glucose. The isolation and quantitative determination of certain metabolic products formed from carbon compounds by crown-gall and

Transcription

1 THE CARBON METABOLISM OF THE CROWN-GALL AND HAIRY-ROOT ORGANISMS' H. A. CONNER, A. J. RIKER AND W. H. PETERSON Departments of Agricultural Chemistry and Plant Pathology, University of Wisconsin Received for publication March 22, 1937 INTRODUCTION The isolation and quantitative determination of certain metabolic products formed from carbon compounds by crown-gall and related bacteria have been studied as part of a larger program (Riker and Berge, 1935) on atypical and pathological multiplication of cells. The general question of the metabolism of these microorganisms has been reviewed in a previous paper (Conner, Peterson and Riker, 1937) dealing specifically with the nitrogen metabolism. The crown-gall and related organisms do not produce carbon dioxide from sugars rapidly enough to be classed in the conventional manner as gas formers (Smith, et al., 1911; Riker et al. 1930; and Pinckard, 1935). Studies on single-cell cultures by Sagen et al. (1934), Wilson (1935) and others have shown that while the hairy-root organsm produces some acid, the crowngall organism produces little or no acid. It seems likely, therefore, that substances other than carbon dioxide and acids are produced in considerable quantities. The present paper reports studies on the carbon metabolism of these organisms with special reference to certain products resulting from their action on glucose. 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. The figures were prepared by Eugene Herrling. 221

2 222 H. A. CONNER, A. J. RIKER AND W. H. PETERSON EXPERIMENTAL WORK Cultures and media. The crown-gall and hairy-root cultures A-1 and C-1 respectively were progenies of single cells isolated by Wright et al. (1930). The identities of the cultures were checked at frequent intervals during the course of the investigation by inoculation of suitable host plants. After some preliminary experiments to determine the effect of various concentrations of yeast infusion and phosphates, a medium was selected which gave good utilization of the sugar. It had the following composition: 20 per cent press-yeast infusion, 50 cc.; calcium sulfate (CaSO4), 0.1 gram; magnesium sulfate (MgSO4.7H20), 0.2 gram; sodium chloride (NaCl), 0.2 gram; sodium phosphate (NaH2PO4), 2.9 grams; potassium phosphate (K2HPO4), 8.8 grams; glucose, 10 to 40 grams; and distilled water to make 1000 cc. A solid medium was prepared by adding 15 grams of agar. Any variations from the above amounts are noted later. Methods of analysis. Sugar was determined by the method of Shaffer and Hartman as modified by Stiles, Peterson, and Fred (1926). Carbon dioxide was removed from the cultures by a stream of sterile, carbon-dioxide-free air, absorbed by ascarite, and determined by direct weighing. Bacterial cells and gum were precipitated by alcohol or barium hydroxide, and removed by centrifuging. Other metabolic products were precipitated by a technique described later. Total carbon of the various fractions was determined by the wet combustion method (Heck, 1929). RATE OF FERMENTATION Rates of fermentation of glucose by the two organisms on agar media containing 1, 2 and 4 per cent of the sugar were determined. Ten cubic centimeters portions of each medium were placed in a number of tubes, with special precautions in sloping so as to insure a surface of uniform area in all tubes. Determinations of glucose were made on the combined contents of three tubes every fifth day and the percentage of glucose fermented was calculated. The results of these determinations are

3 CROWN-GALL AND HAIRY-ROOT ORGANISMS 223 given in figure 1. The hairy-root organism fermented glucose at an appreciably faster rate than the crown-gall organism at all concentrations of glucose studied. Although the percentage of glucose destroyed by either organism was less at the highest concentration, the actual weight of sugar fermented was equal to or greater than that at the lower concentrations. Downloaded from FIG. 1. TIME IN DAYS FERMENTATION OF GLUCOSE BY THE HAIRY-ROOT AND CROWN-GALL ORGANISMS ON AGAR MEDIA MEASUREMENT OF CARBON DIOXIDE PRODUCTION The set up of fermentations in which carbon dioxide was determined is shown in figure 2. Carbon dioxide production was determined daily by weighing the absorption tube. These results are summarized in figures 3 to 6 inclusive. The scales used in figures 5 and 6 are one-fifth and one-tenth, respectively, as large as the scales used in figures 3 and 4. The results given in each figure are representative of 2 to 6 trials. The curves show: (1) that the hairy-root organism produced approximately ten times as much carbon dioxide as the crown-gall organism; (2) that the addition of calcium carbonate to an agar on December 31, 2018 by guest

4 224 H. A. CONNER, A. J. RIKER AND W. H. PETERSON 9 WIO -i.-- m z 28 v m E-4 0 z oo v 0P E-4

5 CROWN-GALL AND HAIRY-ROOT ORGANISMS 225 d u o 0.3 IUe o. o0. 0 x 2 4 vc FIG. 3. CARBON DIOXIDE PRODUCTION OF THE HAIRY-ROOT ORGANISM ON r AGAR MEDIA FIG. 4. CARBON DIOXIDE PRODUCTION OF THE HAIRY-ROOT ORGANISM IN LIQUID MEDIA

6 226 H. A. CONNER, A. J. RIKER AND W. H. PETERSON FIG. 5. CARBON DIOXIDE PRODUCTION OF THE CROWN-GALL ORGANISM ON AGAR MEDIA Downloaded from on December 31, 2018 by guest FIG. 6. CARBON DIOXIDE PRODUCTION OF THE CROWN-GALL ORGANISM IN LIQUID MEDIA

7 CROWN-GALL AND HAIRY-ROOT ORGANISMS medium markedly lowered the carbon-dioxide production of the hairy-root organism, but had no effect on that of the crown-gall organism. The decreased production might be explained in several ways, e.g., less glucose might have been fermented, or less glucose might have been converted to carbon dioxide in the carbonate medium. Other experiments showed that carbon dioxide production did not bear a constant ratio to glucose fermented. IDENTIFICATION OF THE ACIDS PRODUCED BY THE HAIRY-ROOT ORGANISM The hairy-root organism produces small amounts of acid (Riker et al. (1930)) when grown in media containing glucose. The acid was identified by the following procedure. The culture was grown in the basal medium containing 3 per cent glucose. Two liters of medium were placed in three-liter flasks and incubated for at least sixty days in order to have sufficient acid for identification. At the end of this time the cultures were filtered, neutralized, concentrated under reduced pressure, acidified with H2SO4 and extracted for 48 hours with ether. Fifty cubic centimeters of water were added to the extract, the ether was removed, and the aqueous solution decolorized with charcoal and filtered. On distilling the filtrate with steam, both distillate and residue were found to contain acid. The distillate was purified by distilling with steam a second time. Only small quantities of acid were obtained, about 4 to 6 cc. of N/10 per liter of culture. A Duclaux determination on the volatile acid indicated acetic acid. Additional evidence of the identity of this acid was obtained by the preparation of its p-toluidide. For this purpose 50 liters of culture medium were extracted as described, and the volatile acid was converted to its sodium salt. Then 0.4 gram of the sodium salt, 0.4 cc. of concentrated hydrochloric acid and 1 gram of para-toluidine hydrochloride were intimately mixed in a tube and gently heated for 30 minutes. The reaction mixture was cooled, extracted with 95 per cent alcohol and the alcohol poured into 50 cc. of water. The aqueous mixture was evaporated to a volume of about 10 cc., filtered and allowed to cool. The crystals which separated were recrystallized twice from water 227

8 228 B. A. CONNER, A. J. RIKER AND W. H. PETERSON and dried. Their melting point was 1460C. uncorrected. A sample of acet-p-toluidide prepared from acetic acid also melted at 1460C. uncorrected. A mixture of the two showed no depression of the melting point. The residue from the steam distillation was concentrated and used in a number of qualitative tests for the identification of the non-volatile acid. The solution reduced ammoniacal silver nitrate and alkaline potassim permanganate at room temperature. With iodine and sodium hydroxide a precipitate of iodoform was produced in the cold, indicating the presence of a methyl ketonic group in the molecule. Since the tests indicated the presence of a keto acid, the 2-4 dinitrophenylhydrazone of the acid was prepared. After two crystallization from ethyl acetate the hydrazone melted at uncorrected. Since pyruvic acid gives all these reactions the 2-4 dinitrophenylhydrazone of this compound was prepared. This hydrazone also melted at 2150C. uncorrected and a mixture of the two hydrazones showed no depression of the melting point. CARBON BALANCE A comparison of the carbon in the glucose fermented with that in the known products showed that both organisms must produce other products in considerable quantities. These substances were not volatile, since the sulfuric acid through which the air passed after leaving the culture contained no organic compounds as was demonstrated by analysis for carbon. A carbon balance was made to show what percentage of the glucose was accounted for by products. The cultures used in the studies of carbon dioxide production, were also used for the determination of (1) reducing sugars, (2) total carbon, and (3) carbon in cells and gum. The cells and gum were precipitated by alcohol. The results of four representative determinations are given in table 1. The following conclusions may be drawn from these data: (1) The cell and gum production of the crown-gall organism was slightly higher than that of the hairy-root organism. (2) The major part of the metabolic products remained in the culture medium after removal of the carbon dioxide, cells, and alcohol-

9 CROWN-GALL AND HAIRY-ROOT ORGANISMS 229 precipitable gum. Seventy to 80 per cent of the carbon in the fermented glucose formed unknown products. (3) Less glucose was recovered in known products from the cultures of the crowngall organism than from those of the hairy-root cultures. A search for products which, on the basis of the above, must be present resulted in the isolation of two unidentified products from cultures of each organism. An outline of the procedures employed and some of the characteristics of these products follow. TABLE 1 Glucose fermentation by the crown-gall and hairy-root organisms, and carbon distribution of the products ANALYTICAL DETRNINATIONS CROWN-GALL OANISM HAIRY-ROOT ORGANISM Experiment Experiment Experiment Experiment gram e grams r gram p gram. Glucose fermented* '. 0 Carbon in glucose fermented Carbon in CO Carbon in cells and gum Carbon in products determined Carbon not accounted for Carbon in culture at beginning Carbon in culture at end plus carbon in CO * The medium contained 2.0 grams glucose per 100 cc. before fermentation. ISOLATION OF GUM AND GUM-LIKE PRODUCTS Twenty to 40 liters of the basal medium containing 3 per cent glucose were incubated at room temperature for thirty days and then worked up for gum and gum-like products. Gum.2 Precipitation of the gum was effected with alcohol or 2 A different medium in large quantities was used for the preparation of some of the samples of gum. This consisted of magnesium sulfate (MgSO4.7H20), 0.2 gram; sodium chloride (NaCI), 0.2 gram; calcium chloride (CaCl2), 0.1 gram; potassium phosphate (K2HP0), 0.2 gram; potassium nitrate (KNOs), 5.0 grams; and glycerol, 10.0 grams. The bacteria were removed by filtration from several hundred liters and the filtrate was concentrated to about one-tenth its volume. This work was done by Mulford Biological Laboratories, Sharp and Dohme, to whom grateful acknowledgment is made.

10 230 H. A. CONNER, A. J. RIKER AND W. H. PETERSON acetone, and with barium hydroxide. The hydroxide treatment is preferable when it is desired to isolate other metabolic products from the filtrate, whereas the first procedure is more convenient for the isolation of the gum. The gum was purified by dissolving it in water and reprecipitating with alcohol acidified with hydrochloric acid. The excess alcohol was squeezed out and the gum dried over calcium chloride in a vacuum desiccator. The product was white, difficultly soluble in water, non-reducing to Fehling's solution, and precipitable from aqueous solutions by barium hydroxide and basic lead acetate. Such preparations were free from nitrogen but contained from 5 to 30 per cent ash. A preparation containing only 0.36 per cent ash was obtained by electrodialysis. When the gum was hydrolyzed with 2 per cent HCl or 5 per cent H2SO4, reducing values calculated as anhydroglucose accounted for 72 to 98 per cent of the ash-free material. Uronic acid and total pentosan determinations gave figures of about 5 per cent for both components. Qualitative tests on the gumhydrolysate for mannose, fructose and galactose were negative. Evidence that the reducing substance consisted mainly of glucose was obtained by the isolation and characterization of this sugar. Ten grams of crown-gall gum, containing 0.36 per cent ash, were hydrolyzed with 5 per cent H2SO0 at 15 pounds pressure for 5 hours. The solution was neutralized with hydroxide-free barium carbonate, heated for 20 minutes at 60'C., and the barium sulphate filtered off and washed with hot water. The filtrate was concentrated under reduced pressure at 600C. to a thick syrup which was extracted with a number of portions of hot 95 per cent alcohol or hot absolute methyl alcohol. The alcoholic extract was decolorized with norit, concentrated to a thick syrup and two volumes of glacial acetic acid were added. After one week in the refrigerator the crystals which had formed were filtered off, washed with glacial acetic acid and recrystallized from a small amount of water and acetic acid. The yield was about 4 grams. Further concentration of the filtrate yielded about 2 grams more of crystals. The specific rotation of a dried sample of the first crop was a22 = (C. = 65.8 mgm./cc.). The specific

11 CROWN-GALL AND HAIRY-ROOT ORGANISMS rotation of glucose is The osazone of the sugar and that of glucose prepared at the same time melted at 204'C. (uncorrected). From these data it appears that the gum consists mainly of anhydro-glucose units. Gum prepared from the glycerol-salts medium had approximately the same composition and yielded the same products on hydrolysis as that from the glucose-salts medium. Gum-like products. Since such a large portion of the fermented glucose remained unaccounted for, a search for metabolic products, other than carbon dioxide, cells and gum, was made. Substances, as yet incompletely characterized, but which resemble the bacterial gum in certain respects were found. The cells and pellicle were filtered from 20 to 30 liters of culture and the bacterial gum was precipitated by addition of a saturated solution of barium hydroxide. An appreciable excess of barium hydroxide solution (about 1 to 2 liters for every 30 liters of culture medium) was added, and the mixture was allowed to stand for thirty minutes. The bulky precipitate was removed by filtration, washed with water, and the washings added to the filtrate. Besides the barium salts of the gum the precipitate contained phosphate, sulfate and carbonate. The filtrate was treated with carbon dioxide until it was nearly acid to phenolphthalein. The precipitated barium carbonate was filtered off, the filtrate acidified with acetic acid and evaporated under reduced pressure to a thick syrup. An excess of acetic acid was avoided, since it exerted a definite solvent action on the lead precipitate obtained later. A saturated solution of basic lead acetate was added, with agitation, to the thick syrup. A gelatinous precipitate was formed, which on standing in contact with excess basic lead acetate became flocculent. The precipitate was filtered off and washed with water. In order to remove the glucose it had adsorbed, the precipitate was repeatedly suspended in 60 per cent alcohol and settled by centrifuging. Great difficulty was experienced in removing the glucose, and the washing process, though tedious, was more satisfactory than repeated precipitation because the 231

12 232 H. A. CONNER, A. J. RIKER AND W. H. PETERSON latter caused serious loss of material. In later experiments the washing process was shortened by placing the lead salt in collodion bags and dialyzing in running water for two weeks. The washed salt was dissolved in dilute acetic acid and the lead precipitated with hydrogen sulfide. The filtrate and washings were combined and evaporated under reduced pressure until all but a small amount of the acetic acid had been removed. The clear glassy material was dissolved in a minimal quantity of water and the solution saturated with cupric acetate. Ninety-five per cent alcohol was added, which precipitated the copper salt of the product. In the presence of too large an excess of acetic acid the copper salt was precipitated in the form of a heavy syrup. The tendency to precipitate as a syrup was especially marked with material from the crown-gall organism. The precipitate was allowed to settle and the supernatant alcohol decanted. The remainder of the material was filtered and the precipitate washed until the washings gave no test for glucose. The filtrate from the lead precipitation was deleaded with hydrogen sulfide and the filtrate and washings were evaporated under reduced pressure to a thick syrup to remove as much acetic acid as possible. The syrup was diluted until it could be handled conveniently and saturated with copper acetate. On adding alcohol a precipitate formed. This was filtered and washed as described above. The above method of isolation is summarized in figure 7. The metabolic products nos. 2 and 3 have not been completely characterized. They were obtained in the free state in the form of a hard glassy mass by evaporating their aqueous solutions under reduced pressure, or in the form of a sticky gum when concentrated solutions were treated with alcohol. On standing in contact with alcohol the precipitate would slowly redissolve. Oxidation with nitric acid yielded oxalic acid. No reducing sugars were formed on hydrolysis with either hydrochloric or sulfuric acids. Since the substance precipitated by basic lead acetate from cultures of the hairy-root organism (metabolite no. 2) could be isolated and purified with less effort than the corresponding

13 CROWN-GALL AND HAIRY-ROOT ORGANISMS product from the crown-gall organism, it was selected as the most suitable for experiments designed to secure information concerning the nature of the material. Acetylation, benzoylation, and methylation yielded syrups that could not be crystallized. The 3-5 dinitrobenzoate, prepared by heating the metabolic product with 3-5 dinitrobenzoyl chloride, was likewise a syrup and could not be crystallized. These syrups differed from the original material in respect to solubility. The original material was SO MEABOLITE NO.1 GUM FILTERED CULTURE SQallCOa COo ALCOO DiSC*RD ALCOHOL WATER ETC. DISCARD bs ~ DISCARD if_ PbS BASIC LEAD ACETATE II*S AL 0 DISCARD C&('AcqA fas O/SC Cei(OAo)~ ALCt CjIOSL DISCAR$/N2 'O 'E CuS' MSETAOLITE ALCOIfJ NO.2 ETC. Cicu S METABITlE DISCARD DISCARD NO. 3 FIG. 7. DIAGRAMMATIC REPREsENTATION OF THE PROCEDURE USED TO ISOLATE CERTAIN METABOLIC PRODUCTS OF THE HAIRY-ROOT AND CROWN-GALL ORGANISMS soluble in water and only difficultly soluble in alcohol, while the acetyl and benzoyl derivatives were insoluble in water and soluble in alcohol and other organic solvents. Since acid phthalates are crystalline in many cases, an attempt was made to prepare the acid phthalate of the substance by heating it with phthalic anhydride to Nothing could be isolated from the mixture except phthalic anhydride. Reduction with hydroiodic acid and phosphorous yielded only traces of a volatile oil, while the remainder of the material refused to crystallize and could not be distilled. 233

14 234 H. A. CONNER, A. J. RIKER AND W. H. PETERSON The acetone compound of the metabolite was prepared by treating it with purified acetone, containing hydrogen chloride as a catalyst, for eight days at 36TC. At the end of this time the acetone was removed and the residue dissolved in alcohol. The alcoholic solution was boiled with decolorizing charcoal and filtered. Nothing could be crystallized from the solution. Oxidation of the acetone compound with alkaline permanganate yielded no definite product. DISCUSSION While distinct differences exist in their nitrogen metabolism (Sagen, et al. 1934), the close relationship of the crown-gall and hairy-root organisms, as indicated by their similar pathogenicity, is reflected in their carbon metabolism. With the exception of the production of acid by the hairy-root organism, only quantitative differences have been found in their behavior during these studies. The organisms are quite similar in their abilities to ferment glucose. The hairy-root organism is, however, able to ferment high concentrations of glucose more rapidly than the crown-gall organism. In all cases the hairy-root organism produced more carbon dioxide than the crown-gall organism. On agar media the hairyroot organism produced carbon-dioxide rather rapidly for the first seven days, then less rapidly for the remainder of the period. The crown-gall organism produced carbon dioxide at a fairly steady rate during the entire course of the fermentation. The production of acid from glucose by the hairy-root organism is also a useful method of distinguishing it from the crown-gall organism. It is of interest to note that pyruvic acid, which is produced by hairy-root bacteria has been reported (Hitchcock, 1935) to induce a growth response in plants. However, the, evidence is inadequate to determine whether this acid is involi ed in the growth response induced in the host by the hairy-root organism. While no proof exists that the various metabolities discussed are produced in the host plant, it is not improbable that they do occur in plant tissue. It remains for further study to determine

15 CROWN-GALL AND HAIRY-ROOT ORGANISMS 235 whether these substances play a role in the peculiar type of cell division found in the tissue surrounding the bacterial pockets. SUMMARY Quantitative data have been obtained regarding the fermentation of glucose, the rates of fermentation, the amounts of carbon dioxide produced, and the carbon distribution of the metabolic products of the crown-gall and hairy-root organisms. The amount of glucose fermented in a liquid medium by the hairy-root and crown-gall organisms may be raised by increasing the concentration of yeast infusion or by the addition of one per cent phosphates. Agar media were more satisfactory for fermentation than liquid media. The hairy-root organism was able to ferment higher concentrations of glucose more rapidly than the crown-gall organism. The hairy-root organism produces approximately ten-fold as much carbon dioxide as the crown-gall organism. Acetic and pyruvic acids were identified as metabolic products of the hairy-root organism. From 13 to 20 per cent of the carbon of the fermented glucose was recovered as carbon dioxide, cells, and gum from cultures of the crown-gall organism and approximately 30 per cent was recovered from cultures of the hairy-root organism. Seventy to 80 per cent of the sugar fermented goes to form other products, some of which have been isolated and partly characterized. These metabolites differ from the bacterial gum in that they do not yield reducing sugars on hydrolysis. The bacterial gum consisted chiefly of glucose units (72 to 98 per cent) and small amounts (5 per cent) of uronic acid. Qualitative tests for other hexoses were negative. The writers are indebted to Dr. Rudolph Nagy for his collaboration in the preparation and analysis of the gum produced by the organisms. REFERENCES CONNER, H. A., PETER8ON, W. H., AND RIKER, A. J The nitrogen metabolism of the crown-gall and hairy-root bacteria. Jour. Agr. Research 54:

16 236 H. A. CONNER, A. J. BIKER AND W. H. PETERSON HECK, A. FLOYD 12 A method for the determination of total carbon and also for the estimation of carbon dioxide evolved from soils. Soil Science, 28, HITCHCOCK, A. E Indole-3-n-propionic acid as a growth hormone and the quantitative measurement of plant response. Contrib. Boyce Thompson Inst., 7, PINCKARD, J. A Physiological studies of several pathogenic bacteria that induce cell stimulation in plants. Jour. Agr. Research, 50, RIKER, A. J., BANPIELD, W. M., WRIGHT, W. H., KnITT, G. W., AND SAGEN, H. E Studies on infectious hairy root of nursery apple trees. Jour. Agr. Research, 41, RIKER, A. J., Arm Bunou, T Atypical and pathological multiplication of cells approached through studies on crown gall. Amer. Jour. Cancer, 25, SAGEN, H. E., RIKER, A. J., AND BALDWIN, I. L Studies on certain physiological characters of Phytomona. tumefaciens, Phytomona. rhizogenee, and Bacillus radiobacter. Part 1. Jour. Bact., 28, SMITH, E. F., BROWN, N. A., AND TOWNSEND, C Crown-gall of plants: its cause and remedy. U. S. Dept. Agr. Bur. Plant Indus. Bul. 213, 215 p. STILES, H. R., PETERON, W. H., AND FRED, E. B A rapid method for the determination of sugar in bacterial cultures. Jour. Bact., 12, WILSON, A. R The influence of Phytomonas tumefaciens and Phytomonas rhizogenee on the actual acidity of certain liquid and agar substrata. Phytopathology, 25, 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. Jour. Agr. Research, 41,