THE EFFECT OF D-GLUCOSE ON THE UTILIZATION OF D-MANNOSE AND D-FRUCTOSE BY A FILAMENTOUS ' DOROTHY E. SISTROM AND LEONARD MACHLIS Department of Botany, University of California, Berkeley, California Received for publication December 23, 1954 In earlier studies (Machlis, 1953a, b, c) it was inorganic nutrients. DS (dilute salt) solution reported that the Burma lDa strain of the consists of a 1:10 dilution of the inorganic com- filamentous watermold, macrogynus, ponents of the synthetic medium exclusive of the was unable to grow in a synthetic medium in trace elements. which either D-mannose or D-fructose was sub- Cultures consisting of 50 ml of medium in 125 stituted for D-glucose as the carbon and energy ml Erlenmeyer flasks were inoculated with either source. The purpose of the present report is to mitospores or mycelial fragments. The mitospore describe conditions under which the mold does suspensions were obtained by a modification of grow on mannose and fructose in the synthetic the procedure described by Machlis (1953a). medium. Young plants, grown for four or five days in the medium, were separated from the solution by MATERIALS AND METHODS pouring the contents of a culture into a 40 or 60 The organism (Emerson, 1941; Emerson and mesh, Monel metal, wire screen basket. The re- Wilson, 1954) was provided by Professor Ralph tained plants were immersed in 50 ml of DS solu- Emerson. Its life history is pertinent to the later tion for four to twelve hours, sometimes with a consideration of mutation and selection. The renewal of the DS solution, and then transferred mold consists of two coenocytic, isomorphic, into 12 ml of DS solution in a small (6 cm) petri independent generations. The haploid gameto- dish. At the beginning of the process the plants phyte develops from a uninucleate, haploid, bear mitosporangia formed during growth in the motile spore. The diploid sporophyte develops culture flasks. These discharge in the first phase either from a zygote formed by a fusion of male of mitospore procurement and are lost when the and female gametes or from uninucleate, diploid, plants are transferred to fresh DS solution. motile mitospores produced by the sporophytic Subsequently, new mitosporangia are produced plant (Emerson, 1941). Only the sporophytic which release large numbers of mitospores. The generation enters into the present investigation. total time involved was 24 hours or less. Cultures It was originally obtained from a single uni- were inoculated with 0.5 ml of the spore suspen- nucleate, unicellular, haploid spore. This spore sion which contained from 10 to 20 X 103 spores developed into a gametophytic plant bearing per ml as determined by direct microscopic both male and female gametangia. Fusion of a counting of aliquots. male and female gamete resulted in the uni- Mycelial fragments were prepared from plants nucleate zygote from which the sporophyte de- grown four to five days in synthetic medium. veloped. After eliminating the plants larger than 0.5 mm The synthetic nutrient medium and its prepara- in diameter, the remaining plants were trans- tion are fully described elsewhere (medium B of ferred through two DS solution wash baths and Machlis, 1953b). This medium supplies nitrogen then placed in a Waring blendor with 35 to 50 as ammonium, sulfur as , and carbon ml of DS solution. After fragmenting for 25 and energy as 0.5 per cent glucose. It also con- seconds, the suspension was passed through the tains the vitamin thiamin as well as the usual Monel metal wire screen baskets to remove ex- cessively large fragments. Each flask of medium 1This investigation was supported in part by received 2 ml of the final mycelial fragment a research grant (G-2149) from the Division of Research Grants and Fellowships of the National suspension. Institutes of Health, United States Public Health The inoculated flasks were incubated at 25 C Service. on a horizontal shaker previously shown to pro- 50 1955] UTILIZATION OF MANNOSE AND FRUCTOSE BY FUNGUS 51 vide adequate aeration. Each determination of which began growth at the same time. Two of growth was made by combining 5 to 10 replicate these were sacrificed on successive days to obtain flasks. The plants were filtered off through fine- the dry weight data on which curve B, figure 1, mesh toweling, sucked free of excess water on a was constructed. This curve shows the average Buchner funnel, and dried to constant weight at shortest lag period for growth in mannose and 70 C. Growth is reported as mg dry weight per also reveals that once growth is initiated, it pro- flask. ceeds at approximately the same rate as that in glucose. EXPERIMENTAL RESULTS In fructose, the shortest observed lag phase is The experiments to be described are concerned about 20 days, the approximate linear growth with the lag phase of growth under various con- rate is somewhat less than that in either mannose ditions. In the synthetic medium containing 0.5 or glucose, and the maximum dry weight is also per cent glucose inoculated with mitospores, slightly less (curve C, figure 1). growth is characterized by a lag phase of ap- For ease of presentation, the terms mannose- proximately 100 hours (hereafter called the lag and fructose-lag will henceforth be used to glucose-lag) and a linear growth phase of about mean the minimal lag time in these two sugars 50 hours resulting in the production of 60 to 70 less the lag time in glucose. Thus the mannose-lag mg dry weight of mycelium per flask (curve A, is 7 days and the fructose-lag is about 16 days, figure 1). When mannose or fructose was sub- these being equal to the total lag periods of 11 stituted for glucose, Machlis (1953c) observed no and 20 days, respectively, less the glucose-lag of growth after 11 and 14 days' incubation, re- 4 days. These terms imply, in addition, the highly spectively. variable extent of the lag period in mannose and Upon repeating this work but with longer in fructose, but not in glucose. incubation periods, growth occurred in both If a small part of the mannose or fructose is re- mannose and fructose cultures. This growth was placed by glucose (0.05 per cent glucose plus 0.45 characterized by an extremely erratic lag phase. per cent mannose or fructose), then growth For example, 12 days after inoculating 39 flasks proceeds as in glucose (curves E and F, figure 1). containing mannose, no growth had begun. On The 0.05 per cent glucose alone supports only 10 successive days for the next 13 days, growth mg dry weight of growth per flask (curve D, began in 1 to 4 flasks. After 26 days' incubation, figure 1). The inclusion of the small amount of 5 flasks were still devoid of growth. In the course glucose eliminates both the mannose-lag and the of many experiments growth was never initiated fructose-lag. in less than 11 days' incubation. By inoculating a The minimal amount of glucose required for large number of flasks several could be obtained elimination of the mannose-lag was determined to be between 0.04 and 0.05 per cent glucose or X7 essentially that combination of glucose and mannose originally used. A similar test was not c'IC C_ 5 /, made with fructose. ,i 11r I / Mutaion and selection. The remaining experi- I ments were confined to growth in mannose. The * il in the 30 effect of glucose eliminating mannose-lag It 20 * t1 At, ,, - ,, could be either genetic, in the sense of affecting Ji la mutation and selection of mannose-utilizing o00 200 300 400 500 GM 7u HOURS OF INCUBATION strains, or physiological, for example, an effect on Figure 1. Growth in synthetic medium (see metabolism or permeability. A series of experi- text) with sugar supplied as 0.5 per cent glucose ments was performed to distinguish between a (A), 0.5 per cent mannose (B), 0.5 per cent fruc- genetic or nongenetic mechanism. tose (C), 0.05 per cent glucose (D), 0.05 per cent Plants grown in mannose were used to obtain glucose plus 0.45 per cent mannose (E), and 0.05 mitospores by the standard 24 hour treatment in per cent glucose plus 0.45 per cent fructose (F). DS solution. These were seeded into Data points for B and C are omitted because of mitospores the approximate nature of these curves as ex- glucose medium. The resultant plants were sub- plained in the text. jected to the treatment in DS solution to obtain 52 DOROTHY E. SISTROM AND LEONARD MACHLIS [VOL. 70 a suspension of mitospores which were now in- experiments was to obtain inoculum which would oculated into mannose solution. The typical grow in mannose without a mannose-lag. The mannose-lag was observed. Had the original usual procedure for procuring mitospores had plants been mutants, they should have multi- been to hold young plants in the purely inorganic plied in the passage through glucose, so that DS solution for 24 hours. Such mitospores, upon seeding into mannose again, there should whether produced by plants grown in glucose or have been no mannose-lag. in mannose, grow only with the mannose-lag. A more thorough effort was made to obtain When the DS solution was supplemented with mutants capable of using mannose immediately. 0.5 per cent mannose with the purpose of keeping Mitospores from plants grown on glucose were the plants, and the spores they were to produce, streaked onto solidified mannose medium at the continuously in the presence of mannose, spore rate of 100, 500, 1,000, and 5 to 10,000 spores per discharge was almost completely inhibited. How- plate. Similarly, spores were streaked onto glu- ever, in one attempt, a low concentration of cose plates and water-agar plates. Two days later spores was obtained from plants grown in man- minute germlings could be seen on the glucose nose and processed in DS solution supplemented and water-agar plates. Those on the lowest with 0.05 per cent mannose. These were in- density glucose plates grew rapidly, attaining a oculated into a set of mannose cultures. On the diameter of approximately 3 cm in seven days seventh day, growth began in sixteen of the while those on water-agar made no further seventeen cultures and proceeded to completion growth. Observable development of the spores on normally. Although the mannose-lag was not the mannose plates did not begin until the eliminated, it was reduced by about 60 per cent seventh day; moreover, only a very few plants in spite of the exceedingly poor quality of the appeared in contrast to the comparatively inoculum. So far, attempts to obtain normal luxurious and uniform development on the mitospore inoculum in the presence of sugar glucose plates. However, on each successive day, (mannose or glucose) have been unsuccessful. more and more plants appeared on the mannose The preceding experiment suggests, however, plates until, after 20 days, they were equal in that the presence of mannose during sporo- number to those on glucose. There was one strik- genesis leads to spores which can use mannose ing difference. Whereas the plants on glucose without a mannose-lag. were relatively uniform in diameter, those on To obviate sporogenesis with its mandatory mannose were predominantly small, about 1 to sugar-free conditions, mycelial fragments were 3 mm in diameter but with an occasional large, used in a series of experiments. If young plants, rapidly growing individual. grown in glucose, are washed in two to four Entire small plants and parts of the larger ones changes of DS solution over a period of four were transferred to fresh mannose plates and a hours, fragmented in the blendor, and then inocu- week later transferred again. The growth was lated into mannose medium, growth proceeds uniformly good with no reflection of the original without the mannose-lag. Exactly similar results difference in size. Bits of mycelium from each are observed when the same experiment is done subculture were next inoculated into liquid man- using for inoculum plants grown in mannose nose medium. Growth proceeded without the solution. mannose-lag. From the plants so grown, mito- The earlier experiments suggested that man- spores were produced by the standard procedures nose grown mycelial inoculum should continue in DS solution and seeded into glucose medium. growth in mannose without a mannose-lag. On The next generation of mitospores was used to the other hand, the similar behavior of glucose inoculate mannose medium. Sporadic growth grown mycelium was unexpected. The possi- began only after a mannose-lag. The results bility was present that glucose grown mycelium indicate that the plants which grow on mannose contained reserves of glucose sufficient to initiate are not mutants. Therefore, the effect of glucose growth in mannose. Therefore, plants were in eliminating the mannose-lag must be sought grown in glucose medium containing a limiting in some nongenetic change in the physiology of concentration of glucose. With the glucose at a the plants. concentration of 0.05 per cent instead of the usual Effect of previus history. The purpose of these 0.5 per cent, the maximum dry weight per flask 1955] UTILIZATION OF MANNOSE AND FRUCTOSE BY FUNGUS 53 of approximately 10 mg is attained in about 120 inoculated with mitospores suggested a possible hours' incubation. These plants were incubated explanation for the erratic and delayed growth an additional 24, 48, 72, and 96 hours. After each in mannose. Counts of the number of spores in- 24 hours of starvation, plants were well washed troduced into a flask and of the resulting plants in DS solution, fragmented, and inoculated into indicate that under the best of growth conditions glucose and mannose media. As shown by figure only about 10 per cent of the mitospores develop 2, 96 hours of starvation had a negligible effect into plants. The possibility was tested that dur- on growth in glucose. On the other hand, 72 ing the lag in mannose the autolysis of spores re- hours of starvation completely eliminated the leased substances activating the utilization of capacity for mannose-lag-free growth on mannose. mannose and that the critical level of such sub- Of significance to the next experiment is that the stances would be attained at different times de- starvation and the fragmentation did not destroy pending on the actual number of spores intro- the capacity for growth on glucose. duced. This number is known to vary from 5 to If, then, the experiment were repeated, but 10 X 103. with plants grown and starved in mannose The first part of the mannose medium (glucose medium, perhaps the mycelial fragments would and Ca and Mg salts omitted) was prepared, and retain the capacity for mannose utilization with- the usual 0.5 ml of mitospore suspension added out a mannose-lag. Such plants were grown be- per flask and twice this quantity to still other ginning with mycelial fragments from plants flasks. After autoclaving and addition of the grown in mannose. After 36, 60, 84, and 108 sugar-salt solution, the flasks were inoculated hours of starvation (subsequent to exhausting as usual. Growth in these cultures (curves C and the limited supply of mannose), mycelial frag- B, respectively, figure 4) was compared to that ments were prepared with the usual washing in in mannose medium containing 0.05 per cent DS solution and seeded into glucose and mannose glucose (curve A) and in mannose alone (curve media. As figure 3 shows, even though the plants D). The autoclaved inoculum reduced the man- grew in mannose until the supply was exhausted, nose-lag from the normal 200 hours to about 85 the subsequent starvation resulted in the loss of hours. There was no significant difference in the the capacity for rapidly using mannose. effect of 0.5 and 1.0 ml of spore suspension. The mannose-lag. The fact that a small amount Autoclaved mycelial fragments (2 ml per flask) of glucose eliminates the mannose-lag in cultures were almost as effective as the autoclaved spores.

y70 - IN GLUCOSE HOURSOFIN MANNOSE

~30 : .< 20 1 i

60J (B,8 IC,ad18()hus 100 200 300 400 HOURS OF INCUBATION 100 200 300 400 HOURS OF INCUBATION Figure S. Growth in synthetic medium con- Figure S. Growth in glucose or mannose follow- taining glucose or mannose following inoculation ing inoculation with mycelial fragments obtained with mycelial fragments obtained from plants from plants initially grown in medium containing initially grown in medium containing mannose glucose and then held in the absence of sugar for and then held in the absence of sugar for 36 (A), 24 (A), 48 (B), 72 (C), and 96 (D) hours. 60 (B), 84 (C), and 108 (D) hours. 54 DOROTHY E. SISTROM AND LEONARD MACHLIS [VOL. 70 fructose involves induced enzyme synthesis. Consistent with this hypothesis is the required presence of substrate, the loss of activity upon removal of substrate as in the starvation experi- ments, and the demonstrated absence of muta- tion and selection. Should this hypothesis be proven by direct enzymatic analysis, then the starvation experiments indicate a destruction of enzyme rather than a loss of activity by dilution. In terms of induced enzyme synthesis the role of glucose would appear to be that of a carbon and energy source for the enzyme synthesis. That this may be too simple an explanation is indicated by the effectiveness of autolyzed spores HOURS OF INCUBATION in reducing the mannose-lag. Further, the or- Figure 4. Growth in synthetic medium con- ganism can use acetate as a carbon and energy taining 0.45 per cent mannose plus 0.05 per cent source (Machbis, 1953c); yet when acetate was glucose (A), 0.5 per cent mannose plus 1 ml auto- substituted for glucose, it failed to reduce the claved mitospores (B), 0.5 per cent mannose plus mannose-lag. These observations suggest that 0.5 ml autoclaved mitospores (C), and 0.5 per something produced secondarily from glucose cent mannose (D). and also present in autolyzed spores is the active agent in initiating growth in mannose. Approximately 5 mg of glucose per flask are Although induced enzyme synthesis has been necessary to effect a reduction in the mannose- tentatively invoked to explain the above results, lag equal to that effected by the autoclaved we are cognizant not only of the lack of proof spores; yet the dry weight of spores in 1.0 ml of but also of certain major differences between inoculum was only 0.03 mg or about 0.6 per cent the growth behavior of bacteria and yeasts, of the equivalent glucose. Such a low level of upon which most of the work on induced enzyme glucose is without effect. The experiment suggests systems has been done, and that of a filamentous that something in the spores other than glucose fungus like Allomyces. Two differences are par- activates mannose utilization and is effective in ticularly important. A mitospore upon introduc- low concentrations. The release of these sub- tion into a nutrient medium is a motile cell. stances by autolysis might explain the long and Sooner or later, the flagellum is lost and the variable lag in mannose medium. spore develops into a germling consisting of a Other substrates. A long list of sugars and re- rhizoidal system and a hyphal tube. From the lated substances has been shown by Quantz tube develop the typical, dichotomously branch- (1943), Machlis (1953c), and Ingraham and ing hyphae which constitute the bulk of a mature Emerson (1954) to be unsuitable as carbon and culture (Emerson, 1941). This contrasts con- energy sources for the growth of Burma lDa as siderably with the much simpler growth pattern well as other strains of Allomyces. Of interest of a bacterial culture. Secondly, cytological ob- was the possibility that utilization of these sub- servations indicate that active protoplasm is stances could be initiated by a small amount of concentrated in the hyphal tips of the fungal glucose. Cultures containing 0.5 per cent of the culture. Thus, as a culture proceeds through the following substances, with and without 0.05 per linear growth phase, it seems likely that more and cent glucose, were prepared and incubated for 19 more of it consists of empty and dead hyphal days: sucrose, lactose, galactose, arabinose, walls. The relation of these factors to the growth xylose, sorbose, raffinose, rhamnose, dulcitol, and to use of mycelial and mannitol. No significant growth lags in various media the sorbitol, fragments as inoculum is unknown. was observed.

DISCUSSION SUMMARY A possible explanation of the facts is the hy- The Burma lDa strain of the filamentous pothesis that the utilization of mannose and of watermold Allomyces macrogynus grows i 1955] UTILIZATION OF MANNOSE AND FRUCTOSE BY FUNGUS 55

synthetic liquid nutrient medium with mannose cytotaxonomy of Euallomyces. Mycologia, 46, or fructose as the carbon and energy source only 393-434. after a long and variable growth lag. A small INGRAHAM, J. L., AND EMERSON, R. 1954 Stud- amount of glucose makes possible immediate and ies of the nutrition and metabolism of the uniform growth equal to that with glucose as the aquatic Phycomycete, Allomyces. Am. J. Botany, 41, 146-152. carbon and energy source. Autoclaved spores MACHLIS, L. 1953a Growth and nutrition of also reduce the growth lag in mannose. Mutants water molds in the subgenus Euallomyces. for mannose utilization were not found by various I. Growth factor requirements. Am. J. isolation methods. The ability of spores or my- Botany, 40, 189-195. celial fragments to grow in mannose without Fn MACHLIs, L. 1953b Growth and nutrition of extended lag depends on the continuous pres- water molds in the subgenus Euallomyces. ence of mannose. The results are interpreted in II. Optimal composition of the minimal terms of induced enzyme synthesis. medium. Am. J. Botany, 40, 450-460. MACHLIS, L. 1953c Growth and nutrition of REFERENCES water molds in the subgenus Euallomyces. EMERSON, R. 1941 An experimental study of III. Carbon sources. Am. J. Botany, 40, the life cycles and of Allomyces. 460-464. Lloydia, 4, 77-144. QUANTZ, L. 1943 Untersuchungen zur Ernih- EMERSON, R., AND WILSON, C. M. 1954 Inter- rungsphysiologie einiger niederer Phyco- specific hybrids and the cytogenetics and myceten. Jahrb. wiss. Botan., 91, 120-168.