Cytochrome Oxidase Activity in Blastocladiella emersonii

Studies of cytochrome oxidase in isolated mitochondria of Blastocladiella emersonii Cant. and Hyatt show that the enzyme was present in zoospores and throughout the development of ordinary colorless sporangia and of resistant sporangia. The enzyme activity was present in KCl, NaCl, NH₄Cl, and KHCO₃ induced resistant sporangia, and was shown to be as active or more active than the enzyme found in ordinary colorless sporangia and zoospores. Interfering substances causing difficulties in the measurement of cytochrome oxidase activity were found in whole cell homogenates of KHCO₃ grown resistant sporangia, but not in KCl, NaCl, or NH₄Cl grown thalli. These substances could be removed by dialysis or by sedimentation of the mitochondria.     

Gene expression during development of Blastocladiella emersonii

To evaluate gene expression during sporulation and early development of the aquatic fungus Blastocladiella emersonii, the nucleotide sequence complexity of the polysomal RNA has been measured at different stages. To assess the effect of medium composition on gene expression, similar experiments were completed during early development in a range of simple to complex media. The polysomal RNA sequence complexity was measured by hybridization with single-copy tracer DNA and with a complex class-enriched cDNA fraction copied from the stored zoospore poly(A⁺)RNA. Forty-four to eighty-six percent (8.2 × 10⁶ to 16 × 10⁶ nucleotides) of the single-copy DNA sequence complexity was found on polysomes, depending upon the stage examined or the medium used, compared to 42.5% (8 × 10⁶ nucleotides) in the stored RNA pool of zoospores. The highest levels of complexity occurred during the two periods of active differentiation, sporulation and germination. During starvation-induced sporulation, and average of 82% of the total asymmetrically transcribed complexity was expressed; half of this complexity was lost prior to the completion of zoospore differentiation and was missing from the zoospore-stored RNA pool. During the first 30 min of zoospore germination the level of sequence complexity increased by 46 to 66% over the zoospore level, depending upon the medium used. The polysomal RNA complexity then decreased by a nearly equal amount between 30 and 60 min when the cells entered the growth phase. An inverse relationship was found between the richness of the medium and the level of sequence complexity found on polysomes. The data indicate that sequences representative of most of the zoospore-stored poly(A+)RNA were expressed at all other stages and maintained by turnover and resynthesis. In addition, significant numbers of new sequences were also expressed, particularly during stages of active differentiation. Cells that germinated and completed early development in an inorganic starvation medium showed a marked loss of the middle and high abundance classes of poly(A⁺)RNA and slight enrichment for the low abundance class.     

Ionic control of germination of Blastocladiella emersonii zoospores

Encystment and germination of Blastocladiella emersonii zoospores involve a rapid and radical transformation of the motile but nongrowing spore into a sessile, growing germling. Certain inorganic ions, notably 50 mM KCl, are efficient inducers of germination. By use of the carbocyanine dye DiO-C6-(3), we found that KCl depolarizes the plasma membrane of zoospores and noted good correlation between depolarization and subsequent germination. Zoospores avidly accumulated K+ ions from the medium, attaining an internal concentration of over 50 mM and a concentration gradient of 2,500. Sodium ions, by contrast, were expelled. Internal K+ was required for normal germination but its function is not known. Zoospores also took up considerable amounts of calcium; most of this was associated with the external surface and appeared to be necessary for maintenance of zoospore integrity. KCl (50 mM) and other salts displaced surface calcium but this was not in itself sufficient to induce germination. The calcium ionophore A23187, in the presence of external calcium, was an effective inducer of germination, suggesting a possible role for cytosolic calcium in triggering the transformation. We propose that the first step in the induction of germination by salts is depolarization of the plasma membrane; subsequent events require the intervention of cytoplasmic signals.     

Acquisition of thermotolerance during development of Blastocladiella emersonii

In the fungus Blastocladiella emersonii the synthesis of heat-shock proteins is developmentally regulated; particular subsets of heat-shock proteins are induced by heat shock during sporulation, germination and growth and some heat shock-related proteins are spontaneously expressed during sporulation (Bonato et al., 1987, Eur. J. Biochem., in press). Nevertheless, acquisition of thermotolerance can be induced at any stage of the life cycle. The development of thermotolerance is correlated with the enhanced synthesis of some heat-shock proteins: hsp 82a, hsp 82b, hsp 76, hsp 70, hsp 60, hsp 25, hsp 17b. Other hsps are not specifically involved in thermotolerance.     

An analysis of developmental timing in Blastocladiella emersonii sporulation

We propose a model of time regulation for the expression of the Blastocladiella emersonii sporulation phenotypes based on new methods (Soll, 1986) which analyse the effect of temperature on the rate limiting processes, i.e., "timers" of certain events during development. By using reciprocal shift experiments (transferring sporulating cells from 22 to 27 degrees C and vice versa) we characterized the timers of the phenotypes: septate zoosporangium, papillate zoosporangium, cleavage zoosporangium, and empty zoosporangium, considering the number of the components, sensitivity, duration, and the mutual dependency of each limiting factor. The timers for the first three phenotypes started at zero time of sporulation induction and acted in parallel. The fourth phenotype, empty zoosporangium, has a timer which appears to act sequentially to that of the papillate zoosporangium. We also studied the effects of polyoxin D, calcofluor white, and congo red on sporulation. The first drug prevents the appearance of the septate zoosporangium and the other two prevent the expression of the papillate zoosporangium. In spite of the morphological blockage, the zoosporogenesis proceeds, resulting in the formation of normal zoospores. These results are interpreted as additional evidence for the parallel model of control proposed here.     

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.     

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii

The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B.?emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.     

Phenotypic dissections of the Blastocladiella emersonii zoospore's developmental choice

A developmentally homogeneous neural crest cell population has been used to assay the role of environmental factors in regulating crest cell differentiation. If cultured on tissue culture plastic, virtually all of the cells of this population differentiate into melanocytes. In contrast, when these cells are cultured for 3 or more days on substrata “conditioned” by somite fibroblasts, the proportion of cells undergoing melanogenesis decreased and the proportion expressing formaldehyde-induced fluorescence (FIF), characteristic of catecholamine-containing cells, increased. For a limited period of culture on somite-conditioned substrata, some cells in the population exhibit both pigment granules and fluorescence. Collagen-coated substrata decreased the number of cells that formed pigment but did not stimulate FIF. In contrast, optimum doses of exogenous cellular fibronectin mimicked the effect of somite-conditioned substrata, suppressing melanogenesis and promoting FIF. Glycosaminoglycan-derivatized substrata (i.e., hyaluronic acid, various chondroitin sulfate preparations, and heparin) did not alter the differentiative homogeneity of the cultured crest cell populations. The choice and expression of phenotype by some members of a cultured crest cell population can, therefore, be affected by environmental stimuli provided in the form of certain substrate-attached macromolecules. We suggest that optimal concentrations of some extracellular matrix components produced by embryonic tissue and normally encountered by migrating crest cells may elicit the expression of FIF in crest cells that would otherwise follow a different developmental pathway.     

Utilization of endogenous reserves by swimming zoospores of Blastocladiella emersonii

Summary 1. Methods are described for inducing the synchronous release of zoospores from single-generation cultures of Blastocladiella emersonii and preparing washed suspensions of up to 2×10¹⁰ non-encysted zoospores for physiological studies. 2. During 5 h incubations of such zoospores in a buffered CaCl₂ solution, the rate of oxygen uptake was ca. 10 l O₂ x h^-1 x (10⁷ cells)^-1, the respiratory quotient was 0.92, the average dry weight (47 picograms) of the spore decreased 1.5 picograms (pg), and other components (per-spore) decreased as follows: nucleic acid, nil; total lipid, 0,95 pg; phospholipid, 0.80 pg; polysaccharide, 0.5–1.0 pg, depending upon initial (1.5–2.3 pg) intracellular levels; protein, 3.0 pg; and total nitrogen 0.4 pg. During this period, 0.38 pg of NH₃-nitrogen was released per spore. Correspondingly, the lipid bodies decreased in size and number and the SB-matrix became progressively thinner. 3. It was concluded that during the endogenous metabolism of a non-encysted zoospore of B. emersonii, a significant portion (11.5%) of its protein pool, as well as lipid and polysaccharide components, were degraded.Abbreviations OC ordinary colorless - RS resistant sporangial - %NE and %E per-cent non-encysted and encysted, respectively - R.Q. respiratory quotient, Q_{O 2} (cell), l O₂ x h^-1 x (10⁷ cells)^-1 - BP Blastocladiella polysaccharide - MOPS Morpholino propane sulfonic acid - RNA ribonucleic acid - TCA trichloroacetic acid - PYG a peptone-yeast extract-glucose medium from Difco Labs, Detroit, Michigan     

Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii

The enzyme amidotransferase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C.P. Selitrennikoff, N.E. Dalley, and D.R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent Mr 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores.     

Tricarboxylic Acid Cycle Enzymes and Morphogenesis in Blastocladiella emersonii

During exponential growth, ordinary colorless (OC) plants of Blastocladiella emersonii consumed little glucose and produced no lactic acid. Similarly, resistant sporangial (RS) plants did not utilize glucose or produce lactic acid during the first 24 hr of exponential growth. During the next 24 hr of RS development, glucose was consumed with the concomitant production of lactic acid which was then reutilized. Lactic acid gradually accumulated again at maturity. Enzyme studies on cell-free extracts indicated the presence of all tricarboxylic cycle enzymes except α-ketoglutarate dehydrogenase at all stages of development of both RS and OC plants. Included among the enzymes detected were an adenosine monophosphate-stimulated, nicotinamide adenine dinucleotide-isocitric dehydrogenase, and citrate-condensing enzyme. When measured on a per plant basis, tricarboxylic cycle enzyme levels increased during the exponential growth of both kinds of plants. Only after the bicarbonate ceased to have effect on RS plant morphogenesis was there a decrease in the levels of the tricarboxylic cycle enzymes when measured on a per plant basis. Specific activity measurements indicated some differences in the differential rates of synthesis among the enzymes studied previous to 36 hr. Preliminary studies utilizing short periods of ¹⁴C-bicarbonate fixation in young RS plants indicated that during the first 4 min most of the label was located in aspartic acid. These results are discussed in terms of previous results and particularly Cantino's hypothesis concerning the relationship between bicarbonate induction and tricarboxylic-cycle enzymes in the morphogenesis of B. emersonii.     

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K⁺ channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K⁺ selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K⁺ channel and the rhodopsin–guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K⁺ channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.