growth-stimulating factors were added in the yeast extract and CROWN-GALL ORGANISM' tumefaciens (Smith and Town.) Bergey et al., have been examined

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1 THE ROLE OF CERTAIN VITAMINS AND METALLIC ELEMENTS IN THE NUTRITION OF THE CROWN-GALL ORGANISM' F. C. McINTIRE, A. J. RIKER, AND W. H. PETERSON Departments of Biochemistry and Plant Pathology, University of Wisconsin Received for publication July 12, 1940 The growth factors and minor metallic elements required for the best development of the crown-gall organism, Phytomona8 tumefaciens (Smith and Town.) Bergey et al., have been examined in contimuing studies on its nutrition. The importance of sources of nitrogen and carbon and certain physical chemical factors has been shown by several investigators (e.g., Sagen et al., 1934, Pinckard, 1935). They worked mostly with media containing animal or plant extracts which doubtless supplied the materials that were necessary only in minute quantities. With a suitable synthetic medium (Van Lanen, Baldwin and Riker, unpublished) and an adequate means of growing the cultures so as to secure reproducible results (McIntire, Peterson and Riker, 1940) the way was opened to study various growth-stimulating factors and their synthesis by these bacteria. The synthetic medium supported fair growth when suitably aerated, but growth was greatly improved by the addition of yeast extract. The purpose of this study was to determine what growth-stimulating factors were added in the yeast extract and whether any of these substances could be synthesized by the bacteria. 1 Published with the approval of the director of the Wisconsin Agricultural Experiment Station. This work has received support through grants from the International Cancer Research Foundation and from the special research fund of the University of Wisconsin, and has been aided by the personnel in the University of Wisconsin W.P.A. official project no The figure was prepared by Eugene Herrling. 1

2 2 McINTIRE, RIKER AND PETERSON EXPERIMENTAL WORK Materials and methods The culture, P. tumefaciens A-6, was the progeny of a single cell (Wright et al., 1930). Pathogenicity was checked at intervals by inoculation of tomato plants. The basal synthetic medium (McIntire, Peterson and Riker, 1940) contained nutrient salts, glutamic acid, sucrose, and phosphate buffer of ph 7. As supplements thereto the following were used singly or in various combinations: aqueous yeast extract, yeast ash, thiamin, riboflavin, pimelic acid, nicotinic acid amide, glutamine, inositol, vitamin B., biotin2, pantothenic acid2, a mixture of pure amino acids, iron, manganese, zinc, and copper. The amino acid mixture was that of Hutchings and Woolley (1939) and was used at their same concentration. A water extract of 2 grams of fresh bakers yeast per 100 ml. of medium was used as a standard growth-stimulating preparation. This was about the optimum concentration. Yeast ash was prepared by ashing the dry extract in a platinum crucible at dull red heat in an electric furnace. Ferric iron was added as ferric alum, ferrous iron as the ammonium sulphate compound. Solutions of these salts were acidified with hydrochloric acid and sterilized by filtration through a sintered glass disc. Manganese, zinc, and copper sulphates and other supplements were added after autoclaving. Thiamin was sterilized with a Seitz filter. Three types of cultures were used: (1) Ten ml. liquid in ordinary culture tubes, not aerated. (2) Ten ml. liquid in 50-ml. Erlenmeyer flasks, aerated by mechanical shaking at the rate of about 60 complete strokes per minute. To avoid contamination by splashing of the medium on the stoppers, 3-inch extension necks were attached to the flasks. To increase uniformity of aeration, cotton plugs were replaced with a double layer of sixply, oil-treated air-filter tissue. (3) Two hundred ml. liquid in 2 Concentrates of biotin and of pantothenic acid were prepared by Dr. P. M. West (West and Wilson (1939)) and by Dr. E. E. Snell (Snell, Strong, and Peterson, 1939) respectively.

3 NUTRITION OF CROWN-GALL ORGANISM 1-liter Erlenmeyer flasks, also fitted with extension necks and airfilter tissue, were aerated by mechanical shaking. The data are representative of at least three experiments, unless otherwise specified. Each 10 ml. culture was inoculated with 1 ml. of a washed cell suspension containing about 5,000 cells (by plate count) taken from a 24-hour growth on yeast-water-mannitol agar. Some of the 200 ml. cultures received a proportionately larger inoculum of washed cells and others received a 2' per cent inoculum of 24-hour liquid culture. Incubation was at 26 t 1 C. The growth in cultures of types (1) and (2) was measured by determining turbidity with the Evelyn photoelectric colorimeter after about 48 hours incubation. In cultures of type (3) growth was estimated by determining the amount of sugar fermented in 4 days. Sugar analyses were made by the method of Stiles, Peterson, and Fred (1926). Carbon dioxide produced was determined by the difference in total carbon between the beginning and the end of the experiment, since it is the only volatile metabolic product. Gum and cells were precipitated by adding 4 volumes of 95 per cent alcohol; the precipitate was collected by centrifuging and washed twice with 4: 1 alcohol. For carbon analysis, the precipitate was dried in a vacuum desiccator; for nitrogen, ammonia was removed by suspending the precipitate in 2 per cent sodium carbonate solution and evaporating to dryness at reduced pressure. Carbon was determined by the method of Heck (1929), and nitrogen by the Kjeldahl method. Effect of aeration on growth stimulation Stimulation of growth by yeast extract depended upon aeration of the medium as shown in table 1. Without aeration, growth was slow and yeast extract not very stimulative. In the aerated cultures containing yeast extract, growth began sooner and was more rapid than in the controls. Stimulative constituents of yeast extract In an attempt to show whether any of the well-defined bacterial growth factors were responsible for the stimulative effect of yeast 3

4 4 MCINTIRE, RIKER AND PETERSON extract, various known compounds were tested singly and in groups. Thiamin, riboflavin, pantothenic acid, nicotinic acid, biotin, pimelic acid, inositol, glutamine, and vitamin B. were tried. None of these was stimulative alone. Either thiamin or riboflavin with pantothenic acid gave a slight stimulation. These three together gave more stimulation than either pair, and the effect was consistent and appreciable (table 1). Other growth factors tested showed no consistent stimulative effects when added either alone or in various combinations with thiamin, riboflavin, TABLE 1 Effect of growth factors and amino acids on growth in aerated and unaerated cultures SUPI0MUN TO BASAL MEDIUM COLOBUTUB BEAD- ING-PUB CEM OW LIGHT TA1NUsITTID Test-tube cultures, not aerated None Yeast extract Cultures in 50-ml. flasks, aerated None Riboflavin Pantothenic acid Riboflavin + thiamin Riboflavin + pantothenic acid Thiamin + pantothenic acid Thiamin + riboflavin + pantothenic acid Amino acids Amino acids + thiamin + riboflavin + pantothenic acid.. 65 Yeast extract.53 * The smaller the figure the greater the turbidity. and pantothenic acid. Maximum effects were noted with 0.2 microgram of thiamin and riboflavin and 0.4 microgram of pantothenic acid per ml. Because of the slight stimulation obtained it was difficult to determine optimum concentrations with accuracy. A mixture of pure amino acids added to the synthetic medium was also stimulative as shown by table 1. The effects of the amino acids and growth factors were additive and together they probably account for a considerable part of the stimulative action

5 NUTRITION OF CROWN-GALL ORGANISM of yeast extract. Acid-hydrolyzed casein was approximately as effective as the amino-acid mixture. No attempt was made to determine which of the amino acids were most important. According to Van Lanen, Baldwin and Riker, (1940) a number of amino acids were stimulative to this organism at low concentrations but when used at higher concentrations some were stimulative, e.g., aspartic acid, and others were toxic, e.g., alanine. Growth stimulation probably could not be attributed to reducing substances in the supplements, since in no case was the oxidation-reduction potential of the medium appreciably altered. Lowering the oxidation-reduction potential by adding various amounts of sodium thioglycollate had no effect. Following these results with growth factors, studies were made in two directions, viz., (1) whether the bacteria are able to synthesize necessary growth factors in a synthetic medium, and (2) whether inorganic substances have a stimulating effect. Synthesis of growth factors by P. tumefacienm The fact that a few washed cells of P. tumefaciens will grow in a synthetic medium suggests that this organism has the ability to synthesize its own growth factors. This was studied in regard to biotin, pantothenic acid, riboflavin, and thiamin. The data of table 2 show that considerable quantities of these factors were produced when the organism was grown in the synthetic medium. The synthesis of biotin is very striking. Its concentration in composite samples from different groups of 4-day-old cultures was from 3500 to 6300 times that reported by Peterson et al. (1940) as supporting good growth of Clostridium butylicum (Beijerinck, Donker) No. 21. The concentration of pantothenic acid in a 4-day-old culture was approximately equal to that which Snell et al. (1939) found to be optimum for Lactobacillus casei E, Orla- Jensen. The concentration of riboflavin produced was about 60 times that reported by Snell et al. (1937) as optimum for L. casei e. The amount of thiamin in the dried cells of the crown-gall organism was about the same as Pavcek et al. (1938) found in bakers' yeast grown in a synthetic medium. Since certain of these growth factors are important in plant 5

6 6 MCINTIRE, RIKER AND PETERSON growth the possibility exists that their synthesis by these bacteria might contribute to stimulation of cell growth in the host plant. As further evidence of its self-sufficiency, this organism was carried through 31 successive generations in test-tube cultures in the basal medium. Transfers were made every 24 to 48 hours with a small wire loop. The thirty-first generation was as vigorous and pathogenic as stock cultures carried on yeast-water mannitol agar. The only detectable change in carbon metabolism as TABLE 2 Synthesis of growth factors by the crown-gall organism* GROWTH FACTOR QUAVITTY OF GROWTH FACTOR In coils In oel-free fer- in cel plus fer- Inoells mented medium mented modiumt microgrgm8 ams pe r ml microgrs per ml. gram dry weigtmirgrm Biotin Riboflavin Pantothenic acid None found Thiamin t * Biotin assays were made by L. E. McDaniel and J. 0. Lampen with C. butylicum no. 21 as the assay organism. Riboflavin assays were made by R. E. Feeney with the Snell-Strong microbiological method. Pantothenic acid assays were made by A. C. Dornbush with L. casei e as the assay organism. Thiamin was determined by Dr. Aaron Arnold with the chick assay method. These data are supported by at least two experiments. t Calculated from assays on cells and cell-free medium for pantothenic acid, direct determination of biotin and riboflavin. There was 0.69 gram of dry cells per liter of culture. t For thiamin assays the bacterial cells were dried in a vacuum while frozen. a result of the 31 transfers was a slight increase in the rate of sugar fermentation. A very small inoculum of this organism has given good growth in a medium containing only ammonium sulphate, nutrient salts, phosphate buffer, and highly purified sugar. Stimulation by inorganic materials Since the organic constituents of yeast extract which were tested accounted for only part of its stimulative action, the inorganic material in the extract was investigated. Figure 1 shows

7 NUTRITION OF CROWN-GALL ORGANISM the relative effects of yeast extract and ash from the same amount of extract. To account for this effect several metallic elements which might occur in yeast ash were tested, namely, iron, copper, manganese, and zinc. Of these, iron, manganese, and zinc were stimulative at proper concentrations (fig. 1). Approximately maximum 7 MEDIA FIG. 1. GROWTH STIMULATION BY INORGANIC MATERIALS AND YEAST EXTRACT growth was noted with ferric iron at about 5, manganese at 0.1, and zinc at 0.5 micrograms per ml. of medium. Ferric iron was better than ferrous iron. Concentrations of iron greater than 10 micrograms per ml. were often slightly inhibitory, especially ferrous iron. As shown in table 3 the stimulative effects of these metals are

8 8 McINTIRE, RIKER AND PETERSON somewhat interdependent. Zinc was stimulative only when both iron and manganese were also added to the medium. Furthermore, the stimulation by zinc was noted only in young cultures and in about 75 per cent of the trials. Cultures which showed a stimulation when 48 hours old showed no such effect when 4 days old. Thus, if zinc is essential for the growth of this organism the amount present in the basal medium is almost enough for maximum growth. In more recent experiments in which the basal medium was modified to contain only 0.1 per cent phosphate TABLE 3 Growth stimulation by metallic elements and the effect of age of culture* TURBIDITY O CUVTURBS-COLORIMBTXR RUADINGS SUGAR FURMUNTUD IN 4-DAY-OLD CULTURUS- SUP1'LBMBT TO BASA GRAMS N 100 ML. MUDIUR 48 hours old 96 hours oldt Experi- E:peri- Ezp.ri- Experi- Experi. ExperEpi mont ment2 menti ment2 monti ment2 None Fe Mn Zn Fe + Mn Fe + Zn Mn + Zn Fe +Mn + Zn * These were 200-ml. cultures in 1-liter Erlenmeyer flasks, inoculated with about 100,000 washed cells. t Diluted 1:4 for turbidity readings. buffer and 0.1 per cent ammonium sulphate, stimulation by zinc was obtained in 4-day-old cultures. Duplicate cultures supplemented with iron and zinc gave as much as 53 per cent increase in sugar fermentation over cultures supplemented with iron alone. In 48-hour cultures stimulation by manganese was observed in about 100 per cent of the trials when iron also was added but when manganese was added alone the results were irregular. In older cultures the stimulation by manganese was always obtained, even without added iron. This suggests that the stimulation by manganese may be limited by a lack of materials which may be syn-

9 NUTRITION OF CROWN-GALL ORGANISM thesized by the organism and whose synthesis is accelerated by the addition of iron. The stimulative effect of iron was obtained regularly regardless of whether manganese and zinc were added. A comparison of optimum concentrations of these metals for the crown-gall organism and the diphtheria organism (Mueller, 1938) shows that the former requires more iron and less manganese. The results agree with those of Thorne and Walker (1936) for rhizobia in the optimum concentration of iron and the superiority of ferric over ferrous iron. Copper gave no detectable stimulation in a wide range of concentrations. This does not indicate that the organism does not require copper, since the basal medium undoubtedly contained traces of this metal. As shown in figure 1, the stimulation by iron, manganese, and zinc together is of about the same order as that of yeast extract. Yeast ash plus iron and manganese is no more stimulative than just iron and manganese, which indicates that the other inorganic constituents of yeast extract either are not required or are present in sufficient quantities in the basal medium. However, yeast extract plus iron, manganese, and zinc is more stimulative than the three metals alone. This indicates that the organic material in yeast is stimulative even though the medium contains optimum concentrations of the inorganic materials. The data of table 1 suggest that amino acids, thiamin, riboflavin, and pantothenic acid are important in this effect. The influence of iron, manganese, and zinc on the products of metabolism Studies with various molds have shown that their metabolism may be altered by the presence of optimum concentrations of various metallic elements, especially zinc (Foster, 1939). The possibility of such effects with the crown-gall organism was therefore investigated and representative data are given in table 4. In contrast to its influence on certain molds, zinc had no noticeable effect on the metabolism of the crown-gall organism. Iron increased growth but had no apparent effect on the products of 9

10 10 McINTIRE, RIKER AND PETERSON metabolism. Manganese induced very marked quantitative (perhaps even qualitative) changes in the metabolism of this organism. Besides increasing the sugar fermentation, manganese produced roughly a 100 per cent increase in the percentage of fermented sugar converted to gum and cells with a corresponding decrease in the amount of unidentified products. Thus, when TABLE 4 The effect of iron, manganese, and zinc on the metabolism of the crown-gall organism* SUPLMUNT ADDS!D TO B3ASAL MUDIU None Fe Mn Zn Fe + MnF +Mu +Zn Carbon in fermented sugar, grams per 100 ml Total carbon metabolized, t grams per 100 ml Metabolized carbon accounted for as: Carbon dioxide, grams per 100 ml Cells and gum, grams per 100 ml Unidentified products, grams per 100 ml Nitrogen in cells and gum, grams per 100 ml C/N ratio in cells and gum * The figures are averages for duplicate 4-day-old, 200-ml. cultures given a 24 per cent inoculum. t In calculating these values it was assumed that 50 per cent of the glutamic acid was utilized. Unpublished data show that this percentage is probably a minimal value. The figures for carbon in unidentified products (obtained by difference) are therefore probably low for cultures showing especially vigorous growth, e.g. cultures supplemented with manganese. manganese was added to the basal medium the amount of sugar fermented was increased to about 2-fold while the amount of guim and cells produced was increased to about 4-fold. There was no appreciable change in carbon dioxide production. The carbon and nitrogen analyses indicate that the C/N ratio in the gum and cell fraction remained reasonably constant. Since there is no nitrogen in the gum (Conner et al., 1937) a constant C/N ratio

11 NUTRITION OF CROWN-GALL ORGANISM indicates a constant ratio between gum and cells. Manganese, therefore, causes an increase in the yield of both gum and cells. This conclusion is substantiated by direct cell counts made together with carbon and nitrogen analyses on the gum and cell fraction, table 5. The direct counts, done only once, should be considered not strictly quantitative but only relatively so because there were many clumps of cells which could not be broken up. However, they correlate well with the carbon and nitrogen analyses. The cell count increases at roughly the same rate as the amount of carbon or nitrogen in the gum and cell fraction. TABLE 5 Correlation between direct cell count and carbon and nitrogen analyses on gum and cells* GUM ND CELLS SUPPLEMENT TO BASAL MEDIUM Carbon Nitrogen Grams RerRatio Grnmer Ratio CULLS PER ML. None X Mn X Fe + Mn + Zn X * These were 200 ml. cultures in 1-liter Erlenmeyer flasks given a large inoculum. Direct counts with a Petroff-Hausser counter were made on mixed samples of duplicate cultures 3 days after inoculation. Carbon and nitrogen analyses were made when the cultures were 4 days old. SUMMARY Iron, manganese, and zinc are important in the nutrition of the crown-gall organism. This organism will grow very well in a simple synthetic medium containing proper amounts of these elements. Growth in this medium could be increased only moderately by the addition of yeast extract. Evidence presented indicates that the stimulation by organic material in yeast extract may be attributed to thiamin, riboflavin, pantothenic acid, and amino acids. This organism was shown to synthesize unusually large quantities of biotin, large amounts of riboflavin, and moderate amounts of thiamin and pantothenic acid. It also apparently synthesizes 11

12 12 McINTIRE, RIKER AND PETERSON any other such factors necessary for its growth in the synthetic medium. This character needs consideration in relation to the cell-stimulating property of these bacteria. Supplementing the synthetic medium with manganese had very interesting effects upon the carbon metabolism: The rate of sugar fermentation was increased. The percentage of fermented sugar converted to gum and cells was increased. Less fermented sugar was accounted for as unidentified products. REFERENCES CONNER, H. A., RIKER, A. J., AND PETERSON, W. H The carbon metabolism of the crown-gall and hairy-root organisms. J. Bact., 3, FOSTER, JACKSON W The heavy metal nutrition of fungi. Botan. Rev., 5, HECK, A. FLOYD A method for the determination of total carbon and also for the estimation of carbon- dioxide evolved from soils. Soil Sci., 28, HUTCHINGS, BRIAN L., AND WOOLLEY, D. W Growth of some hemolytic streptococci on a chemically defined medium. Science 90, MCINTIRE, F. C., PETERSON, W. H., AND RIKER, A. J Factors influencing the carbon metabolism of the crown-gall organism (in press). MUELLER, J. HOWARD A synthetic medium for the cultivation of C. diphtheriae. J. Bact., 38, PAVCEK, P. L., PETERSON, W. H., AND ELVEHJEM, C. A Factors affecting the vitamin B1 content of yeast. Ind. Eng. Chem., 30, PETERSON, W. H., McDANIEL, L. E., AND McCoy, ELIZABETH Biotin requirements of clostridia and assay of biological materials for biotin. Proc. Am. Soc. Biol. Chem., New Orleans, March, 1940, p. lxxv-lxxvi. PINCKARD, J. A Physiological studies of several pathogenic bacteria that induce cell stimulation in plants. J. Agr. Research, 50, SAGEEN, H. E., RIKER, A. J., AND BALDWIN, I. L Studies on certain physiological characters of Phytomonae tumefaciens, Phytomonas rhizogenes, and Bacillus radiobacter. J. Bact., 28, 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, 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, STILES, H. R., PETERSON, W. H., AND FRED, E. B A rapid method for the determination of sugar in bacterial cultures. J. Bact., 12, THORNE, D. W., AND WALKER, R. H Physiological studies on rhizobium: VI. Accessory factors. Soil Sci., 42,

13 NIUTRITION OF CROWN-GALL ORGANISM 13 VAN LANEN, J. M., BALDWIN, I. L., AND RIKER, A. J Attenuation of cell-stimulating bacteria by specific amino acids. Science, 92, WEST, P. M., AND WILSON, P. W The relation of "coenzyme r" to biotin. Science, 89, WRIGHT, W. H., HENDRICKSON, A. A., AND RIKER, A. J Studies on the progeny of single-cell isolations from the hairy-root and crown-gall organisms. J. Agr. Research, 41,