PHOTOMORPHGGENI’ESIS EN A NEW AQUATEC E’UNGUS BLASTQCLQMELLA BRHANNECA That. {01’ Hm Degree a? DB. D. MICHEGAN STATE UNIVERSITY Evelyn Anne Horenstein 1965 THESIS LIBRARY Michigan Statfl University This is to certify that the ' thesis entitled Photomorphogenesis in a new aquatic fungus, Blastocladiella britannica presented by Evelyn Anne Horenstein has been accepted towards fulfillment of the requirements for Eh . D. degree in _an:an.y_ WOW Major professor Date May 6, 1955 0-169 .3 r“ v. r. I s. L. 3. a. .. A, r. t . a. E a. v. .. .7. f. C E f. .«u .rw .au a? L; “A «C a; no ~‘ ‘ , AC ABSTRACT PHOTOMORPHOGENESIS IN A NEW AQUATIC FUNGUS BLASTOCLADIELLA BRITANNICA by Evelyn Anne Horenstein The developmental history in pure culture of a single— spore isolate of a Blastocladiella was investigated in some detail. No other species in the Blastocladiaceae which has been subjected to rigorous examination displays such a high degree of morphological variability--a variability which is probably phenotypically controlled. This characteristic led to the isolation of several distinct substrains, one of which was incapable of producing resistant sporangia and another (strain B 101) produced them in abundance. As a consequence of these and other observations, this single- celled fungus was designated a new species, B. britannica° In strain B 101, formation of resistant Sporangia was affected by several environmental factors, but most strik- ingly by white light. When grown on agar media, it produces: (a) in the dark, about 90-100% brown, thick-walled resistant sporangia (RS) with a generation time of about 65 hr.; (b) in white light, nearly colorless, thin-walled (TW) Sporangia with a generation time of about 30 hr. However, neither the absence nor the presence of light is required continuously 5r” RV‘ v..- ... o i. _. 1 1. 24 ...t c. S, 5 we‘— 1-. v.4 _: Evelyn Anne Horenstein throughout the entire growth period for the genesis of one or the other of these morphological forms° The organism's early stages of development are quite plastic; cells which start their growth in the light, and which are, therefore, on the TW pathway, can be induced to revert to RS types by eliminating the light. Conversely, cells which start growth in the dark (i.e. along the RS pathway) can be transformed into TW types by exposure to light. In both instances, however, a stage in development is reached beyond which addition or withdrawal of illumination can no longer effect morphogenetic reversal. At this point of no return, the cells have become committed to one pattern or the other. To study photomorphogenesis at a chemical level, it was necessary to grow the organism in large quantities under conditions which induced reproducible morphological uniform- ity and which could be controlled as precisely as possible. Consequently, a method was devised for growing synchronized, single generations of B. britannica (a million or more cells at a time) uniformly suspended in agitated media, wherein the effects of light and dark on morphogenesis were demon- strable as well as the reversal of morphogenesis by altera- tion of the light and dark regime before a point of no return. Up to a stage just preceding the end of the generation time of a TW sporangium, no morphological differences are discernible under the light microscope between it and a ’1 (-u r12?" U» o I0. {I u o '0 a \ I. . 1 c. .3 E C f I C x... c. r; .. Y a... a: v. C C. E Q . ... a. .t .. 3. K I C S E E C C E . In _. v e T. a” C E L a. f C. 7. T. f 3L w. .. i n. e a. C. L... r. . . S a L 3. a, .r“ .3 L” L... nun s; «a Co at a» . . To .3 3 . C .C .C s. E C n. C ‘5 I 3. C. Evelyn Anne Horenstein dark-grown developing RS at a corresponding chronological age. Yet, within the next 1-2 hr., the entire protoplast of a TW is cleaved into hundreds of uninucleate, uniflagellate motile spores, and this new generation of cells is then dis- charged. On the other hand, the thalli growing in the dark have reached only the half-way point in their ontogeny; they continue to enlarge for several hours thereafter and then gradually differentiate into mature RS. In synchronous cultures, dry weight/cell increases exponentially at the same rate in light and dark. On the other hand, the capacity for uptake of glucose by cells of various ages grown in the dark exceeds that of light-grown cells. Furthermore, just as the course of development can be reversed by excluding or supplying light before their respective points of no return, so, too, their capacities for glucose uptake can be similarly reversed. However, the point of no return for glucose uptake precedes the point of no return for morphogenesis by several hours. The light- sensitive glucose uptake by B. britannica may be a factor in the determination of the ultimate morphology of this organism. PHOTOMORPHOGENESIS IN A NEW AQUATIC FUNGUS BLASTOCLADIELLA BRITANNICA BY Evelyn Anne Horenstein A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1965 ACKNOWLEDGMENTS I am very grateful to Dr. L. G. Willoughby who graciously sent us the culture of Blastocladiella britannica, thus affording me the opportunity to study this organism. Thanks are due to Professor William M. Seaman, who kindly provided the Latin translation for the diag- nosis of Blastocladiella britannica. For his capable guidance, his never-failing en- couragement and expressions of confidence, and his enthusiastic support, I should like to express my very deepest gratitude to Professor Edward C. Cantino who, more than anyone, is responsible for the successful completion of this thesis. *************** ii To Mother, Dad, and Don iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . 4 Description of the Blastocladiaceae with Emphasis on Unique Characteristics of the Family. . . . 4 The motile spore . . . . . . . . . . . . . . . 6 The resistant sporangium . . . . . . . . . . . 10 Life Cycles in the Blastocladiales . . . . . . . . 12 Brachyallomyces type . . . . . . . . . . . . . 12 Cystogenes type. . . . . . . . . . . . . . . . 12 Euallomyces type . . . . . . . . . . . . . . . 15 Differentiation of the Resistant Sporangium in Blastocladiella emersonii. . . . . . . . . . . 15 Induction of resistant sporangium formation. . 15 Formulation of a hypothesis. . . . . . . . . . 15 Biochemistry of morphogenesis. . . . . . . . . 19 Period of exponential growth . . . . . . . 22 Period of differentiation. . . . . . . . . 27 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . 32 Culture Procedures . . . . . . . . . . . . . . . . 52 Analytical Procedures. . . . . . . . . . . . . . . 37 OBSERVATIONS AND EXPERIMENTAL. . . . . . . . . . . . . 41 Conditions for Optimal Growth. . . . . . . . . . . 41 Morphological Variability. . . . . . . . . . . . . 42 Pure Strains of TW Colonies. . . . . . . . . . . . 47 The RS Colonial Strain . . . . . . . . . . . . . . 50 Life history . . . . . . . . . . . . . . . . . 50 Effect of temperature on RS formation. . . . . 51 Effect of bicarbonate on RS formation. . . . . 51 Effect of glucose on RS formation. . . . . . . 52 Effect of light on RS formation. . . . . 54 Isolation of RS strains yielding 100% indi- vidual RS plants . . . . . . . . . . . . . 54 Development of Synchronous Cultures of Resistant Sporangia. . . . . . . . . . . . . . . 60 Growth and Morphological Characteristics of Syn- chronized Single Generation Cultures . . . . . 68 iv TABLE OF CONTENTS - Continued Growth pattern in the dark Growth pattern in the light. Point of no return in development. Glucose Uptake Capacity. . . . Reversal of GUC. . . . . . The point of no return for GUC DISCUSSION . . o . . . . . . . . . The Taxonomic Position of Blastocladiella britan- nica O O O O O O I O O O O The Aquatic Phycomycetes and Biological Research Morphogenesis in Blastocladiella britannica. Morphological variability. Photomorphogenesis . . . . SUMMARY. . . . . . . . . . . . . . LIST OF REFERENCES . . . . . . . . APPENDICES . . . . . . . . . . . . Page 70 71 76 79 81 82 85 95 95 97 101 102 108 TABLE II III IV VII LIST OF TABLES Page Lactic acid production by B. britannica. . . . 41 Morphological types produced by B. britannica. 46 Relationships among volume per cell, weight per cell, and weight per unit volume of TW cells at maturity. . . . . . . . . . . . . . . 76 GUC and intracellular pools. . . . . . . . . . 117 Effect of phosphate on GUC . . . . . . . . . . 119 Effect on GUC of different sugars during growth . . . . . . . . . . . . . . . . . . . . 120 Effect on GUC of the presence of other sugars. 122 vi LIST OF FIGURES FIGURE 1. Life cycles in the Blastocladiales . . . . . . . 2. Enzyme reversal in B. emersonii. . . . . . . . . 5. Comparative activities of isocitritase in syn— 10. 11. 12. 15. 14. chronously developing OC and RS plants of B. emersonii during ontogeny . . . . . . . . . . Transformations in total RNA during morpho- genesis of RS. . . . . . . . . . . . . . . . . . Transformations in composition of RNA during . sol morphogene51s of RS. . . . . . . . . . . . . . . Changes in protein nitrogen, plant volume, and dry wt. during RS morphogenesis in B. emersonii. Increase in chitin and melanin content during RS differentiation in B. emersonii . . . . . . . Changes in lactate pool, glucose-6-ph05phate dehydrogenase, glucose~consumption, and poly— saccharide pool during RS differentiation in B. emersonii . . . . . . . . . . . . . . . . . . Developmental potentialities in B. britannica. . Size of spores from the TW and RS strains. . . . Effect of different concentrations of PYG (2% agar) on the composition of populations derived from spores of TW colonies . . . . . . . . . . . Effect of different concentrations of PYG (2% agar) on the composition of populations derived from spores of RS colonies . . . . . . . . . . . Effect of different concentrations of glucose (in PYG) on RS formation . . . . . . . . . . . . Effects of semi-anaerobic conditions on the composition of populations derived from spores of RS colonies . . . . . . . . . . . . . . . . . vii Page 14 24 26 28 28 50 50 50 45 48 55 55 55 HFA 2.. ‘r— .r. §\.V _./L I: r: .3 .1 ../~ LIST OF FIGURES - Continued FIGURE 15. 16. 17. 18. 19. 20. 21. 22. 25. 24. 25. 26. 27. 28. 29. 50. 51. Effect of light on RS plant development. . . . . The relation between duration of light and RS formation on medium PYG. . . . . . . . . . . . . Results of attempts to select populations of vigorous RS producers. . . . . . . . . . . . . . Growth curves of individual RS plants. . . . . . Incidence of RS after transfer from liquid to solid media. . . . . . . . . . . . . . . . . . Growth of RS cells in the dark . . . . . . . . . Effect of pH upon the growth rate of TW cells in the light. . . . . . . . . . . . . . . . . . . . Effect of population density upon the growth of TW cells . . . . . . . . . . . . . . . . . . . . Effect of population density upon the volume of TW cells at generation time. . . . . . . . . . . Effect of population density upon the generation time of TW cells . . . . . . . . . . . . . . . . Relationship between the volume of the TW cell at generation time and its dry wt. per unit volume at this time. . . . . . . . . . . . . Points of no return for RS and TW cells. . . . . Cell types resulting from exposure to different light-dark regimes . . . . . . . . . . . . . . . Increase in dry wt. of synchronous cultures. . . The glucose uptake capacity of light-grown and dark-grown cells at different stages in ontogeny at 24 . . . . . . . . . . . . . . . . . . . . . The reversal of glucose uptake capacity. . . The point of no return for glucose uptake capacity . . . . . . . . . . . . . . . . . . . . viii Page 55 56 59 59 64 72 72 74 74 75 75 78 78 80 80 84 84 PLATE II III LIST OF PLATES Page Blastocladiella britannica . . . . . . . . . . . 45 B. britannica grown in light and dark. . . . . . 61 Selected stages in development of TW and RS cells. . . . . . . . . . . . . . . . . . . . . . 69 ix LIST OF APPENDICES APPENDIX Page I List of Abbreviations. . . . . . . . . . . . . 107 II Attempts at RS Germination . . . . . . . . . . 110 III Ballons d'essai. . . . . . . . . . . . . . . . 116 INTRODUCTION As a result of an ecological investigation of the lower saprophytic fungi from Esthwaite Water in the Lake District of England, Willoughby (1959) isolated the first species of Blastocladiella from Great Britain or the continent of Europe; all previously reported species were obtained in the Western Hemisphere. He suggested that we might be interested in this fungus and sent us pure cultures from one of his single spore isolates. Because I had been associated with him for a number of years in his research on Blastocladiella emersonii, Dr. Cantino thought I might enjoy and profit from the experience of working with a different organism. The idea was that I would have a look at this new water mold; that is, grow it, observe its characteristics, and determine if, indeed, it was a new Blastocladiella. At the outset, I expected that this new creature would be thoroughly domesticated and all pertinent observations would be completed within a few months, after which time I would resume my work with B. emersonii. My naiveté has long since vanished; there were very few observations that could be made without qualifications. As a result, I have been 'looking at' this organism ever since. Along with these qualifications came questions, aris- ing at an ever accelerating rate. I was very soon forced into making a choice concerning what aspects of these riddles I was going to pursue. Being especially intrigued by the degree of variability in developmental potential displayed by this microfungus--the frustrations far exceeding the intrigue at times--I chose to exploit this particular attribute for morphogenetic studies. Past experience with B. emersonii was also a significant influencing factor in that I had acquired an appreciation for these tiny plants as valuable tools in such investigations. Notwithstanding the fact that the Blastocladiellas, for example, are one- and two-celled plants and have been rele- gated to a poSition on one of the lower branches of the phylo- genetic tree, they exhibit what appears to be a highly developed system of regulatory mechanisms enabling them to alter their morphogenetic pattern in response to various environmental conditions. Massive cultures of synchronously growing plants can be obtained with ease and almost any phase of their development can be followed throughout a single generation. Transitions from one moment to the next, includ- ing many of the dynamic aspects attached thereto, can be analyzed in a complete organism--not merely an amputated organ or tissue. Those of us who have worked with the water molds feel that they have much to offer to studies of growth and differentiation. This thesis represents essentially a progress report on what has been accomplished to date on Blastocladiella britannica, including its establishment as a new species. Only a begin- ning has been made, for after a number of years an under- standing of its nature and behavior has but advanced to the point where it now provides a more enlightened notion of what questions to ask of it. LI TERATURE REVI EW Description of the Blastocladiaceae with Emphasis on Unique Characteristics of the Family The group commonly referred to as the aquatic Phycomy- cetes comprises the most primitive of the true fungi. In spite of the fact that they are such an ubiquitous and myriad lot (see Sparrow, 1960), relatively little, aside from taxonomic descriptions, is known of most of them. There are only a few genera, primarily in the family Blastocladiaceae, about which an abundance of information has become available. The genus Blastocladia was first described by Reinsch (1878) with the discovery of B. pringsheimii. In the original diagnosis, he placed this organism in the Saprolegniales prob— ably because of the presence of what he mistakenly thought were oogonia, the female reproductive organs which are regu- larly formed in this group. In 1909 the order Blastocladiales was proposed by Petersen (1909) to incorporate this single genus. This decision was based on two characteristics-~the lack of cellulose in the walls of Blastocladia and the absence of sexual reproduction, both well established characters of the Saprolegniales. Since that time, three additional genera have been added to the family Blastocladiaceae which typifies the order. They are Allomyces (Butler, 1911), Blastocladiella (Matthews, 1957), and Blastocladigpsis (Sparrow, 1950). These are all saprophytes and can be found in damp soils and slow moving bodies of fresh water, growing on organic debris of a diverse variety (Sparrow, 1960). It is not unusual for them to be associated with great numbers of microflora and microfauna, sometimes forming dense pustules surrounded by a slimy layer of microorganisms. The family is characterized by the following general features of morphology and growth habit. The very distinctive motile, uninucleate spore (swarmer), after a period of swim- ming, settles down and germinates. A delicate germ tube emerges and proliferates into a branched system of rhizoids which seem to serve, in the main, to anchor the growing plant to its substratum.* Shortly after the initiation of the rhizoidal system, the chitinous-walled thallus begins to develop. The extent of this development is highly variable and, like the final architecture, is dependent upon the genus- For the simplest type of morphology we look to ELEEEQ‘ cladiella, which displays a determinate system of growth. At the end of its growing period (i.e. when there is no fur— ther increase in size), the greater part of the plant body becomes delimited from a basal portion by a septum and is converted into a single reproductive structure: either a *- It has often been stated that the rhizoids also serve as nutrient gathering devices although no evidence has ever been offered to substantiate this assertion. colorless, thin-walled eporangium or a brown, thick-walled resistant sporangium. Blastocladia, which is also characterized by a determin— ate growth pattern, does however, develop into a more complex structure which may be branched or lobed and on which are usually formed several thin-walled sporangia and/or resistant sporangia. Blastocladiopsis does not differ radically from Blasto- cladia and is differentiated from it by certain features of its resistant sporangium. The genus Allomyces, on the other hand, manifests an indeterminate system of growth characterized by extensive dichotomously branched hyphae, on the ends of which develop the reproductive organs. None of these fungi is septate (although Allomyces dis- plays 'pseudosepta'). except when the reproductive structures become partitioned from the rest of the plant; thus, they are coenocytic organisms, with their many nuclei dispersed through- out the cytoplasm of the thallus. At maturity, the entire protoplasmic content of the multinucleate sporangium under— goes progressive cleavage into uninucleate cells which are then liberated through one or more papillae or discharge tubes as motile spores, ready to begin the cycle once again. The motile spore The spore is a motile cell measuring about 5 to 12 u in length and slightly less in width, the exact dimensions varying with the species. It possesses a single, posterior, whiplash flagellum (Sparrow, 1960), the length of which is several times that of the spore body. Numerous observations made with the light microscope resulted in the following composite picture of the Blastocladiaceous spore. 1) Near the posterior end of the spore is found the single prominent nucleus with its nucleolus. 2) Surrounding the apical portion of the nucleus and extending much beyond, making it the most conspicuous inclu— sion in the spore, is the nuclear cap. This organelle, which is typical of the Blastocladiales and some Chytridiales, has long been referred to as a 'foodbody' (another example of mycological guesswork) although its function was not under- stood (Barrett, 1912; Emerson, 1941; Sparrow, 1960). 5) In some Species of Blastocladiella, a body along one side of the nuclear apparatus is consistently observed. Harder and Sargel (1958) first referred to it as the 'seitenkorper' in their description of B. variabilis. 4) There are also many smaller organelles, including refractile lipid granules and vacuoles (and in Blastocladiella emersonii, some very small 'gamma' particles which stain with * the Nadi reagent ; Cantino and Horenstein, 1956a). More recent studies employing the electron microscope, cytochemical techniques, and chemical analyses have elucidated *- This color reaction results from the oxidation of di- methyl-p-phenylenediamine in the presence of ornaphthol and is catalyzed by oxidative enzymes. the situation enormously and provided us with a more sophisti- cated picture of the spore. The results of these investi- gations are summarized below. 1) Electron photomicrographs of gametes of Allomyces macrogynus and spores of Blastocladiella emersonii have shown the flagellum of both species to be structurally similar to that of many other motile cells (Hoffman-Berling, 1959). There are two central fibrils surrounded by nine outer ones, all enclosed within the flagellar sheath (Blondel and Turian, 1960; Cantino et al., 1965). Fourteen years ago, Cantino (1951) observed the retraction of the flagellum into the body of the spore upon germination of B. emersonii. More recently, electron photomicrographs verified the presence of the flagellum within the cell after retraction, where it was ex- tended along almost the entire inner periphery of the spore wall (Lovett, unpublished). 2) Results obtained with various cytochemical tech- niques led Turian (1955; 1958) to conclude that the nuclear caps of Allomyces gametes represent a localized accumulation of RNA. Additional evidence was offered by Blondel and Turian (1960), who reported that RNA-containing particles (presumably ribosomes) concentrated in the areas surrounding the nuclei in the mature gametangia of A. macrogynus during the genesis of the caps, just as gametes were being cleaved. Electron photomicrographs suggested that the nuclear caps of Allomyces gametes and Blastocladiella spores are similar. Virtually all of the-electron dense particles in the swarmers of B. emersonii are contained within its nuclear cap (Cantino et al., 1965; Lovett, 1965). Chemical analyses of 'clean' nuclear caps isolated from the spores of B. emersonii cor- roborated what was implied by the electron photomicrographs (Lovett, 1965). The caps represented 69% of the total RNA of the cell and they, themselves, were composed of 57% protein and 65% RNA. These data, in addition to other evidence, support the concept that this organelle is a unique package of ribosomes. However, relative to ontogeny, this aggregation of ribosomes is a temporary phenomenon; at the onset of germination of both the spore of Blastocladiella and the zygote of Allomyces (in which the nuclear caps from the two participating gametes have fused), the membrane bounding the nuclear cap disintegrates and the ribosomes are dispersed throughout the cytoplasm (Turian, 1958; Blondel and Turian, 1960; Lovett, 1965; Cantino and Lovett, 1964). 5) Electron microscopy has revealed another structural similarity between Allomyces gametes and Blastocladiella spores. The double membrane which separates the nuclear cap from the nucleus is perforated by pores, thus possibly perm mitting exchange of materials between these two structures (Turian and Kellenberger, 1956; Cantino et al., 1965). These seem to be the first documented instances of such intimate contact between the bulk of the DNA and RNA of a cell (Cantino and Lovett, 1964). 10 4) These same studies (Cantino et al., 1965) provided evidence that the everpresent 'sidebody' in B. emersonii is a giant mitochondrion, the gnly_mitochondrion in the spore: This somewhat cup-shaped body extends upward from the vicinity of the flagellum at the base of the cell to about 2/5 of the distance on one side of the swarmer. The mito- chondrion is penetrated by the flagellum which appears to be attached to it yia_one or more rootlet-like appendages. In view of the fact that the flagellum propels the spore while it is actively swimming, one would expect that a great deal of energy is being expended during this process. As a matter of fact, the endogenous 002 of the spores is close to 100 at this time (McCurdy and Cantino, 1960). This, then, would be a most convenient location for the energy source needed for motility. The revelation of a solitary mitochondrion in the spore of B. emersonii is a deviation from the situation in the gamete of Allomyces macrogynus where several mito- chondria, of smaller dimensions, have been detected (Turian and Kellenberger, 1956; Blondel and Turian, 1960). The resistant sporangium It is not exceptional that aquatic microorganisms have evolved an expedient, usually in the form of a specialized resistant structure, to 'guarantee' their survival under otherwise intolerable environmental situations. The Blasto- cladiales have evolved the resistant Sporangium. Instead of 11 developing a thin—walled sporangium, the fungus produces one possessing a thick chitinous wall (Lovett and Cantino, 1960a) which becomes impregnated with melanin (Emerson and Fox, 1940; Cantino and Horenstein, 1955). After its fabrication, the resistant sporangium remains in a state of dormancy until conditions become favorable for the release of spores. Resistant sporangia of Allomyces have retained their viability after twenty years of desiccation (Emerson, 1954), and B. emersonii for almost as long. It had been assumed by some for many years, but never shown experimentally, that meiosis occurred in the zygote of the aquatic fungi. In 1949 Emerson and Wilson (1949; Wilson. 1952) made the first detailed study of meiosis in a water mold and offered conclusive evidence that in Euallomyces, meiosis occurs in the resistant sporangium. This demonstration of the site of meiosis placed Allomyces in a unique category of filamentous fungi, in which vegetative hyphae are truly diploid. To distinguish between the two types of swarmers liberated from the sporophytic plant, Emerson (1950) proposed the term 'mitospores' for diploid spores resulting from mitotic divisions and liberated from thin-walled sporangia, and 'meiospores' for haploid spores resulting from meiotic divisions and liberated from resistant sporangia. The site of meiosis, if it occurs at all, has not been established for either Blastocladia or Blastocladiella. 12 Life Cycles in the Blastocladiales Emerson (1941) assigned subgeneric names to Allomyces on the basis of three different types of life cycles which it displayed. Inasmuch as these three patterns circumscribe all the known life cycles of the other genera as well, Sparrow (1960) adopted Emerson's nomenclature to include the entire order Blastocladiales. This precedent will be con— tinued in the following description. Brachyallomyces type The simplest of the life histories is the Brachyallomyces type (Fig. 1). This comprises an asexual (or sporophytic) generation only, and is exhibited by such Species as Blasto- cladia pringsheimii, Blastocladiella emersonii, and Allomyces anomolous. Spores released from both thin-walled sporangia and resistant sporangia develop directly once again into a new generation of sporophytes. Cystogenes type The organisms displaying the Cystogenes life cycle under~ go both sexual and asexual reproduction (Fig. 1). The asexual is much the dominant phase and is similar to the Brachy- allomyces type as far as the Spores released from the thin- walled sporangium are concerned. But the spores from the re~ sistant sporangium encyst shortly after discharge; the cyst constitutes the gametophyte. Isogametes emerge from the cyst, and the zygote resulting from sexual fusions develops once 15 again into a sporophyte. Organisms of this sort have not been studied extensively; therefore, more detailed information is lacking. Allomyces cystogenus and Blastocladiella gystogena are two species displaying such behavior. Euallomyces type This third type is one in which there is an alteration of equivalent Sporophytic and gametophytic generations (Fig. 1). The sporophyte is morphologically the same as in the two previous types. The basic growth pattern of the gametophyte is similar to the sporophyte except when the reproductive cells are differentiated. In Allomyces, gametangia are formed in pairs on the same thallus. One member of the pair develops into a female gametangium, much like the colorless thin-walled sporangium in appearance. The other member, the male, in addition to being different in size than the female (smaller or larger, depending on the species), reveals, at maturity, orange protoplasm resulting mainly from the presence of gamma carotene in lipoidal bodies (Emerson and Fox, 1940). The arrangement of these paired gametangia is species specific. In A. macrogynus, the orange male gametangium is terminal (epigynous) and in A. arbuscula, the male is subterminal (hypogynous). The motile female gametes liberated from color- less gametangia resemble mito— or meiospores in morphology, while male gametes are orange in color, somewhat smaller in size, and more rapid in motion. Male and female gametes (often from the same plant) fuse to form a biflagellate zygote. SfiAv Afi" an- r" . _ 3.. 3. VI 3 1| H. v. Z a. “a Cu l. .l..4. li.~v:- iilu‘l 75.1.. ‘ IA 14 » Spores L i Thin-walled sporangium Sporophyte~<:::::::: Brachyallomyces Resistant sporangium t 4, Spores - Spores l, Thin-walled sporangium Sporophyte <::::::; T Resistant sporangium Zygote Spores Cystogenes 1 Gametes Gametophytic cyst MitOSpores Thin-walled sporangium Sporophyte <:::::::; Resistant Sporangium (K i, Meiospores Euallomyces Gametophyte 3' Gametangium 3 Gametangium 51 Gametes 9 Gametes Zygote Fig. 1. Life cycles in the Blastocladiales 15 Following a brief swimming period, the zygote develops into a new sporophytic generation. Copulation between two aniso- planogametes was described by Kniep (1929) in the first accounts of sexuality in Allomyces. This is a phenomenon that hitherto had not been known to occur among the fungi; up to the present, such fusions are, to my knowledge, still un- known outside of the Blastocladiales. The description of Blastocladiella variabilis (Harder and Sérgel, 1958) renders the life cycle of this organism similar to Euallomyces; however, the male and female gametangia are on individual thalli by virtue of its monocentric nature. This type of life cycle, then, is representative of_a group of Blastocladiales which is distinctive among the aquatic fungi: (a) the sporophyte is an independent diploid generation; (b) the resting stage is an asexually formed resistant sporangium; (c) the zygote germinates immediately after its formation. Differentiation of the Resistant Sporangium in Blastocladiella emersonii Induction of resistant sporangium formation Emerson (1954) has said of resistant Sporangia (RS). "Observing their highly modified structure, noting their special function, and recalling that they are the locus of the critical reduction divisions in Allomyces, one can immediately recognize their intrinsic interest." But until relatively 16 recent times, not much attention was focused on this alternate developmental structure. Perhaps one of the reasons is that when some of these fungi were brought into the laboratory and divested of all other creatures commonly sharing their habitat, RS were rarely seen. This is what happened when Emerson and Cantino isolated Blastocladia for the first time, preparative to carrying out studies on nutrition and physiology (Emerson and Cantino, 1948). When grown in pure culture under aerobic conditions, RS of B.Apringsheimii were unobtainable. It wasn't until pure tank C02 was bubbled through the medium, in which the pH was maintained between 5.5-5.8, that these structures were produced. A few years later a new species of Blastocladiella, B. emersonii, was isolated by Cantino (1951). With this organism too, difficulty was met in attempts to obtain RS. When swarmers were inoculated onto agar media, the first generation population consisted solely of thin-walled, ordi- nary colorless (OC)* plants. When each of these OC plants matured and discharged swarmers, hundreds of new plants grew, clustered around the now evacuated parent Sporangium. Among each of the second generation clones, varying numbers of RS appeared. It turned out that C02, as bicarbonate, played a fundamental role in RS induction in B. emersonii, just as * 0C sporangia are equivalent to thin-walled sporangia, but since ’OC' is entrenched in the literature on B. emersonii, its use will be continued in the discussion of this species. t ”.6" .pb“ rr * .. b 15" av“ ' fin. V Us. ‘ n v... - v - i 7‘ f ‘ Au“ 0V .4 {jvqfi n s-.u» V a .-,\. -VV'I . 17 with Blastocladia pringsheimii, although its effective concen- tration was of an entirely different magnitude and the opti- mum pH range was not so narrow. Blastocladiella did not tolerate C02 concentrations above 5%; however, by incorporat— ing 10'2 M bicarbonate into the nutrient agar, the resultant first generation populations consisted of 95-100% RS (Cantino, 1952). When the bicarbonate was omitted, the population invariably consisted, instead, of 100% OC plants. Here, then, was an organism whose genotype endowed it with the capacity to develop into one of two distinct morphological types--either a smooth, thin-walled OC sporangium or a pitted, thick-walled, brown RS (with a longer generation time)--depending on the absence or presence of bicarbonate, respectively. Bicarbonate acted in some way to effect a profound modification in a morphogenetic pattern which, in nature, could literally Spell the difference between perpetuation or extinction of the Species. Formulation of a hypothesis In the earliest studies, it was found that under certain nutritional conditions where bicarbonate alone was insufficient to induce RS in B. emersonii, the addition of obketoglutarate in conjunction with bicarbonate did induce their formation. Subsequent experiments, centered around the possible involve- ment of orketoglutarate, produced the following results (Cantino, 1951): (a) biotin, which has been implicated in l . c 1. .C 1: 1: C 3 C T. a... 3. C C C. S .3 a . . 18 oxidative decarboxylation reactions (Lichstein, 1960), pre- vented RS formation in the presence of afiketoglutarate; (b) arsenite (an inhibitor of oxidative decarboxylations) and semicarbazide-(a keto reagent) augmented the effect of orketoglutarate in RS induction. These same metabolic in- hibitors prevented the germination of mature RS at the identi- cal concentrations that facilitated their formation; (c) when OC plants were bathed in bicarbonate solution, their internal pool of diketoglutarate increased. These observations led to the notion that bicarbonate somehow interfered with the normal operation of the Krebs cycle by preventing the oxidative decarboxylation of orketoglutarate and thereby caused it to accumulate within the plant (Cantino, 1955). When OC plants are grown in the presence of glucose, under certain conditions, fermentation to lactic acid is the predominant dissimilatory mechanism (Cantino, 1951). Although homogenates of these same cells reveal the presence of all the enzymes associated with the Krebs cycle, it seemed unlikely that it functioned as the major energy source for the plants' requirements under these conditions. Cantino suggested that in B. emersonii, the role of this cycle lay elsewhere, such as the focal point for shunt mechanisms emenating from the accumulated oPketoglutarate and ultimately leading to the genesis of an RS. This notion was strengthened by the exami- nation of a mutant strain of B. emersonii which was incapable 19 of forming RS, even in the presence of bicarbonate. This strain was devoid of aconitase and orketoglutarate dehydrogen- ase, thus the two successive decarboxylations in the Krebs cycle could not take place (Cantino and Hyatt, 1955a). As a consequence of the preceding discoveries, the following hypothesis was formulated: 1) Increased concentrations of bicarbonate interfere with the normal operation of the Krebs cycle. 2) The effect of this obstruction is to set in motion shunt mechanisms leading to the genesis of RS. 5) The primary locus for the triggering of these mechanisms, at the biochemical level, is at the orketoglutarate area of metabolism. Biochemistry of morphogenesis Intensive investigation was now directed toward the elucidation of the "biological Significance of the 'trigger mechanism' in terms of causal biochemical reactions involved” (Cantino, 1955). Comparative studies revealed the following distinguishing features associated with mature RS plants, as compared to mature OC plants: (a) de novo synthesis of y-carotene, melanin, and at least one new soluble protein fraction (Cantino, 1961a); (b) deposition of increased amounts of chitin and fat (Lovett and Cantino, 1960b): (c) disappear- ance of cytochrome oxidase and two soluble protein fractions (Cantino, 1961a); (d) drastic reduction, if not a complete loss, of enzymatic activity associated with the Krebs cycle 20 except for a TPN—specific isocitric dehydrogenase which, in the presence of bicarbonate, mediates the reductive carboxyl- ation of orketoglutarate (Cantino and Horenstein, 1955): (e) decrease in the free amino acid pool, particularly a sharp drop in tyrosine (Lovett and Cantino, 1960b). To test the hypothesis that new pathways, leading to the genesis of RS, derived from orketoglutarate, the melanin synthesizing system was selected for examination. The ob- jective was-to look for the presence of a polyphenol oxidase as well as a possible coupling to orketoglutarate. Such an enzyme was found in homogenates of RS plants; cell-free preparations oxidized tyrosine, catechol, and dihydroxyphenyl- alanine (Cantino and Horenstein, 1955). These activities were not present in preparations made from CC plants. More- over, this polyphenol oxidase, which seemed to function as a terminal oxidase in lieu of the cytochrome system which. dis- appeared in RS, could be coupled to either oxygen or TPN (but not DPN). The addition of drketoglutarate to the reaction mixture accelerated the enzymatic oxidation of catechol and tyrosine. It was postulated that the TPN, which served as a hydrogen acceptor in the tyrosinase reaction, was generated during the carboxylation of onetoglutarate to isocitrate. amino acidypool \ isocitrate tyrosine fumarate TPN succinate «ATPNH orketoglutarate melanin block C02 21 From the inception of this Work, it had been known that in order for B. emersonii to develop along the RS pathway, bicarbonate need not be present throughout the entire growth period. There is a point of no return in ontogeny--during the exponential growth period--before which the dual potenti- ality of the fungus is maintained. This means that plants which have been growing in the presence of bicarbonate, and therefore developing along the RS pathway, can be reversed to the OC pathway by removal of the bicarbonate. Beyond this point, the plants' fate has been sealed. This point of no return, therefore, signifies that the changeover to the meta- bolic machinery responsible for the fabrication of RS is com- plete and that differentiation is irrevocable. During the first 40% of their generation time, thalli developing along the RS pathway appear to be morphologically identical to OC thalli during a comparable period in their ontogeny. However, it seemed plausible to assume that certain mechanisms are put into play ty exogenous bicarbonate long before the morphological changes become discernible. In order to determine exactly what changes were occurring, methods were developed for obtaining synchronously developing mass cultures of both OC plants (McCurdy and Cantino, 1960; Goldstein and Cantino, 1962) and RS plants (Lovett and Cantino, 1960b). This means of growing populations of B. emersonii, in which 108 to 1010 plants are all precisely the same age and at the same developmental stage, made it possible to follow the 22 dynamics-of-various systems throughout a single generation. Investigations-of the relation between early biochemical events and later morphogenetic events were undertaken. In such RS culturesf-the-point of no return occurs at 45% of its 84 hour generation time (56 hr. at 240). This time also de- notes the termination of exponential growth (increase in Size) and the concurrent formation of a crosswall which delimits the sporangium from the basal portion of the thallus. The final 57% of the generation time is the period of RS differentiation. Period of exponential growth. Compared to an 0C plant, an RS plant exhibits a 46% decrease in its exponential growth rate (based on dry wt./cell) and an abrupt drop in Qoe—-the latter, by the end of exponential growth, amounts to a 90% reduction. These changes are, in themselves, not the cause of RS morphogenesis Since decreases in both growth rate and respiration can be brought about by other means without con- comitant RS production. Nevertheless, they are clearly effects induced by bicarbonate. One would expect that oxygen consumption would drop, for during this period the Krebs cycle comes to a virtual standstill (judging from changes in enzyme activity), no glucose is being consumed, and a reserve pool of polysaccharide is being accumulated. It was assumed a priori that any metabolic systems inti~ mately involved in RS morphogenesis and turned on by bicarbonate would be operative before the expression of the morphogenetic events they regulated, and that these systems would be 25 reversible before the point of no return but not afterwards. Cultures of RS were harvested at various ages, and two relevant enzyme systems were examined during the exponential growth period. There was a 6500-fold increase in isocitric dehydrogenase but-only a 650-fold increase in orketoglutarate dehydrogenase-activity; that is, the activity/cell of the latter enzyme rose to only 10% of that of isocitric dehydro— genase. This, considered together with the 90% reduction in oxygen consumption, is evidence for the presumed mode of operation of the bicarbonate trigger mechanism. Moreover, when bicarbonate was removed before 45% of the generation time had elapsed, orketoglutarate dehydrogenase increased the iso- citric dehydrogenase activity decreased, both of them approach— ing the level found in OC plants (Fig. 2). After the point of no return, the activities of these two enzymes remained un— affected by the removal of bicarbonate. McCurdy and Cantino (1960) demonstrated the operation of a previously postulated isocitritase system in B. emersonii, as well as a glycine-alanine transaminase. These two systems apparently operate in sequence to remove isocitrate as it is formed and thus prevent the carboxylation of asketoglutarate from bogging down. TPNH +-orketoglutarate + C02 -—fi>—isocitrate + TPN isocitritase glycine g1 oxylatSZ/+ succinate >' tr ansaminase ‘cmmouco weapon Hacomumsm .m mo mucmad mm new no mcHaon>mn xamaocoucocsm ca mmapuuuaoomw mo mmwuw>auoo m>eumumasou NEE. zo_._.04 «Dome .pum>04 pco ocaucmu scum pmpcaaoamomu mums pump m>onrmp muccemocutwtmmoonam .Amvaoou mumuoma cw mmmcmzu .wficomuweelqm ca cowuoaucmumumwp mm mCHuau pccaa\ucmucoo Anchcmame can Apvcwuficu CH mmmouocH A.oumumzu umumamu pump umcuo Ham new .00? up now mes Emu“ Loco uoe Hm>ma Enstce .mmuzmwu ucmnemmnam can has» nouv .«wcomumsm .m ca mammcmmozauoe mm mcwuan ucmaa\fiov.ua xup ecu .Anvmeaao> pecan .Amvcmmonuwc camuoue cw momenta wit. ZO_._.wumu mEOADmH canoe mo cowuwmoasoo on» co Anson RNV u>e Lo wcowumuucmocoo accumuewp Lo unmeuu .F..aau ¢>1 no xhczuzh. 00.x. Ox. Ox. N) 9954453 q q . q _ a I a -0 I I o .I/ a I 10 I . I . I. I II It II o O I npzaua 3» /.A\\ I ’ 00/ II ’ “v I I ’ 0 $9201.00 3... I I I "" l OOI NOIIV'IndOd d0 $ .mmuoam m mucmmmuamu mwxm aeoauum> mc» co coamw>qn comm A.»maow> cmwucmm cues pmcaanm ecu neon owemo me cues pmxfluv .mcaeupm me see an are seen mouse. to swam .oe.oau .4. .a<:..>< 0.9 ..O to FF OK ”.0 0.0 0.0. d u q s \ " Q Q Q 0' Q o \o O O \ a \ o .250 ‘a.on.>< z.<¢hm 3h 49 a fact quite consistent with the known distribution of such pigments in the Blastocladiaceae (Emerson and Fox, 1940; Cantino and Hyatt, 1955b; Cantino and Horenstein, 1956b; Turian and Cantino, 1959). Inasmuch as first generation plants of Blastocladiella emersonii could be induced to form RS by the addition of bicarbonate to PYG agar (cf. LITERATURE REVIEW), attempts were made to induce B. britannica to do the same.— In spite of repeated trials with a variety of bicarbonate concentrations, the results were uniformly negative. Then, because the concentration of PYG had affected the incidence of resistant sporangia in the RS strain (yiQ§_ infra), the pure TW colonial strain was grown on serial dilutions of the basal PYG medium. Although the number of TW clones increased as the concentration of PYG decreased (Fig. 11), neither RS plants nor RS clones were ever produced. Similarly, modifications of the glucose concentration in PYG (from 0.5% to zero) did not induce RS formation. Finally, because of the profound effects of illumination upon the RS strain (vide infra), cultures of the TW colonial strain were incubated on PYG in light and dark. The compo- sition of populations grown in the light was 40% TW colonies, 17% TW plants, and 45% non-viable, as compared with 10%, 90%, and nil, respectively, for those grown in the dark. Once again, no RS plants were produced. Thus, it was concluded that this pure TW colonial strain, incapable of producing RS 50 plants even though it was derived from a parental culture which had this-ability, had suffered a permanent metabolic lesion of some sort in its cytoplasmic and/or chromosomal machinery. For this reason, it Should serve as a valuable tool in studies on morphogenesis in B. britannica. The RS Colonial Strain Life histopy As was Shown in Table II, an assortment of progeny can be derived from an RS colony which is composed of varying proportions of RS plants and TW plants (Fig. 9). It should be emphasized at the start that there has been a total lack of success in inducing even one RS plant to discharge spores. Innumerable attempts have been made, using sporangia of many ages (5 days to 14 months) grown under a wide variety of conditions and maintained on wet agar media, soil, desiccated filter paper strips, and liquid cultures (nutrient media, soil media, hemp seeds in water, etc.). Furthermore, RS were subjected to all of the usual treatments (heat shocks, cold treatments, wetting agents, fat solvents, nutrients, etc.) plus many unusual treatments, but with a singular lack of success (see APPENDIX II). Thus, in the sections which follow, any reference to spore discharge from an RS colony will always mean that it is only the TW plants in such a colony which liberate spores. 51 Effect of temperature on RS formation The earliest Observations with the original culture and its progeny suggested that temperature had a differential effect on RS formation. Subsequent experiments revealed that when spores derived from RS clones were incubated on PYG agar at 12, 16, 19, 22, and 250 until all first generation plants had reached maturity, individual RS plants were seldom produced at 12, 16, or 190. However, at 22 and 250, up to half of the total population consisted of RS plants: Clearly, RS formation was favored by elevated temperatures. Effect of bicarbonate on RS formation Reasoning once again from the studies of the bicarbonate trigger mechanism in Blastocladiella emersonii, B. britannica was grown on various concentrations of bicarbonate, along with B. emersonii for comparison, and incubated for 10 days at 220. The results revealed two important facts. First, the tolerance of B. britannica for bicarbonate was quite different from that of B. emersonii; between 6 x 10‘3 M and 2 x 10‘2 M, the range within which B. emersonii grows well and is induced to produce RS, growth of B. britannica was inhibited com— pletely. Second, at 4 x 10'3 M bicarbonate and below, B. britannica did grow well, but RS formation was greatly de— layed rather than promoted in comparison with the time . required for RS development in the absence of added bicarbon- ate. Finally, even total lack of atmospheric C02, which does affect the development of B. emersonii (Cantino and 52 Horenstein, 1959), had no detectable effect on populations of B. britannica. Just as with the TW colonial strain, progressive dilu- tion of the PYG medium caused a Shift in the composition of populations derived from the RS colonial strain of B. britan- gig§_(Fig. 12). But once again, when bicarbonate was incorporated into 1:10 PYG, the higher concentrations of bicarbonate as used above inhibited growth, while lower con- centrations reduced RS formation. Thus, in its response to bicarbonate,EL britannica differs strikingly from B. emersonii. Effect of_glucose on RS formation Alteration of the concentration of glucose in PYG had a remarkable effect upon RS formation. The number of indi- vidual RS plants rose from 26% at double-strength glucose to ca. 90% when glucose was entirely eliminated; conversely, individual TW plants dropped from 56% to ca. 10%. There was little change in the usual small number of colonies (Fig. 15). On the other hand, alteration of the concentration of peptone and/or yeast in PYG had no pronounced effect on the incidence of RS plants. Because of the effect of glucose, cultures were grown under reduced oxygen tensions. For example, under a flowing stream of nitrogen (part. pres. of 02, ca. 0.1 mm. Hg) ngbritannica exhibited no detectable growth. Under somewhat higher partial pressures of 02 (e.g. under mineral oil), A.H\.sm n n u>a cw .cocoo mmooaam HpEpOZV .mcowuaawp Ham cw accumcoo xamuo5wxonaao pecans Imp .nouuoae up: was scans .mmwcoaoo mo mocmpao Ice 30a och .cowuanom mm co Au>a cav mmoozam .mmficoaoo mm mo mmuoam acne pm>aump mcowuodaaoe Lo couuwmoeeoo on» co Anode mm 55 to neonbnneeoocoo prostates to beaten .ne.onu use so necessnscmocoo broadcast to screen .NF. an .na no :Pozucha 0 mm N2 0921.32: . . Jo 24:4. £0 4 IIIIIIIII II .. c. .I.I.I.I.I.I....I. IIIIIII a o o IIIIIIII OIIII. I III! 3 .NI 0 I/ o I” O 321:. 3c. // o o . M../.. I ...I. v m . Il'll o x x 1 mwz<4a we -.-- -u .... m. 0 . 9 J . 9 o .a o O M n . 9 V O .I .. mh241E 3h 0 I. 0 V . m II. a N o N mhz<4a mm 1 OOI muio 400 w c OOI 54 growth did occur, but relative to aerobic controls individual RS plants decreased while non-viable plants and RS colonies increased (Fig. 14). Effect of light on RS formation Finally, because-visible light accelerated RS formation in B. emersonii (Cantino, 1957) when bicarbonate was present, B. britannica was also grown in the presence and absence of light. Fig. 15 illustrates the dramatic effect obtained. B. britannica, grown in the absence of light, responded morpho- genetically as B. emersonii does in the presence of bicarbOnate; i.e., by differentiating into resistant sporangia. Consequently, the question immediately arose: throughout what period in ontogeny was light needed for formation of TW plants (or, conversely, inhibition of RS plants)? The results (Fig. 16) of an experiment designed to test this revealed that plants could be exposed to light for about 28 hrs. and still retain their capacity for developing into RS when placed in the dark. Beyond this point of no return, a change in the light regime had no effect. _;§olation of RS strains yielding 100% gguiividual RS plants As was mentioned earlier, attempts to effect germination 0f mature RS plants have been totally frustrated. On the other hand, it ya§_possible to induce Spore discharge in 'YOung', potential, RS plants; the methods evolved to accom- Plish this were essential to subsequent experimental studies 55 Fig.14 SHADED - CONTROL CO '- SOLID - UNDER OIL % OF POPULATION :3 ' a: 0- a 3 = 2! 3: J j 2 )4 a 0 g‘ o. J a v3 3 O 03 c .- 0 t.- Fig.15 g- :I § 3 .: SHADED-DARK " 0_ some -LIeNT _ 7 \7 \ ‘\ 2- \ \ _ a M J \ .\ Fig.14. Effects of semi-anaerobic conditions on the composition of populations derived from Spores of RS colonies. 24 hr. following inoculation, plates were covered with mineral oil 5 mm. deep and in- cubated for 5 days. Fig.15. Effect of light on RS plant develOpment. Inoculated plates of PYG were incubated in the light (or dark) for 3 days. Each bar represents the percentage of RS plants in a population de- rived from Spores of a single RS colony. 56 BO 80 % RS PLANTS 4!) 20 H30 I IN POPULATION I I l l l 0 IO 20 30 4O 50 Fig.16. EXPOSURE TO LIGHT (HRS) The relation between duration of light and RS formation on medium PYG. Control plates were incubated in the dark for 50 hr.; all others were eXposed to 180 f.c. of white light for different times and then covered with aluminum foil for the remainder of the 50 hr. growth period. Each point is an average value for duplicate cultures. 57 of B. britannica in particular, and they may be applicable to other members of the Blastocladiaceae in general. It has already been stressed that the usual population of mature plants on PYG agar is a heterogeneous and variable one. However, when this population is still young, i.e., 50 to 40 hr. old at 22°, all plants have essentially the same appear- ance; it is possible to induce spore cleavage and discharge in many of them if they are simply removed from the agar and placed in water. Thus, after isolating individual thalli in this age group, inducing spore discharge, and inoculating these spores onto PYG agar plates, the population which re- sulted was, when mature, usually as variable as the parent population. During the course of many such experiments, however, populations derived from spores of single plants occasionally consisted of a very high percentage of RS plants. This suggested that the young isolated plants from which these spores had come may well have been potential RS plants, and that they would have developed thus had they been permitted to reach maturity. Having assumed that it might be possible to obtain a pure RS strain (i.e., populations of 100% indi- vidual RS plants) if the 'right' plants were selected, this approach was continued on an expanded scale. Many 55 hr. Old plants were isolated at random, placed individually into water in depression slides, and the dis- charged swarmers inoculated onto PYG agar. Then, when these first generation populations were 55 hr. old, 5 to 10 58 individual thalli were again isolated from each plate, and the process was repeated. And, indeed, populations were finally obtained which, when grown in the dark to maturity, consisted of 100% RS individuals! Tabulation of the progeny derived from a random sample of such individual plants (Fig. 17) revealed that selection from one end of the distribu— tion curve was being achieved. Typical growth curves at 16 and 220 for such RS strains which produced only individual RS plants are shown in Fig. 18. The maintenance of these strains presented a serious problem because populations had to be subcultured every 55 hr., i.e., before they developed into mature RS which, as has been pointed out, do not germin- ate. However, the difficulty was partially circumvented by maintaining populations of these potential RS plants in an immature state for several weeks through the simple expedient of incubating them at 50. Thus, plants were isolated from the 50 population whenever they were needed for initiation of subcultures of the pure strain of RS plants. At this time it is not known if the swarmers derived from young, potential RS plants are the same, genotypically or phenotypically, as those which should emerge from the same plant in its 'fully mature' state, that is, a resistant Sporangium. Until the means for germinating mature RS plants is at hand, this question will remain unresolved. 59 .mmcaa umuuopv uuoacfl xcwucw o» mcflmmb Aocaa neaomv Haws mm are .Aommv .sr on .no on statesman .nbcnsa ON to condense mmoum>o or» mucmmmuomu ucfioe comm .mucoaa mm Honpw>wpcfi Lo mm>uao cuaouu .mw.o«u .mmzs sz Lo mcowuoaauoo uomamm o» nauseous mo muaammm 20.h<4:10a 2. mhz<4a mcfl oh—‘omfik AON3003UJ 60 Development of Synchronous Cultures of Resistant Sporangia To recapitulate, a strain of Blastocladiella britannica was selected from the various morphological types which, when grown on PYG agar medium, culminates in the formation of either a hyaline thin-walled cell or a thick—walled, brown, pitted resistant Sporangial cell. Unlike its near relative, B. emersonii, these alternate morphogenetic pathways in B. britannica are not controlled by the presence or absence of exogenous bicarbonate. However, an environmental factor which does have a profound effect on differentiation on solid media is white light; TW cells are formed in its presence and RS cells are formed in its absence (Plate II). In order to study this phenomenon at a biochemical level, it was essential to learn to grow the organism in large quantities under conditions which permitted reproducible morphological uniformity, and which could be precisely controlled. The following section is devoted to the elaboration of methods for growing synchronized, single generation, mass cultures of B. britannica, uniformly suspended in agitated media wherein the effects of light and dark on morphogenesis were demonstrable. It soon became apparent that to procure mass populations of RS cells in liquid culture, vastly more was entailed than simply growing plants in PYG broth in flasks instead of on PYG agar in Petri dishes; Special methods had to be devised. 61 Plate II B. britannica grown in light and dark. A plate of PYG agar was inoculated with spores, covered with black paper from which the letters "BB” had been cut out, and exposed to 500 f.c. of white light. The white "dots" represent TW cells and some small, second generation colonies derived therefrom. The dark RS cells occur in great abundance in the darker areas in the picture but are not visible be- cause of the black background. Stray light diffusing beyond the confines of the letter cutouts, was responsible for the occurrence of some scattered TW cells among the RS cells. 62 It was not until 22 months and 72 experiments later (involv- ing some 500 cultures) that this goal was achieved to any degree Of satisfaction. When liquid PYG was used, light- and dark-grown cul- tures behaved in identical fashion; i.e., they discharged swarmers. The first goal, then, was to find a medium in which at least §gme_plants would develop into RS. This, it was reasoned, would provide a clue and a starting point. It was known from work already described that aeration of the cultures was desirable for two reasons: first, growth was much more prolific and second, plants were maintained in suspension, resulting in more uniformity. Over a two to three day incubation period, there was considerable evapor- ation of the medium; this led to adoption of a system whereby incoming air was first passed through water, thus saturating it and reducing evaporation to a minimum. B. britannica grew well enough under these conditions; but, no RSI It was recalled from work with static cultures that RS occasionally were seen in flasks which had NaOH in the side arm (used for neutralization). Perhaps alkali absorbed metabolic C02 which might have been inhibitory to RS morpho- genesis. Experiments in which NaOH was used as a C02 trap indicated that this was not so. Then, in attempts to extrapolate from observations made from plate cultures, two flasks of FY broth (PYG minus glucose) were inoculated with spores and incubated, one in the 65 light and one in the dark. At the end of the growth period, there was no evidence of RS in either one of these flasks. From these same cultures however, small samples had been removed at various times during growth and transferred onto PYG agar plates which, in turn, were incubated in the dark. This was done to ascertain if B. britannica, by growing under these conditions, had actually lost the potential to develop along the RS pathway. The plates made from illuminated liquid cultures produced the expected results. The proportion of RS in the mature populations on these plates was reciprocally related to the duration of previous exposure to light; the longer the exposure, the fewer the RS. Unfortunately, the plants transferred from dark—grown cultures to plates responded in the same manner (Fig. 19): Cells removed from liquid cul- tures (both light and dark) during the first 15 hr. of growth produced high percentages of RS plants on agar media. But beyond 15 hr. the number of RS on plates rapidly decreased. These results seemed to indicate complete independence of any influence of light. The same kind of experiment was carried out using PYG broth + bicarbonate (5 x 10"3 M). In this instance the re- sults were quite different (Fig. 19). The dark-grown plants retained the ability to develop into RS when transferred to agar plates, while light-grown plants gradually lost this capacity. And yet, those cells which were left to mature in unilluminated liquid cultures did not form RS; instead, they 5 o (I) O IN POPULATION 40 RS 20 9:, 64 "' g a x x ‘- -------------- .-‘\O r dz/I”””””T”——--. \ DM:*’I”( I - | ° ‘. PYG-H4003 I " 'I I LT >DK _ I’Y IAL ‘5 > I IDK DUI T7, \ " LT r on . l L l lrn_____4. Fig.19. O 5 IO I5 20 25 AGE OF PLANTS AT TRANSFER Incidence of RS after transfer from liquid to solid media. Small samples of cells from light- and dark-grown broth cultures (DY and PYG + HCD ') of various ages were inocu- lated on PYG plates which were then incubated in the dark. when mature, pOpulations were scored for R8. 65 behaved like the light-grown cells by developing into TW plants and discharging Spores at about 50 hr. of age. To eliminate the contingency that metabolic products, inhibitory to RS morphogenesis, accumulated in liquid cul- tures, the following test was made. Spent media, in which B. britannica had produced only mature TW sporangia in the dark, were filtered and autoclaved. Agar plates prepared therefrom were inoculated with Spores and incubated in the dark. The resulting populations consisted of 95% RS plants; therefore, inhibition by non-volatile, heat-stable metabolic products was at least ruled out. Aerating cultures with various concentrations of gases and mixtures of gases with air, including N2 and C02, was tried; none of these induced RS formation. The possibility that the agar component in PYG plates was exerting some physical effect which might be responsible for the photomorphogenetic response of B. britannica was con- sidered next. Small quantities of agar (.01 - .1% y§_2% on plates) were incorporated into liquid media. Only TW plants developed (in light and dark), but at the higher concentra- tions, agar tended to extend the generation time of dark- grown TW cells. However, a 58 hr. old culture containing .05% agar did contain some RS. With reference to this particu- lar experiment, it must be added that no attempt had been made to maintain neutral conditions, and by the end Of the growth period, the pH in all the flasks had dropped from 6.8 66 to ca. 4.85. Although only a few RS were observed, this was a straw to grasp at--low pH with agar added to the medium. But very soon thereafter, it was concluded from further experiments that agar was not a physical factor in photo- morphogenesis, and its use was discontinued. On the other hand, reduced pH was still a condition to be reckoned with. Incorporation of a citric acid-phosphate buffer into the medium to lower the pH proved to be extremely satis- factory. At the concentration most favorable for growth, its buffering capacity was quite adequate for maintaining a constant pH (i.0.2 unit) during the entire incubation period, thus eliminating the necessity for adjusting it periodically. An unexpected but important fringe benefit accrued when the citrate-phosphate buffer was used. Plants remained nicely suspended as individual cells rather than in small clumps which often was the case in all of the unbuffered media used theretofore. Notwithstanding the lower pH values, the appearance of RS was still an inconsistent if not infrequent event. All during this exploratory period, the RS strain continued to produce ca. 100% RS when grown in the dark on solid media. Once again, the possible involvement of agar came to mind, but this time in terms of chemical effects. Plates made with purified agar (Difco) failed to alter the composition of mature populations. This indicated the improbability that impurities in agar were responsible for RS induction; 67 however, the chemical constitution of agar itself was questioned. Because it is composed of a sulfuric acid ester of a linear galactan, the unlikely possibility was con- sidered that B. britannica might utilize the galactose moiety of this polymer in preference to the glucose in the medium. This idea was contemplated because in some cases more RS had been produced on plates in the complete absence of glucose than in its presence (Fig. 15). A new series of experiments was launched, and for the first time reproducible results were obtained. Liquid cultures in which 0.5% galactose was added to PYG broth, as well as those in which 0.5% galactose was added in place of the glucose, gave 50-70% RS. These media were buffered with citrate-phosphate to different levels over a pH range of 4.9-5.5. After some modifications in buffer concentration and determination of the most suitable inoculum size (number of swarmers), a system was at hand wherewith Single generations of synchronously developing thalli of B. britannica could be mass produced. By exposing cultures to light, populations consisting of 100% TW Sporangia resulted; elimination of light resulted in 90-100% RS. At this point, galactose was replaced by glucose and the same satisfactory results were obtained. Although it served in working out the details of the conditions necessary for RS cultures, galactose was not a requisite for their production. Instead, it was a critical pH range that was required in liquid culture: The procedure ultimately adopted for 68 cultivating large numbers (107 — 109) of B. britannica is described in MATERIALS AND METHODS. Unless otherwise indi- cated, RS substrain B101wwas used in all subsequent work. Growth and Morphological Characteristics of Synchronized Single Generation Cultures The growth and morphology of Blastocladiella britannica in liquid culture did not differ perceptibly from that on agar media. The uninucleate spore (ca. 5 x 7 u), following a short Swimming period, rounded up and began to germinate (PLATE III). A slender germ tube emerged which branched in root-like fashion culminating in an extensive system of rhizoids. Concomitantly, the thallus enlarged symmetrically into a single-celled spherical plant.* Until shortly before the end of the generation time of a light-grown TW sporangium, no morphological differences were discernible microscopically between it and a dark-grown potential RS at a corresponding chronological age. Yet, within the next 1-2 hr., the entire protoplast of a TW plant cleaved into hundreds of uninucleate, uniflagellate motile spores and a new generation of cells was then discharged through newly formed discharge tubes. On the other hand, the thalli grown in the dark had reached only the half—way point in their ontogeny; they continued to enlarge * In these respects the morphology of B. britannica dif- fers from that of B. emersonii. The latter is characterized by a somewhat cylindrical form as well as septation of the basal portion as the plant approaches maturity (PLATE I), thus making B. emersonii a two- celled organism. 69 .. it? an? 'r’ of" Plate III Selected stages in development of TW and RS cells. Up to 28 hr., cells grown in light or dark were identi- cal in appearance. After 28 hr., morphological differ- ences began to appear. Light-grown thalli developed into typical TW cells with discharge tubes (51 hr.), and dark-grown thalli developed into thick-walled, brown RS (55, 50, and 65 hr.). Note that in these synchronized cultures the rhizoidal system arose from one locus; this contrasted sharply with results obtained when B. britannica was grown on solid media, where rhizoids arose at several places on the surface of the cell. 70 for several hours thereafter and then gradually differentiated into mature RS. For TW sporangia in liquid culture, generation time is defined as the time at which 20% of the population of such cells produce discharge tubes. Essentially all of the cells in a population produce such tubes within one hour. This re- flects the high degree of synchrony obtainable in a single generation culture. The generation time of RS grown in liquid culture is defined as 65 hr. Since RS cells do not discharge spores, this time was selected because it represents the point at which no further change in morphology and degree of pigmentation is microscopically evident. Growth pattern in the dark In the absence of illumination, a population of Spores gave rise to 90-100% RS cells. The volume/cell increased exponentially (Fig. 20) up to about 50% of the generation time of the cell. After this, the chitinous wall quickly thickened and, simultaneously, the cell began to decrease slightly in diameter (compare photographs of 65 hr. cell and 50 hr. cell in PLATE III). Glucosamine was the only hexos- amine found in chromatograms of cell wall hydrolysates. This was true for plants grown in media containing glucose as well as those grown with galactose as the sole carbohydrate source; acid hydrolysates of the latter contained no detectable galactosamine. For a mature TW cell which weighed 1.68 x 10‘2 ug, the chitin content was 9.28 x 10‘4 ug or approximately 5% 71 of its dry weight. RS cells contained about 10% more chitin/ mg. dry wt. than TW cells. Concomitantly with increased chitin synthesis, a melanin-like pigment was deposited in the wall; its absorption spectrum was linear between 400 and 600 mu with a slope of 0.00298. This compared well with the slope of 0.00285 for B. emersonii (Cantino and Horenstein, 1955) and 0.0027 for Neurospora crassa (Schaeffer, 1955). At maturity, the cell was a typical blastocladiaceous, brown, pitted, resistant sporangium. The 2 hr. delay before an increase in volume began (Fig. 20) is a real lag; it involves, among other things, a brief swimming stage, followed by loss of the spores' single flagellum. The growth pattern shown in Fig. 20 was obtained with population densities of 2 x 104 to 9 x 104 cells/ml.; outside of this range, complications may arise (vide infra). Growthgpattern in the light In the presence of continuous illumination, only TW cells were produced. Within the population density range mentioned above for RS cells, and at a suitable pH (e.g., pH 5.6 along plateau in Fig. 21), the volume/cell appeared to increase exponentially (Fig. 22) after a lag of 2 to 5 hours. It is worth noting that this growth rate was 40-fold greater than that of similar cells grown in the same medium, but with- out aeration and agitation. Although the growth rate was not affected by variations in population density (within the range shown in Fig. 22), the final volume of the mature TW cell was . fiq-“uii' 5O 45 b 0 L06 VOLUME (u3)/CELL U U 0‘ O 25 72 b RELAnVE GROWTH RATE 5 ; 00 A o / c) o] 09’ o/ Fig.20 3:) :5 nevus PLATEAU; /"0 ‘—57.r4 ss.—5»9 1 Fig.21 5.5 Fig.20. Growth of RS cells in the dark. Each point is an average for 50 measurements derived from 5 experiments. Fig.21. Effect of pH upon the growth rate of Tw cells in the light. Relative rates were derived from the linear slope of plots of 109 volume per cell vs. time; that obtained at pH 4.85 was set at 1.0, and other rates were related to it. 'AW 75 affected; note the arrows in Fig. 22, which indicate cell generation time. This phenomenon is seen more clearly in Fig. 25, where the relationship between population size and the volume of a mature TW cell is delineated (the greater the population size, the smaller the volume of the cell at maturity). This appeared to be due to the fact that the generation time of the cell (but not the rate of volume change during exponential growth as in Fig. 22) was a function of population size (Fig. 24); in general, the smaller the population size, the greater was the generation time. This being the case, one might expect to find that the dry weight per unit volume would be constant, irrespective of the final size of the mature TW cell. This however, was not so (Fig. 25); the greater the volume of a mature TW cell, by virtue of an extended generation time, the smaller was its dry weight per unit volume. In fact, the total dry weight of a mature TW cell tended to be constant, regardless of its final volume (TABLE III). Thus, within the range of the population densities studied, increasing the number of plants per unit volume of medium did not affect the exponential rate at which such cells increased in volume, nor did it greatly affect the final weight of the mature cell. However, it did decrease the dura- tion of the final enlargement stage of the cell and, therefore, the size of the cell at maturity. 74 55 - Fig.22 o ‘— I 99 x no4 CELLS/ML 0/ so - go ‘_ 3.5 x lo“ CELLS/ML 4— 5.79mo4 CELLS/ML 45 p- 4 +— 9.9 xno CELLS/ML o . A .1 0 In 40' / 0 \ .~ 7‘ 3 o LIJ z / 35 3 " ° ° / > o (D O . 3,, , / o/° 2.5 I- 20 L 1 A J A o no 20 30 40 so ”Ow-s O Fig.23 0 U 2 '7 5 o b 2 II] o O .— ‘ o .J .J O 3 \ o ”i .. 45- “I g 0 .1 o > g o .1 b ‘0 l l I n . L . o s x 10‘ 105 |.5 x105 2an‘ POPULATION DENSITY (CELLS/ML) Fig.22. Effect of population density upon the growth of Tw cells. Within the range shown, increases in population density extended the generation time (see arrows) of the cell but not the rate at which it increased in volume during exponential growth. Fig.23. Effect of pOpulation density upon the volume of Tw cells at generation time. TIME (HRS) GENERATION 3 - VOL/CELL AT 0594.1le ()1 x :03) 75 o 35 D o 30. o O\ o 25!- Fig.24 l I A L l A I 0 5x no‘ :05 :25)ons 2x105 robuuhou cum" tCCLLS/nu D 0 Av. or 2 on > EXPTS. IOO' SHOWING LIMITS I 0 SINGLE exprs. D b I so- I D D . F19.25 0 A A A A A A A A A A A A A A A Fig.24. Fig.25. 20 25 50 any wr/p’ AT can. 1m: (”4): no”) Effect of pepulation density upon the generation time of TN cells. Relationship between the volume of the Tw cell at generation time and its dry weight per unit volume at this time. 76 TABLE III. Relationships among volume per cell, weight per cell, and weight per unit volume of TW cells at maturity rr 1 :_= Volume per cell Weight per cell Weight per unit ( x 108 ) volume ( x 1013) H3 gm . gm o/ua 42,800 1.07 2.5 47,100 1.10 2.5 50,800 1.10 2.2 61,500 1.15 1.9 63,000 1.14 1.8 76,000 1.12 1.5 *Grown at 240 on the standard medium with population densities varied over the range of 2 x 104 to 9 x 104 cells/ml. to induce the differences in the final volumes per cell. Point of no return in development In synchronized single generation cultures of B. britan- piga, containing between 3 x 104 and 5 x 104 cells/ml., the generation time of light-grown TW sporangia occurred at ca. 50 to 52 hr. at 240 (Fig. 24). The generation time for dark- grown RS has been defined as 65 hr. However, neither the presence nor the absence of light was required continuously throughout the entire growth period for genesis of one or the other of these morphological forms. The organism was quite plastic during the early stages of development. When a syn- chronized population of illuminated cells growing in the standard medium was transferred to complete darkness before a 77 critical period in development, all cells on the photomorpho- genetic pathway leading to TW cells up to that time developed into brown, pitted RS cells. If the culture was transferred to the dark environment after this period, inevitably only TW cells formed. This point of no return was rather sharp and occurred at ca. 65% of the normal generation time of the TW plant (Fig. 26). Similarly, synchronized populations of cells growing in the dark (and thus on the way to RS plants) could be induced to develop into TW cells only if they were subjected to illumination before a point of no return t45-50% of the normal generation time of an RS cell). However, if illumination was delayed until the last possible moment—- i.e., until this point of no return was reached--the generation time of the induced TW plants was usually prolonged by a few hours; if it was delayed beyond this point, TW plants were no longer formed while RS appeared instead. Thus, on the basis of the combinations tried, there were three different regimes of light and dark which yielded only TW sporangia and three which yielded only RS (Fig. 27). In effect, there appeared to be two non-overlapping periods (18—20 hr. and 28-50 hr.) in the life span of B. britannica during which light exerted its sharp effect on morphogenesis.‘ Light was obligatory for TW sporangia formation only during one or the other of these periods--or alternatively, RS formation was incumbent on the absence of light during either one of these two periods. 00 IN PO PU LATION Q 0 0) O 7. RS. CELLS 4O 20 78 Fig.26 1 k 20 4O 60 80 I00 _ lJGHT CELLS anuse T0 ow-o- 35 .TM AT me. GE" ' E C DARK CELLS TRANSF.TO Lt-o- RS - L . . 1" 40 50 60 AGE (HRS) Fig.2? Fig.26. Points of no return for R8 and TN cells. Beyond these points, transfer of cells to an illuminated en- vironment or a dark environment, reSpectively, can no longer cause the cells to revert to the alternate morphogenetic pathway. Fig.27. Coll types resulting from exposure to different light-dark regimes. The generation times of the TMS (thin-walled Sporangia) are represented by the heavy vertical bars. (See text for details.) 79 Although the first visible signs of photomorphogenesis were not detectable microscopically until just before the generation time of a TW cell was reached, it was clear from the above reversal studies that light modified the cells of B. britannica in some manner during an earlier stage in their ontogeny. Since a parameter associated with growth rates might have reflected these changes, a detailed search was made for possible differences in cell weights. However, in both light- and dark-grown cultures, the dry weight/cell in- creased exponentially at an identical rate (following the 2-5 hr. lag after spore inoculation) up to 50 hr.; i.e., the pattern was not affected by illumination (Fig. 28). These data complemented the earlier experiments (Fig. 20, 22) which indicated that the rate of exponential increase in the volume of a growing plant was also independent of light and darkness. Glucose Uptake Capacipy Because the aforementioned growth indices were not photo- responsive, a physiological criterion was selected for examini- nation. X-ray diffraction studies, electron microscopy, and chemical analyses (Nabel, 1959; Frey, 1950; Aronson and Preston, 1960; Fuller, 1960) have established that the cell walls of the Blastocladiales consist predominantly of chitin. Thus, since the glucose chain is a building block in the chitin backbone, glucose or glucose derivatives must play a role in the metabolism of Blastocladiella britannica. 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Cam Hoomz so. Coca mm , ............................. .1 Hoomz em.o once we CH0 meme mm Cam .mm .He mmumHm Own Eoum CHO mhmp cm H Camp me How CH um poquSCCH muemHm CHOIhmp hm pHC wmmp we n mmmp we mom H um pmquchH mmcmHm CHOIwmp m mm NO mm NH mm mm mm nN mm MN OOH h mhmp OOH mwmp d pmummnu pmumoonCp CC£3 Com CC£3 Com mcoHqueCO pcmaummua snowmen mm AUCSCHDCCCV HH xHozmmmm APPENDIX III Ballons d'essai GUC and its relation to certain cellular components A few years ago, there was found in Blastocladiella emersonii a considerable quantity of an ethanol-insoluble, glycogen-like polysaccharide which yielded only glucose upon acid hydrolysis (Cantino and Goldstein, 1961). B. britannica possesses a similar polysaccharide. For this reason, in one of the GUC experiments plants were analyzed for intracellular polysaccharide as well as free glucose, both before and after incubating in glucose. As part of the same experiment, the spent media in which the cells were grown were analyzed for glucose disappearance during growth. The results are tabulated in Table IV. The data are suggestive but until the results of later experiments with C14-labeled glucose have been scrutinized, no un- equivocal conclusions can be drawn. However, some of the suggestions calling for further consideration are as follows: 1) B. britannica does not appear to accumulate a large free glucose pool, either during growth with glucose as the carbohydrate source or during incubation of pre- grown cells in a glucose solution. The increase in free glucose following GUC experiments accounted for only 0.5- 5% of the glucose consumed. 116 117 .pommmumxm mH CmmuCCCCCm CHCCB ummuxm .vOH x m: CH mCCHm> HHm umHmmfl HHCCICCQ C CO Cum mCCHm> HH4 CmCCCHm CCHCMCCCmm em.m- ee.em ee.me em.m ee.oa em.me Isaom an mmmmwocH CmCCCHm em.m o.m mm.a mm.m mo.m me.H maflwmeuommsaom HCCQ CwCCCHm Rmm eemH Race some Rm.em em.m mmnm CH mmmmnocH HHHO. mHmO. mmmo. HnH. ammo. thO. HCCQ CmCCCHm Comm Om.m Hm.m nH.H mm.m mm.N mom. ODO CCHqueCCH Cmousfim kumd CmCCCHm No.5 >H.M mN.H Nm.m m>.N NN.H CCHCMCCCmmeCm Nmmo. mmHo. @500. sHmo. hmmo. ONNO. HCCQ CmCCCHm Cmum Cp3owm OCHHCU ECHCCE m.mHH m.>¢ N.Hm O.m> m.om m.mm ECCN CCECmCCC CmCCCHO CCHDMQCCCH CmCCCHm,CHCMCm .ws om .wn mm .un mm .w: on .w: mm .ws mm muamam Co was mquHQ C3owmrxnmo mpCmHQ,CBCHquCmHA mHCCm HCHCHHCCMHCCH pCm ODO .>H mqm¢8 118 2) The polysaccharide content of B. britannica accounts for a substantial portion of the dry weight of the fungus. This level does not remain constant throughout ontogeny; it might be related to GUC and, by extension, to photomorphogenesis. Effect of phosphate on GUC Another aspect that deserves in-depth investigation involves the function of inorganic phosphorous in glucose uptake. Certain experiments intimated that it might play a role of primary importance in the mechanism of glucose consumption by B. britannica. In these studies, some cells were grown in the usual manner in PYG broth made up in citrate-phosphate buffer, pH 5.6; other cells were grown in PYG broth minus the phosphate, where the pH was adjusted with NaOH. Growth was equivalent in both media. With such cells GUC studies were done in the citrate-phCSphate buffer and in citrate minus the phosphate. The results of two of these experiments, in which 29 hr. old dark-grown cells were used, are shown in Table V. The presence of inorganic phosphate during growth enhanced GUC when these cells were placed in a non-growing situation. Inclusion of phosphate during GUC incubations augmented this effect. In what capacity this influence was exerted on GUC remains to be fathomed. 119 TABLE V. Effect of phosphate on GUC GUC (pg x 105) Grown in PYG + Incubated in glucose + Exp.#1 Exp.#2 citrate citrate 47 56.5 citrate citrate-phosphate 59 79 citrate-phosphate citrate 69 54 citrate-phosphate citrate-phosphate 88 105 Effect of different sugars on GUC Effect duringpgrowth. It has already been seen that certain conditions during growth affected the GUC of B. britannica. One of these conditions was the presence or absence of illumination; another was the presence or absence of inorganic phosphorous. What effect, if any, would qualitative and/or quantitative variations in the carbohydrate source during growth have on the fungus' capacity to consume glucose under non-growing conditions? A first attempt to answer this question indicated that the effects were considerable (TABLE VI). In addition, galac- tose and glucosamine markedly reduced the size of the cells (TABLE VI). Although the plants grown in the presence of galactose had a normal appearance, those cells grown in glucosamine appeared very abnormal. This latter 120 .uCCEHCmeC C>Cnm CH Com: CCC3 mquHm CBCHmeCmp CH0 .HC ON IIII IIII m.O mw.o OmnH CCHEmmCCsHm &mm.o mm.O N.m O.H m>.m mHHN CmCquHmm Rm.o O.H m.ow m.m m.mH HHnm CmCCCHm &M0.0 m.H H.em m.m m.mm oeme Lacuucoov mmousflm em.o AmOH x 01V AmOH x miv ACOH x mav AmOH x miv Audv quHm mo CHSCHO OCHHCC m:\.m>HCm HHCC\.m>HCm N1\UDO ODO memo Comwusm CCHHmmCm Cumuphnonumu nuBCHO OCHCCC mummsm uCCCCmeU mo UDO CC pummmm . H> mqmfie 121 observation could well account for the very low GUC dis- played by these cells. Plants grown in the presence of 1/10 the standard glucose concentration were slightly larger than the control cells but otherwise had a normal appearance. Because of the difference in size of the plants grown with different sugars, GUC was also calcu- lated on a surface area basis (TABLE VI). This indicated that the cells which grew with galactose as the sugar source did not have their GUC impaired as much as it seemed at first glance. The values obtained from polysaccharide analyses of these cells following incubation in glucose are also listed in TABLE VI. Effect duripg GUC incubations. When an equimolar amount of galactose, glucosamine, of oemethyl-D-glucoside was added simultaneously with glucose to a suspension of 28 hr. old dark-grown plants, there was no inhibition of GUC during the course of a one hr. incubation period (TABLE VII). In fact, there was a ca. 20% stimulation of GUC by ofimethyl-D-glucoside. This observation is not without precedent; Rogers and Yu (1962) reported that certain non- inhibitors of galactose uptake in Escherichia coli strain A increased its uptake by as much as 15%. However, when B. britannica was preincubated in the presence of glucosamine, there was complete inhibition of GUC. Smith (1960), in describing hexokinase inhibition in .mes CHC3 Hmmsm comm mo 21 om ** .HC m .mu How Umuoum CCCQ pm: mHHwU CwCCu .CCHCQCCCH mm3 mensu NC How uan CCu CEHu wCu >9 umnu mCmCE mHCB .CEHHCMCE Cnu CH HCHMHCOHHNCH CCu CH pmuoum CHC3 wCCHmCCmma HHCC ecu .CHCNCCCCu “CEHH CCC um CDC CHCB mCCHquCC N wHCO .UDO HHCC» CCCmHCHEHp C>MC Cu wECCm wCCHHmQCCCH USO CHCMCQ Cm um mHHCC CBCHOICHQ OCHHCum* .HC ON How xump CCu CH C3Cum CCCQ pm: mHHCO 122 O CmCCCHm .HE O.H pmppm .CHE Om Hmuwm .CCHEmmCCsHm .HE O.H ¢.HH CmCCCHm .HE O.H pmppm .CHE Om Cmumm .Omm .HE O.H m.MH CwCquHmm .HE O.H + CwCCCHm .HE O.H w.MH CmCCCHm .HE O.H #3 meflmooflmuaummob .He O.H + $851.. .2. O.H 83 383m .1: O.H ¢.mH CCHEmmCCCHm .HE O.H + CmCCCHm .HE O.H ** ¢.ON CmCCCHm .HE O.H AmOH x 01v ODO CueuxHE CCHquCH CH mummsm * mummsm CCCHC NC CCCCmCCm CCC NC 020 CC HCCNNN .HH> mqmde 125 Spirochaeta recurrentis, noted that glucosamine inhibited enzyme activity and furthermore, for maximum inhibition, it had to be added before the glucose. It would be out of place to draw any conclusions on the basis of these few experiments, but this does seem to be an area which might prove fruitful upon intensive in- vestigation.