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THE EFFECT OF D-GLUCOSE ON THE UTILIZATION OF D-MANNOSE AND D-FRUCTOSE BY A FILAMENTOUS FUNGUS' 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, Allomyces 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 methionine, 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.

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