Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii.

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

Evolutionary conservation of a core fungal phosphate homeostasis pathway coupled to development in Blastocladiella emersonii.

The model yeast Saccharomyces cerevisiae elicits a transcriptional response to phosphate (Pi) depletion. To determine the origins of the phosphate response (PHO) system, we bioinformatically identified putative PHO components in the predicted proteomes of diverse fungi. Our results suggest that the PHO system is ancient; however, components have been expanded or lost in different fungal lineages. To show that a similar physiological response is present in deeply-diverging fungi we examined the transcriptional and physiological response of PHO genes to Pi depletion in the blastocladiomycete Blastocladiella emersonii. Our physiological experiments indicate that B. emersonii relies solely on high-affinity Na+-independent Pho84-like transporters. In response to Pi depletion, BePho84 paralogues were 4-8-fold transcriptionally upregulated, whereas several other PHO homologues like phosphatases and vacuolar transporter chaperone (VTC) complex components show 2-3-fold transcriptional upregulation. Since Pi has been shown to be important during the development of B. emersonii, we sought to determine if PHO genes are differentially regulated at different lifecycle stages. We demonstrate that a similar set of PHO transporters and phosphatases are upregulated at key points during B. emersonii development. Surprisingly, some genes upregulated during Pi depletion, including VTC components, are repressed at these key stages of development indicating that PHO genes are regulated by different pathways in different developmental and environmental situations. Overall, our findings indicate that a complex PHO network existed in the ancient branches of the fungi, persists in diverse extant fungi, and that this ancient network is likely to be involved in development and cell cycle regulation.

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.

Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii.

HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii.

Observations on the life cycle of Coelomomyces indicus (Blastocladiales: Coelomomycetaceae) in anopheline mosquitoes from the Philippines and Thailand.

The water mold Coelomomyces indicus Iyengar is a widespread pathogen of anopheline mosquitoes in Asia and Africa, and it infects the copepod Microcyclops varicans Sars as its crustacean alternate host. This was determined by direct observation of field-infected copepods, selective meiospore encystment on M. varicans, and experimental infections of the copepod with zoospores from both thick and thin-walled meiosporangia. The physiological conditions governing germination of the 2 sporangial types were determined. The gametothallus in the copepod displays a light yellow pigmentation at maturity, and gametogenesis in both field and experimentally infected copepods occurs just at night fall, or 24 h after dark induction. In vivo culture was attained with the mosquito host Anopheles culicifacies Giles. Attempts to infect Anopheles stephensi Liston and Anopheles gambiae Giles, reported hosts of C. indicus, were unsuccessful.

Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii.

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.

A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42 degrees C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Sp1 and two metal-responsive elements.

Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii.

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.

Effect of heat shock on S6 phosphorylation during the development of Blastocladiella emersonii.

Changes in phosphorylation of ribosomal protein S6 during heat shock, induction of thermotolerance and recovery from heat shock at different stages of Blastocladiella emersonii development were investigated. Independently of the initial state of S6 phosphorylation (maximal or intermediate), a rapid and complete dephosphorylation of S6 is induced by heat shock and S6 remains unphosphorylated during the acquired thermotolerance. During recovery from heat shock rephosphorylation of S6 occurs always to the levels characteristic of that particular stage, coincidently with the turn off of heat shock protein synthesis.

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.

Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii.

The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.

Selective transport of nutrients via the rhizoids of the water mold Blastocladiella emersonii.

Previous work in this laboratory demonstrated that the rhizoids of Blastocladiella emersonii grow chemotropically toward a source of Pi and thus provided preliminary evidence that, in addition to serving as a holdfast, the rhizoids absorb nutrients. To further examine the role of the rhizoids in nutrient uptake, we devised a technique to introduce a barrier between the rhizoids and the thallus to that these cell compartments could be studied independently. Cells were grown on polycarbonate membrane filters in such a way that all of the thalli were on one side of the filter and essentially all of the rhizoids were on the opposite side. Nutrient uptake into the rhizoids and the thallus was measured by floating the filters bearing cells on radioactive medium so that only one side of the filter contacted the label. Mineral oil was used to block the diffusion of the label through the unfilled pores in the filter. This technique permitted us to establish clearly that the rhizoids absorb all seven of the nutrients tested. In addition, we found that some nutrients, specifically Pi and amino acids, appeared to be preferentially taken up via the rhizoids, whereas K+, Rb+, and Ca2+ entered the thallus and rhizoids equally. Cells grown in the presence of the microtubule synthesis inhibitors nocodazole and carbendazim elaborated only a stunted rhizoid system, so we examined their ability to accumulate the two classes of compounds. As expected, these cells were severely inhibited in Pi and amino acid uptake but retained normal uptake of K+, Rb+, and Ca2+.

Circulation of potassium across the plasma membrane of Blastocladiella emersonii: K+ channel.

A previous paper reported that the water mold Blastocladiella emersonii generates a transcellular electrical current, such that positive charges enter the rhizoid and leave from the thallus (Stump et al., Proc. Natl. Acad. Sci. U.S.A. 77: 6673-6677, 1980). To begin to understand the genesis of this current we investigated ionic relationships in this organism by use of intracellular microelectrodes. In cells suspended in buffered CaCl2, the membrane potential could be accounted for as a K+ diffusion potential; no evidence for an electrogenic pump was obtained. Potassium ions diffuse outward by a pathway that also carries Rb+ and Ba2+, but excludes both smaller and larger ions (Li+, Na+, Cs+, Mg2+, Ca2+, and choline). Chloride and other anions make little contribution to the potential, but the presence of Ca2+ in the external medium is required for successful potential measurements. In growing cells, the internal K+ concentration is generally somewhat higher than would be expected if the K+ distribution were determined entirely by the membrane potential. Under certain conditions, net uptake of K+ against the electrochemical potential gradient was observed. We suggest that K+ is actively accumulated by a primary transport system that may exchange K+ for H+, and that K+ leaks passively outward through the K+ channel. The K+ circulation across the membrane amounts to about 2% of the K+ pool per min, or 4.5 microA/cm2 of surface area. We propose that this K+ circulation is one arm of the transcellular current, carrying positive charge out of the thallus.

Stage-specific synthesis of proteins complexed to ribonucleoprotein particles and ribosomes in zoospores of Blastocladiella emersonii.

In Blastocladiella emersonii zoospores, a set of proteins was found associated with the ribosomes and free ribonucleoprotein particles distinct from the ribosomes and polyribosomes. These proteins were designated P120, P105, P64, P56, and P42 based on their molecular weights determined by gel electrophoresis. Synthesis of these proteins was detected only during late sporulation just before the time polyadenylated ribonucleic acid accumulates in the sporangia. These proteins banded in isopycnic metrizamide gradients at densities of 1.31 and 1.27 g/cm3, which corresponded to the densities of the ribosomes and free ribonucleoprotein particles, respectively. Comparison of the distribution of the proteins in sucrose versus metrizamide gradients suggested that P105 was removed from the free ribonucleoprotein particles before complexing with the ribosomes. During germination, these proteins disappeared from the ribosomal fractions, with kinetics corresponding to the resumption of protein synthesis. Another protein (P178) was observed to bind to the ribosomes before the onset of protein synthesis during germination. Cycloheximide did not block the addition of this protein to the monoribosomes.

Oriented growth of Blastocladiella emersonii in gradients of ionophores and inhibitors.

To investigate whether ion currents help to localize growth and development of Blastocladiella emersonii, we grew the organisms in gradients of various ionophores and inhibitors. Gradients were generated by placing into the culture fine glass fibers coated with insoluble inhibitors; in some cases, inhibitors were adsorbed onto beads of ion-exchange resin. Organisms growing in many of these gradients exhibited a striking tendency for the thalli to grow toward the fiber. This proved to be misleading; the cells grew not toward the source of the ionophore but into the unoccupied zone of inhibition adjacent to the fiber. Fibers coated with gramicidin-D induced marked effects on the growth of the rhizoids, which were greatly enlarged and grew toward and onto the fiber. None of the other inhibitors produced such effects, except for beads coated with the proton conductors tetrachlorosalicylanilide and compound 1799. The results suggest that orientation of rhizoid growth results from enhancement of proton flux across the plasma membrane. Growth of the rhizoids was also strongly oriented by gradients of inorganic phosphate and an amino acid mixture; gradients of glucose, K+, Ca2+, and glutamate were ineffective. We propose that a major physiological function of the rhizoid is to transport nutrients to the thallus. Finally, we examined the effects of a series of benzimidazole antitubulins as well as the cytochalasins. These did not orient growth but grossly perturbed the pattern of cellular organization, producing small spherical cells with multiple stunted rhizoids. The findings are interpreted in terms of the interaction of an endogenous transcellular proton current with elements of the cytoskeleton in the determination of form.

Endogenous electrical currents in the water mold Blastocladiella emersonii during growth and sporulation.

We have explored the pattern of electrical currents generated by single cells of the water mold Blastocladiella emersonii at several stages of its life cycle. Extracellular currents were measured with a vibrating probe constructed after the design of Jaffe and Nuccitelli [Jaffe, L. F. & Nuccitelli, R. (1974) J. Cell Biol. 63, 614-628]. In growing cells positive current, of the order of 1 microA/cm2, enters the rhizoid and leaves from the thallus; circumstantial evidence suggests that protons carry much of the current. Sporulation is associated with reversal of the current pattern, such that positive current enters the thallus and leaves from the rhizoidal region; the ions that carry the current have not been identified. These current patterns appear to play a role in the spatial localization of fungal growth and development.