JOURNAL OF BACTERIOLOGY, June 1972, p. 1065-1072 Vol. 110, No. 3 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of Allomyces arbuscula DANIEL J. BURKE,I THOMAS W. SEALE,2 AND BRIAN J. McCARTHY Departments of Biochemistry and Genetics, University of Washington, Seattle, Washington 98105 Received for publication 22 December 1971 The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium forma- tion. Spore induction, germination, and mitosporangium formation are insensi- tive to inhibition of actinomycin D, probably indicating that stable, pre-ex- isting messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an ap- preciable change in the rate of macromolecular synthesis. Several investigators have emphasized the invaluable background for the present bio- potential usefulness of aquatic fungi for studies chemical studies (2, 5, 9, 12, 19, 21, 25, 28, 30). of the mechanisms of cellular differentiation Our initial studies with this organism were and morphogenesis (6, 14). Among the phyco- designed to determine the characteristics of mycetes, Allomyces arbuscula is an especially macromolecular synthesis associated with de- appropriate model system for the correlation velopment and differentiation and to begin to of cytological and biochemical changes occur- identify those points during the life cycle ring during differentiation. Allomyces pos- which are the most critical for the initiation of sesses stable alternation of haploid and diploid these processes. life cycles (Fig. 1), a property common to The studies to be reported were concerned members of the family Blastocladiaceae (5). with the temporal variation of the rate of ribo- The haploid and diploid stages seem to be nucleic acid (RNA) and protein synthesis identical in nutritional requirements and phys- during the diploid stage. In particular, this iology; they apparently differ only morphologi- communication examines the point in the life cally, the haploid producing paired gametan- cycle at which the genome is activated or at gia, and the diploid producing mito- and least genome activity is dramatically increased meiosporangia (20). Both the diploid and the and the several times during the life cycle haploid stages produce several differentiated when it is probable that development is con- cell types: mitospores (2N) and meiospores trolled by pre-existing stable messenger RNA (1N) in the diploid, male and female gametes (mRNA). In many of these instances differen- in the haploid, and an encysted single cell tiation occurs without profound changes in the stage, rhizoidal growth, hyphal growth, and rate of synthesis of RNA or protein. reproductive structures in both. The life cycle can be conveniently reproduced on defined MATERIALS AND METHODS medium, and large amounts of the various cell Organisms and media. All experiments were types can be obtained after proper manipula- performed with a stock of Allomyces arbuscula tion of growth conditions (8, 20, 21). Further- strain Brazil 2 G derived from a single meiospore more, the numerous cytological and physio- (1N) isolate of the original Brazil 2 G strain that was logical studies already published provide an kindly provided by Ralph Emerson. The gameto- phyte plant developed from this meiospore was in- ' Present address: Department of Microbiology, Univer- duced to release gametes at maturity, and the zy- sity of Illinois, Urbana, Ill. 61801. gotes obtained from this were used as the initial 2Present address: Department of Biological Sciences, source of diploid material. This strain has been des- University of Illinois at Chicago Circle, Chicago, Ill. 60680. ignated B2G-15-D. The preparation of YpSs me- 1065 1066 BURKE, SEALE, AND McCARTHY J. BACTERIOL. incubating them with label for either 5 or 10 min. Samples were precipitated with cold 10% trichloroa- cetic acid containing 200 gg of base or amino acids, or both, per ml as carrier, filtered on Whatman GFC filters, and counted in a Packard Tri-Carb scintilla- tion counter. Staining. Mitospores and rhizoid stage plants were stained by mixing a volume of culture with an equal volume of 2% toluidine blue in water. Chemicals. Actinomycin D and actidione (cyclo- heximide) were purchased from Calbiochem, Los Angeles, Calif. 3H-adenine, "4C-phenylalanine, 3H- phenylalanine, and l4C-glutamic acid were pur- chased from New England Nuclear Corp., Boston, Mass. The specific activities of the isotopes are given with each experiment. RESULTS Induction of mitospores. The diploid life (r) cycle can be conveniently divided into four FIG. 1. Life cycle of Allomyces arbuscula. (mt) parts: induction of the mitospore, spore en- Mitospores, (c) cyst, (r) rhizoid growth, (h) hyphae growth, (mts) mitosporangium, (mis) meiosporan- cystment and germination, rhizoidal and hy- geum, (mi) meiospore, (mg) male gametangium, (fg) phal growth (vegetative), and the formation of female gametangium, and (z) zygote. mitosporangia. On flooding of an agar plate culture with DS, the multinucleate cytoplasm in the sporangium cleaves into individual uni- dium, Machlis B medium (MB), and dilute salt solu- nucleate motile spores, and within 2 hr the tion (DS) has been described elsewhere (7, 17). MB are medium was modified by the addition of 15 g of sol- motile spores released through discharge uble starch per liter in place of glucose, 400 gg of L- papillae. The uninucleate nature of the spores leucine/ml, and 400 jig of L-lysine/ml. In some cases, was confirmed by microscopic examination of 1.0 to 4.0 mg of an equal mixture of Casamino Acids stained material. (Difco) and tryptone (Difco) was added per ml to the To determine whether the induction is MB medium. merely a rearrangement of existing elements or Growth conditions. Mitospores were obtained in represents new synthetic events, the uptake of the following manner. YpSs agar plates of 5- to 10- RNA and protein precursors into acid-precipi- day-old plants were flooded with 10 to 20 ml of table material was measured in the presence sterile DS. Motile mitospores were released within 2 hr. For germination and growth experiments, mito- and absence of inhibitors (Table 1). RNA syn- spores in DS were inoculated to a final concentration thesis appeared to be unnecessary for induc- of between 5 x 104 to 2 x 10S spores/ml into YpSs tion: the number of spores induced in the pres- broth or MB broth plus additions and incubated at ence of actinomycin D was the same as that 28 C. The inoculum was never more than 10% of the induced in its absence. The RNA synthesized final volume. The growth vessel was either a 125-ml during induction in the absence of actino- Erlenmeyer flask with a stirring bar for agitation or mycin D might result from some residual vege- a 500-ml Bellco spinner flask. tative synthesis of some RNA species which Spore formation was also studied by growing are subsequently incorporated into the spore plants (this refers to either the rhizoidal or hyphal growth stage) (Fig. 1) for various lengths of time in during cleavage. In any case, it is not neces- liquid YpSs or MB broth. The mycelium was har- sary for further growth since spores induced in vested by centrifugation and resuspended in a the presence of actinomycin D developed nor- volume of DS equal to that of the original culture mally when compared to spores induced in the and incubated until mitosporangia were formed (20). absence of actinomycin D. Labeling. Mitospores were labeled by the addi- Protein synthesis does, however, seem to be tion of 3H-adenine and "4C-Phenylalanine along necessary for spore induction. Cycloheximide with the DS when plates were flooded. Germinated at concentrations as low as 0.1 ug/ml inhibited spores and the plants were labeled in two ways. (i) mitospore induction to a level of 0.01% or less Total uptake into trichloroacetic acid-precipitable of material, was determined by adding a labeled base than that control cultures. The protein syn- or amino acid, or both, to the medium and removing thesis taking place appeared to be directed by samples at various times. (ii) Determinations of the stable mRNA since the amount of synthesis rate of macromolecule synthesis were made by re- was the same in the presence or absence of ac- moving samples from a spinner flask culture and tinomycin D. The time at which this synthesis VOL. 110, 1972 DIPLOID LIFE CYCLE OF ALLOMYCES ARBUSCULA 1067 TABLE 1. Effect of inhibitors on mitospore cysts, and germlings was present for a 7-hr inductiona period after inoculation of the medium. The rate of synthesis of RNA and protein Incorporation was measured by means of the total uptake .No. of Additios (pmoles/ml) into trichloroacetic acid-precipitable material Additions spores/mispr/m Phenyl- Adenine of precursor by germinating spores (Fig. 3a). alanine Zero time was taken as the inoculation of of None 105 19 20 spores into medium capable supporting Actinomycin D, 105 <0.3 16 growth. Synthesis of RNA and protein com- 20 ug/ml menced at about 20 min, at which time encyst- Cycloheximide, 103 <0.3 <0.2 ment was complete. The rate of incorporation 20 Ag/ml of adenine, as determined in a similar experi- ment, revealed some interesting characteristics (Fig. 3b): it increased with time to a maximum a Ten milliliters of DS solution containing 20 gCi value, decreased, and then reached a constant of 3H-adenine (505 gCi/,umole) and 1 gCi of 14C- rate of followed phenylalanine (16.5 qCi/,qmole) was added to each value.