium on histidine decarboxylase activity of Lactobacillus SOa Initial ph Qco, Cell yield 2 (mg/6 ml) * * An additional experiment in which the ph was varied between 5.0 and 6.0 fixed the optimum at sonic irradiation showed only about 10% increase in Qco2 values on cell breakage. The effects of nutrients on QcO2 values noted herein thus represent actual effects on enzyme production, and not on cell permeability. ACKNOWLEDGMENT This study was supported in part by grant A-1448 from the U.S. Public Health Service. LITERATURE CITED AUCLAIR, J. E., AND A. PORTMANN Formic acid as a growth stimulant for Lactobacillus lactis in autoclaved milk. Nature 179: BRIGGS, M An improved medium for Lactobacilli. J. Dairy Res. 20: BROQUIST, H. P., AND E. E. SNELL Biotin and bacterial growth. I. Relation to aspartate, oleate, and carbon dioxide. J. Biol. Chem. 188: DEIBEL, R. H., AND C. F. NIVEN, JR Reciprocal replacement of oleic acid and CO2 in the nutrition of the "minute" streptococci and Lactobacillus leichmanii. J. Bacteriol. 70: FRIEDEMANN, T. E The identification and quantitative determination of volatile alcohols and acids. J. Biol. Chem. 123: GOULD, I. A The formation of volatile acids in milk by high temperature heat treatment. J. Dairy Sci. 28: GUIRARD, B. M., AND E. E. SNELL Pyridoxal and metal ions as cofactors for histidine decarboxylase. J. Am. Chem. Soc. 76: HOLDEN, J. T., AND E. E. SNELL The relation of D-alanine and vitamin B6 to growth of lactic acid bacteria. J. Biol. Chem. 178: KENNEDY, E. P., AND H. A. BARKER Paper chromatography of volatile acids. Anal. Chem. 23: MACLEOD, R. A., AND E. E. SNELL The effect of related ions on the potassium requirement of lactic acid bacteria. J. Biol. Chem. 176: RODWELL, A. W. 1953a. The occurrence and distribution of amino acid decarboxylases within the genus Lactobacillus. J. Gen. Microbiol. 8: RODWELL, A. W. 1953b. The histidine decarboxylase of a species of Lactobacillus; apparent dispensability of pyridoxal phosphate as coenzyme. J. Gen. Microbiol. 8: SHOCEMAN, G. D The acetate requirement of Streptococcus faecalis. J. Bacteriol. 72: SNELL, E. E Microbiological methods in vitamin research, p In P. Gy6rgy [ed.], Vitamin methods, vol. 1. Academic Press, Inc., New York. WILLIAMS, W. L., H. P. BROQUIST, AND E. E. SNELL Oleic acid and related compounds as growth factors for lactic acid bacteria. J. Biol. Chem. 170: