INTRODUCTION

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Calmodulin (CaM) is a small acidic protein present in all eukaryotic cells and shown to be highly conserved both functionally and structurally. Its primary role is to serve as an intracellular Ca²⁺ receptor, participating in signaling pathways leading to proliferation, motility, and cell cycle progression, to name a few of its numerous functions (4, 35). Ca²⁺-CaM complexes have been shown to act by modulating the activity of numerous intracellular proteins, including phosphodiesterase, Ca²⁺-ATPase, Ser/Thr protein kinases, and protein phosphatases (39, 51).

In lower eukaryotes amenable to genetic studies many insights into CaM function have been obtained. For instance, the Ca²⁺-binding function of CaM is dispensable for cell growth and division in Saccharomyces cerevisiae. Mutant CaMs in which the Ca²⁺-binding sites are inactivated support growth, and neither Ca²⁺-CaM-dependent protein kinases nor the Ca²⁺-CaM-dependent phosphatase calcineurin is required for cell proliferation (10, 11, 15). However, Ca²⁺-CaM and the Ca²⁺-CaM-dependent enzymes are required for survival of pheromone-induced growth arrest and for maintaining ion homeostasis (9, 28). In contrast, Aspergillus nidulans requires Ca²⁺-CaM for cell cycle progression, since CaMs mutated in the Ca²⁺-binding sites do not support cell growth and division (37). Furthermore, the single gene encoding A. nidulans Ca²⁺-CaM-dependent protein kinase is also essential (27).

The involvement of Ca²⁺ in fungal cell differentiation has also been investigated in several instances. Calcium has been shown to be important to mycelial dimorphism in Ceratocystis ulmi (29) and to appressorium formation in Metarrhizium anisopliae (46) and Colletotrichum trifolii (49) and serves as a branching signal in Fusarium graminearum (40) and Neurospora crassa (38).

Despite the considerable amount of data concerning the function of Ca²⁺ and CaM in higher fungi, no such studies have been carried out with the more primitive fungal representatives such as the chytridiomycetes, which are situated at the base of the fungal tree (48). In this sense, the chytridiomycete Blastocladiella emersonii constitutes an excellent system to investigate the role of Ca²⁺ and CaM during growth and differentiation in lower fungi. Its developmental cycle presents two stages of cell differentiation, germination and sporulation. During germination, the zoospore, a motile uninucleated nongrowing cell, goes through many important morphological changes, including the retraction of its flagellum, the biogenesis of a cell wall of chitin and a germ tube, and the fragmentation of a single giant mitochondrion, giving rise to the germling cell which is capable of vegetative growth (22). During growth, intense nuclear division unaccompanied by cell division is observed, producing a multinucleated cell, the sporangium. At any time during exponential growth, nutrient starvation induces the other transitional stage, sporulation, which culminates in the intracellular formation of zoospores, which are then released into the medium (22).

A possible role for Ca²⁺ during B. emersonii germination has been previously suggested when it was found that zoospore encystment is accompanied by an efflux of calcium, and that lanthanum, which blocks both the uptake and efflux of calcium, completely inhibits germination when added at the time of induction (18). Furthermore, it is known that calcium is both necessary and sufficient for sporulation of B. emersonii (44) and that low levels of Ca²⁺ enhance the stability of zoospores (45).

These results and the presence of a CaM-like protein in B. emersonii, identified by its ability to activate bovine cAMP-phosphodiesterase (17), have led us to isolate the gene encoding CaM and to study its expression throughout the fungal developmental cycle. Furthermore, we have used several compounds which affect Ca²⁺ homeostasis or CaM activity to investigate the role of Ca²⁺-CaM during the B. emersonii life cycle.

	MATERIALS AND METHODS

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Cloning of B. emersonii CaM gene. To isolate the complete CaM gene, a genomic library constructed in the vector -DASH (Stratagene) with fragments (9 to 20 kb) obtained from a partial digestion of total B. emersonii DNA with restriction enzyme Sau3A was analyzed using the central portion of the CaM gene from C. trifolii (GenBank/EMBL Data Bank) accession number AAA51652) obtained by PCR as a probe. A 240-bp DNA fragment was amplified using two primers (sense, 5'-CGGATCCAGGACATGATCAAC-3', and antisense, 5'-CGGGATCCGCCTCGCGGATCATCT-3') and plasmid pUC19 containing the C. trifolii CaM gene. The genomic library was analyzed by hybridization under low-stringency conditions, with the 240-bp PCR fragment labeled with ³²P by random-primed synthesis (13). The nitrocellulose filters were prehybridized for 2 h at 37°C in 60 mM potassium phosphate buffer (pH 6.2) containing 4× SSC (1× SSC is 15 mM sodium citrate plus 150 mM NaCl), 10 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), 30% formamide, and 5% nonfat dried milk. Hybridization was performed overnight at 37°C in the same solution after addition of the denatured probe (10⁶ cpm/ml). The filters were sequentially washed at 37°C in 4× SSC and 0.1% SDS four times for 30 min each, air dried, and exposed to Kodak X-Omat film with an enhancing screen at 80°C. Approximately 14,000 recombinant phages were analyzed, and a single genomic clone containing an insert of 11 kb was isolated. A complete cDNA encoding the B. emersonii CaM protein was obtained by screening a cDNA library constructed in the gt11 vector, as previously described (24). The probe was the 1.8-kb PstI/SalI fragment from the B. emersonii CaM gene. Analysis of recombinant phages under high-stringency conditions (hybridization solution of 60 mM potassium phosphate buffer [pH 6.2] containing 1× SSC, 10 mM EDTA, 0.4% SDS, 50% formamide, and 5% nonfat dried milk and washing solutions of 1× SSC-0.1% SDS, 0.5× SSC-0.1% SDS, and 0.1× SSC-0.1% SDS, incubated for 30 min at 42°C) led to the isolation of eight identical positive clones presenting an insert of 0.7 kb.

DNA sequence analysis. The 11-kb genomic insert was isolated and subjected to endonuclease restriction analysis, followed by Southern blot analysis revealing a 1.8-kb PstI/SalI fragment which hybridized to the 240-bp PCR-amplified fragment. This PstI/SalI fragment was further digested with restriction endonucleases, and several restriction fragments were subcloned into M13mp18 and M13mp19 (Bethesda Research Laboratories) for DNA sequence analysis of both strands. The nucleotide sequence was obtained by the dideoxynucleotide chain termination method with the Sequenase DNA-sequencing kit (Amersham). The complete nucleotide sequence of the 0.7-kb cDNA clone was determined after subcloning into the pUCBM21 vector, using universal forward and reverse primers and the Delta Taq cycle-sequencing kit (Amersham). Analysis of sequence data and sequence comparisons were performed with programs Blast-X from the National Center for Biotechnology Information and PileUp from the GCG package (Genetics Computer Group, Madison, Wis.).

Primer extension mapping of the transcription start site. An 18-nucleotide (nt) primer complementary to nt 15 to +3 of the CaM gene was 5' end labeled with [-³²P]ATP and T4 polynucleotide kinase and hybridized to 100 µg of total B. emersonii RNA isolated from vegetative cells and zoospores. The annealing reaction was carried out in 25 µl of 100 mM piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.0)-1 M NaCl-5 mM EDTA at 50°C for 16 h. The nucleic acids were ethanol precipitated and resuspended in 100 µl of a solution containing 50 mM Tris-HCl buffer (pH 8.3); 3 mM MgCl₂; 75 mM KCl; 20 mM dithiothreitol; 1 mM (each) dATP, dCTP, dTTP, and dGTP; and 40 U of RNase inhibitor (Boehringer Mannheim). The annealed primer was extended with 200 U of SuperScript RNase Hreverse transcriptase (GibcoBRL) at 42°C for 90 min. The RNA was digested by the addition of 50 µg of RNase A (Pharmacia) and incubation at 37°C for 30 min, and the extended products were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) (7 M urea-7.5% polyacrylamide) followed by autoradiography. The fragments were sized by comparison to a dideoxy sequencing ladder of pUCBM21 containing the 5' region of the CaM gene, using the same 18-nt oligonucleotide as primer.

Preparation of antigen and immunization. The 0.7-kb cDNA fragment encoding the complete B. emersonii CaM protein was cloned in frame into the expression vector pGEX-2TK (Pharmacia). A fresh colony of Escherichia coli BL21 containing the pGEX-2TK CaM plasmid was grown at 37°C in 2× TY (16 g of tryptone per liter, 10 g of yeast extract per liter, 5 g of NaCl per liter) medium supplemented with 100 µg of ampicillin per ml to an optical density at 600 nm of 0.7. Expression of the fusion protein CaM-glutathione S-transferase (GST) was induced by the addition of isopropyl-1-thio--D-galactopyranoside (IPTG) to a final concentration of 1 mM, and incubation was continued for up to 2 h. Cells were then harvested by centrifugation at 4°C for 15 min at 5,000 × g, and the cell pellets were frozen and stored at 20°C. A bacterial lysate was prepared by thawing and resuspending the cells in phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3) containing 1 mM phenylmethylsulfonyl fluoride and 25 mM benzamidine. The suspension was sonicated on ice with a Branson sonicator, and Triton X-100 was added to a final concentration of 1%. After centrifugation for 10 min at 3,800 × g to remove insoluble material, the supernatant was transferred to a fresh tube and the fusion protein was purified by affinity chromatography using glutathione Sepharose 4B from the GST purification modules (Pharmacia). The recombinant B. emersonii CaM was cleaved from GST using a site-specific protease whose recognition sequence is located immediately upstream of the multiple-cloning site on the pGEX-2TK vector. One female rabbit was immunized with approximately 500 µg of the purified CaM in phosphate-buffered saline and 0.5 ml of Freund's complete adjuvant. After 4 weeks, the rabbit received a second injection containing 1 mg of the antigen in Freund's incomplete adjuvant, and 9 days after that, the rabbit was bled from the ear and the antiserum obtained was tested in Western blots and immunoprecipitation assays.

Western blot analysis. Western blots were performed according to the method of Towbin et al. (47), with modifications as follows. Synchronized cells from different stages of B. emersonii development were collected from liquid culture, suspended in cold 10% trichloroacetic acid, and incubated for 30 min at 4°C. After centrifugation at 1,500 × g for 15 min, the precipitated proteins were resuspended by sonication, washed with cold chloroform and methanol (1:1), dried, and resuspended in Laemmli buffer (21) for SDS-PAGE. After electrophoresis the proteins were transferred to a nitrocellulose membrane, and the blot was incubated for 24 h in blocking buffer (10 mM Tris-HCl, pH 7.5) containing 150 mM NaCl, 5% nonfat dried milk, and 0.05% sodium azide. The polyclonal antiserum against B. emersonii CaM was diluted 1:500 in blocking buffer containing 0.02% Tween 20 and 0.03% Triton X-100 instead of nonfat dried milk, and the blot was incubated for 16 h at 4°C. The protein blot was then washed with TBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing 0.05% Tween 20 followed by TBS alone and was incubated with anti-rabbit immunoglobulin G antiserum conjugated with alkaline phosphatase (Sigma). Nitroblue tetrazolium and BCIP (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) were used as substrates to visualize the reaction.

Immunoprecipitation assays. Immunoprecipitation of cell extracts with polyclonal anti-CaM antiserum was carried out as previously described (20) except that labeled cells were lysed by sonication in wash buffer (50 mM Tris-HCl [pH 8.3], 450 mM NaCl, 0.5% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 25 mM benzamidine).

Northern blots. Total RNA, isolated from synchronized cells at different stages of B. emersonii development, was prepared by the Trizol method (Life Technologies, Inc.). RNAs were then resolved by electrophoresis on a 1.5% agarose-2.2 M formaldehyde gel and blotted onto Hybond N⁺ membranes (Amersham). The blots were prehybridized for 2 h at 37°C in 120 mM sodium phosphate buffer (pH 7.2) containing 250 mM NaCl, 7% SDS, and 1 mM EDTA and hybridized for 16 h in the same solution with the 1.8-kb PstI/SalI fragment as probe (10⁶ cpm/ml). The probe was labeled by random-primed synthesis (13). The membranes were sequentially washed under high-stringency conditions, as described above. As a control, the Northern blot was also hybridized to a ³²P-labeled cDNA clone encoding the -subunit of the mitochondrial processing peptidase (MPP) from B. emersonii, which was previously shown to be constitutively expressed in this fungus (41). The membrane was air dried and exposed to X-ray film at 80°C in the presence of an intensifying screen.

CaMKII autophosphorylation assay. All phosphorylation reactions were conducted at 35°C for 10 min in a 20-µl reaction volume containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.5 mM dithiothreitol, 0.1 mM Na₂EDTA, and 100 µCi of [-³²P]ATP/µmol, either in the presence of 2 mM CaCl₂ plus 2.5 µg of CaM from B. emersonii or bovine brain or 5 mM EGTA. Reactions were started by the addition of 50 ng of the -subunit of CaM-dependent protein kinase II (CaMKII) purified from rat forebrain (kindly provided by R. E. Larson, Universidade de São Paulo) and terminated by the addition of 4 µl of Laemmli sample buffer and boiling, followed by SDS-PAGE and autoradiography.

Inhibitor studies. To evaluate the effect of different drugs on B. emersonii sporulation, 1.6 × 10⁵ zoospores were inoculated into tissue culture dishes (35 by 10 mm) containing 1.6 ml of DM3 medium (23) and incubated for 14 h at 17°C. To induce sporulation, vegetative cells adhered to the bottom of the dishes were starved by withdrawal of the growth medium and washing of the cells three times with 5 ml of sporulation solution (SS; 1 mM Tris-maleate buffer [pH 6.8], 1 mM CaCl₂). The cells were then incubated in SS at 27°C for 4 h. Drugs were added separately to the culture dishes containing the induced cells at different times during sporulation. After 7 h of induction of sporulation the amount of empty zoosporangia was determined by observation of the cells under a light microscope. The effect of antagonists of B. emersonii germination was examined by inoculation of 4.3 × 10⁵ zoospores in 1.6 ml of germination solution (1 mM Tris-maleate buffer [pH 6.8], 1 mM CaCl₂, 10 mM MgCl₂, 50 mM KCl) or DM3 medium in tissue culture dishes containing different inhibitors. The effect of the drugs was examined by determining the percentage of germinating cells formed after 5 hours of incubation at 27°C. To examine the role of Ca²⁺-CaM during B. emersonii vegetative growth, 1.6 × 10⁵ zoospores were inoculated into tissue culture dishes containing 1.6 ml of DM3 medium without drugs, and the cells were incubated at 27°C for 60 min, by which time all zoospores had differentiated into germinating cells. Then, different inhibitors were added separately to the tissue culture dishes, followed by incubation at 17°C for 14 h. The resulting vegetative cells were then induced to sporulate in the absence of inhibitors by withdrawal of the growth medium and washing of the dishes three times with SS and incubation in the same solution at 27°C for 4 h. The number of zoospores obtained at the end of this period was determined under a light microscope using a chamber of Neubauer.

Nucleotide sequence accession number. The nucleotide sequence of the B. emersonii CaM gene was deposited in the GenBank/EMBL Data Bank and assigned accession no. AF264065.

	RESULTS

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Isolation and characterization of cDNA and genomic clones for B. emersonii CaM. A 240-bp DNA fragment encoding the central portion of the C. trifolii CaM protein (accession number AAA51652) amplified by PCR was used as a probe to screen a B. emersonii library, which was constructed in the vector DASH with genomic DNA fragments (9 to 20 kb) obtained from partial digestion with restriction enzyme Sau3A. A single genomic clone containing an insert of approximately 11 kb was isolated. Digestion of the insert with several restriction enzymes followed by Southern blot analysis revealed a 1.8-kb PstI/SalI fragment which hybridized to the 240-bp PCR probe and was shown to contain the entire B. emersonii CaM gene by DNA sequence analysis. The coding region of the gene is interrupted by three small introns (ranging from 63 to 268 bp) which were identified by comparison of the genomic DNA sequence with a complete cDNA isolated from a B. emersonii gt11 cDNA library (24), using the 1.8-kb PstI/SalI genomic fragment as probe. Both the 5' and 3' splice junctions of the three introns follow the consensus identified for B. emersonii introns (12), and all introns contain an internal sequence related to CTRAC, which is present in fungal introns (26). Concerning the location of these introns, only the first and the last are in conserved positions relative to those of vertebrate CaM genes, which in vertebrates correspond to the first and the fifth introns (30). The second B. emersonii intron, however, is located in a position close to that of the third intron of the A. nidulans CaM gene (36).

Mapping of the transcription initiation site and analysis of the 5' noncoding region. To determine the start site of transcription of the B. emersonii CaM gene, primer extension experiments were performed. An 18-nt primer complementary to nt 15 to + 3 of the CaM gene (Fig. 1) was 5' end labeled with [-³²P]ATP and hybridized with total RNA isolated from Blastocladiella vegetative cells and zoospores. The hybrids were then extended with Superscript RNase H reverse transcriptase, and the extension products were resolved by PAGE with urea. The extension fragments were sized by comparison to a dideoxy sequencing ladder of pUCBM21 plasmid containing the 5' region of the CaM gene, using the same 18-nt oligonucleotide as the primer. A single start site of transcription was observed at position 85 from the ATG encoding the initiator methionine using either RNA from vegetative cells or zoospores. Nuclease S1 protection assays were also performed to determine the transcription start site, and the protected fragments obtained using RNA from both cell types confirmed that the initiation site was at position 85 (data not shown).

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Transcription initiation site of B. emersonii CaM gene. (A) Primer extension mapping of the transcription start site. An 18-nt primer complementary to nt 15 to +3 was 5' end labeled with [-32P]ATP and hybridized to 100 µg of total RNA from zoospores (lane 1) or B. emersonii vegetative cells (lane 2). The hybrids were then extended with reverse transcriptase, and the extension products were resolved by denaturing gel electrophoresis and autoradiography. The sequencing ladder was generated with the same 18-nt oligonucleotide as primer and pUCBM21 containing the 5' flanking sequence of the CaM gene. (B) Nucleotide sequence of the 5' region of the CaM gene. The nucleotide +1 is the A of ATG encoding the initiator methionine. The underlined sequence is complementary to the oligonucleotide used in the primer extension experiment. The transcription start site is indicated by an arrow. The putative core sequences representing the binding sites for the TATA-binding protein and TFIID (TBP/TFIID), Sp1 (GC-box), CTF/NF1 (CAAT-box), and helix-loop-helix transcription factor (E-box) are indicated.

Expression of the CaM gene at the mRNA and protein levels. Northern blot analysis was performed to investigate possible changes in the amount of the mRNA encoding CaM during the B. emersonii life cycle. Total RNA isolated from synchronized cells at 0, 60, 90, 120, and 180 min of sporulation, zoospores, or cells at 45 and 90 min of germination was resolved by agarose gel electrophoresis, transferred to a Hybond N⁺ membrane, and probed with the ³²P-labeled fragment from the B. emersonii CaM gene. A single 0.7-kb transcript encoding the CaM protein was observed whose levels increased drastically during sporulation (approximately 10-fold), reaching maximum levels just prior to the release of the zoospores at the end of this stage (Fig. 2). At the zoospore stage, 30 min after the CaM mRNA reached its highest amount very low levels were detected (Fig. 2), suggesting a very short half-life for this mRNA. Nevertheless, during germination the amount of the CaM mRNA increased again, reaching maximum levels at 90 min of germination (Fig. 2). After 120 min, CaM mRNA levels start to decrease (data not shown). As a control, the same filter was hybridized to a probe corresponding to a portion of the gene encoding MPP, which is constitutively expressed during the life cycle of the fungus (41).

View larger version (36K): [in this window] [in a new window]   FIG. 2.   CaM mRNA levels during B. emersonii development. Total RNA isolated from cells at different stages of the B. emersonii life cycle was resolved by electrophoresis on a 1.5% agarose-2.2 M formaldehyde gel. The RNA was then blotted onto a Hybond N+ membrane and probed with the 1.8-kb PstI/SalI genomic fragment under high-stringency hybridization conditions. Lanes: 1 to 5, sporulating cells 0, 60, 90, 120, and 180 min after starvation, respectively; 6, zoospores; 7 and 8, germinating cells 45 and 90 min after inoculation in DM3 medium. As a control, the membrane was also hybridized to the MPP gene from B. emersonii (41). (A) Autoradiograms of the blot. (B) Relative amount of CaM mRNA, determined by scanning of the autoradiograms, normalized with respect to the MPP signal.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Immunoprecipitation and Western blot assays using antiserum against B. emersonii CaM. (A) Synchronized cultures of B. emersonii were pulse labeled with [35S]methionine at the indicated times of the fungus life cycle. Cell extracts were prepared, the proteins were immunoprecipitated with anti-CaM antibody, and the immunoprecipitates were resolved by SDS-PAGE and autoradiography to analyze the rate of CaM synthesis during B. emersonii development. Lanes: 1 to 5, sporulating cells 30, 90, 120, 150, and 180 min after starvation, respectively, 6 to 10, germinating cells 30, 60, 90, 120, and 150 min after inoculation in DM3 medium, respectively. (B) Western blot analysis of total protein extracts from cells at different stages of the B. emersonii developmental cycle. Protein extracts from cells at the indicated stages of the fungal life cycle were resolved by SDS-PAGE, and the proteins were blotted onto a nitrocellulose membrane. The membrane was then incubated with anti-CaM antiserum followed by anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. Nitroblue tetrazolium and BCIP were used as substrates to visualize the reaction. Lanes: 1 to 6, sporulating cells 0, 60, 90, 120, 150, and 180 min after starvation, respectively; 7, zoospores; 8 to 10, germinating cells 45, 90, and 120 min after incubation in DM3 medium, respectively. The relative amounts of CaM protein and the relative rates of CaM synthesis were determined by densitometry scanning of the blot and the autoradiogram, respectively.

B. emersonii CaM expressed in E. coli binds Ca²⁺ and activates vertebrate CaMKII. The same recombinant form of B. emersonii CaM used to obtain a specific antiserum was tested for its capability of binding Ca²⁺ and activating CaMKII purified from rat forebrain (7). Ca²⁺ binding to recombinant B. emersonii CaM was investigated by incubating the protein in the presence or absence of Ca²⁺ followed by SDS-PAGE. The protein incubated in the presence of Ca²⁺ exhibited the expected shift observed with vertebrate CaM (33), suggesting that all four Ca²⁺-binding domains may be functional (Fig. 4A). B. emersonii CaM was also assayed for its ability to activate CaMKII. As bovine brain CaM does, B. emersonii CaM was able to activate CaMKII, as determined by autophosphorylation of the enzyme in the presence of Ca²⁺, CaM, and [-³²P]ATP (Fig. 4B). Addition of the CaMKII inhibitor KN93 drastically decreased ³²P incorporation into the enzyme, confirming the specificity of the phosphorylation reaction.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Recombinant B. emersonii CaM is a functional protein. (A) Binding of Ca2+ to B. emersonii CaM and bovine brain CaM. Bovine brain CaM (lanes 1 and 2) and B. emersonii CaM (lanes 3 and 4) were preincubated in the presence of 2 mM CaCl2 (lanes 2 and 4) or 5 mM EGTA (lanes 1 and 3), and the proteins were subjected to SDS-PAGE and visualized by Coomassie blue staining of the gel. (B) Autophosphorylation of CaMKII is activated by Ca2+ and CaM from B. emersonii or bovine brain. Phosphorylation reactions were carried out as described in Materials and Methods, with the additions depicted at the top of the figure.

Effect of Ca²⁺-CaM antagonists during B. emersonii development. To examine the role of Ca²⁺-CaM during the B. emersonii life cycle, the germination, vegetative growth, and sporulation stages were carried out in the presence of various drugs which disturb calcium homeostasis and inhibit CaM activity or CaM-dependent proteins.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Effect of Ca2+-CaM inhibitors during the course of B. emersonii sporulation. Cells grown in DM3 medium for 14 h at 17°C were washed (four times) in SS and incubated in the same solution at 27°C. At the indicated times different drugs were added separately to the tissue culture dishes, and the percentage of empty zoosporangia was determined after 7 h of incubation at 27°C. In the control cells, sporulating in the absence of any drug, 100% empty zoosporangia was observed after 4 h at 27°C. Values are means of at least three independent experiments. Symbols: , TMB-8 (200 µM); , KN-93 (60 µM); ×, compound 48/80 (15 µg/ml); , calmidazolium (10 µM); , W7 (40 µM). A schematic representation of the various cell phenotypes, observed under light microscopy during the course of sporulation, is shown at the top of the figure.

View larger version (78K): [in this window] [in a new window]   FIG. 6.   Effect of Ca2+-CaM antagonists on B. emersonii vegetative growth. Zoospores were inoculated into tissue culture dishes containing DM3 medium and incubated at 27°C for 1 h. After germination, different Ca2+-CaM antagonists were added separately to each culture dish followed by incubation at 17°C for 14 h. The figure shows phase-contrast photographs of vegetative cells grown in the absence of drugs (1A) or in the presence of 10 µM calmidazolium (2A), 80 µM KN93 (3A), 15 µg of 48/80 per ml (1B), 60 µM W7 (2B), and 200 µM TMB-8 (3B).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Full-length cDNA and genomic clones encoding the single CaM gene in the aquatic fungus B. emersonii have been isolated and characterized. Multiple CaM genes have been described for vertebrates, such as humans (14, 50), rats (30), and teleost fish (25), encoding, however, an identical CaM molecule. In nonvertebrate species such as Drosophila melanogaster (52), Candida albicans (42), A. nidulans (36), and Aspergillus oryzae (53), a single gene has been identified, as is the case for B. emersonii.

The coding region of the B. emersonii CaM gene is interrupted by three introns, ranging in size from 63 to 268 bp, with all intron-exon junctions following the so-called GT-AG rule. The localization of introns in CaM genes has been maintained almost perfectly during evolution, although the number and length of the introns seem to be subject to variation. Most higher eukaryotes (34) and some lower eukaryotes (36, 53) present five introns in the CaM gene. The first intron in the B. emersonii gene, separating the ATG translation start codon from the remaining coding sequence, is present in the great majority of the CaM genes described to date. The second and third introns in the B. emersonii CaM gene interrupt the coding region in positions similar to those where the A. nidulans gene is interrupted by its second and fifth introns, respectively.

Primer extension experiments and S1 nuclease protection assays have indicated a single transcription start site for the B. emersonii CaM gene located at position 85 relative to the ATG of the initiator methionine. Analysis of the region upstream of the transcription initiation site revealed no sequences resembling a TATA box. However, in agreement with the proposed role of transcription factor Sp1 in tethering the basal transcription machinery to TATA-less promoters via interaction with the TFIID complex (31, 32), three putative Sp1-binding sites and a sequence following the consensus for the binding site of the TATA-binding protein TFIID were identified upstream of the CaM gene transcription start site. A putative E-box, the binding site for helix-loop-helix transcription factors, and a possible CAAT box were also found in the 5' region of the gene.

The amino acid sequence of B. emersonii CaM, deduced from the nucleotide sequence, presents a high degree of similarity with its homologs from other organisms. The highest percent identity was found with CaM from the oomycete P. infestans (93%), which is a fungal-like protist (48). Interestingly, B. emersonii CaM presents a higher degree of similarity to vertebrate CaM (91% identity) than to its counterpart in filamentous fungi like C. trifolii (83% identity).

A series of in vitro studies have established that CaM is altered conformationally by the binding of Ca²⁺ and in that form becomes capable of modulating its target proteins. Recombinant B. emersonii CaM produced in E. coli showed a shift in apparent molecular mass similar to that observed with bovine CaM in the presence of calcium. Since all four potential Ca²⁺-binding sites are structurally quite similar in both proteins, we can predict that B. emersonii CaM is able to bind four calcium ions, as the protein from vertebrates does. Furthermore, B. emersonii CaM was able to activate the autophosphorylation of the -subunit of CaMKII purified from rat forebrain, which indicates that the protein from B. emersonii is functionally homologous to vertebrate CaM (33).

Changes in CaM and its mRNA have been shown to occur during the cell cycle in animal cells, with CaM levels increasing twofold as cells enter S phase and remaining constant during the remainder of the cell cycle (5, 6, 36). During the B. emersonii developmental cycle, nuclear division is not accompanied by cell division; thus, it was important to investigate possible variations in the levels of CaM and CaM mRNA throughout the fungal life cycle. Northern blot analysis revealed that CaM mRNA levels are developmentally regulated in B. emersonii, being low in vegetative cells, increasing drastically (about 10-fold) during sporulation, and reaching maximum levels just prior to the liberation of the zoospores to the medium at the end of this stage. Zoospores presented very low amounts of CaM mRNA, suggesting that its half-life is short. During germination, CaM mRNA levels increase again. The relative rates of CaM synthesis were also determined during B. emersonii sporulation and germination, and the results showed that changes in the velocity of CaM protein synthesis parallel the variations in CaM mRNA levels, with higher rates of protein synthesis observed when higher amounts of the corresponding mRNA were present.

The changes in the rate of CaM synthesis resulted in variations in CaM protein concentration, which was shown to increase about threefold during sporulation, when cell division occurs. The amount of CaM decreased in the beginning of germination, increasing again when cells started nuclear division.

In view of the fact that B. emersonii is not a genetically tractable organism, to investigate Ca²⁺-CaM function in vivo we have made use of several pharmacological agents which affect calcium and/or CaM activities. The data obtained indicated that during sporulation Ca²⁺ and CaM have important roles. The Ca²⁺ channel blocker TMB-8 completely inhibits sporulation if it is added up to 60 min after induction of this developmental stage, indicating that entry of calcium or a calcium gradient is necessary during the first hour of sporulation. Similar conclusions were reached in a recent report by Coutinho and Corrêa (8), showing that vegetative cells do not sporulate in the absence of external calcium.

Among the pharmacological agents known to antagonize CaM action, W7 was determined to be the most effective in inhibiting B. emersonii sporulation. Even when added 3 h after induction, this antagonist was capable of blocking sporulation in 100% of the cells. Calmidazolium and compound 48/80 were also shown to be quite effective in inhibiting this morphogenetic transition. The CaMKII inhibitor KN93, which blocks CaM binding to the enzyme, was also shown to prevent sporulation, but its action seems to be most important in the first 90 min after induction of this process.

During B. emersonii germination the effect of Ca²⁺-CaM antagonists is less striking. TMB-8, which blocks the entry of Ca²⁺ into the cells, showed no effect on this differentiation process. This observation was not unexpected, since it was previously shown that large quantities of Ca²⁺ are released during the course of germination (18). Nevertheless, W7 and calmidazolium were determined to be effective inhibitors of germination if added at the time of induction, indicating that CaM could have an important role during this morphogenetic transition.

B. emersonii vegetative growth was also shown to be independent of external calcium, since vegetative cells growing in the presence of TMB-8 behaved exactly as control cells. However, the presence of CaM antagonists or CaMKII inhibitor KN93 during growth resulted in a strong decrease in the size of the cells and in the number of zoospores obtained after sporulation, with calmidazolium being the most potent antagonist.

The use of antagonists in vivo must be carefully interpreted. For instance, all drugs tested in this work are known to have secondary effects. Compound 48/80 is reported to be the most specific CaM antagonist, whereas W7 and calmidazolium are the least specific (1, 16). Even though it is considered to be the most specific, compound 48/80 has been shown to inhibit phosphatase A2 and phosphatidylinositol-specific phospholipase C (3). However, the fact that the phospholipase inhibitor U73122 (200 nM) produces no effect on the B. emersonii developmental cycle (data not shown) indicates that compound 48/80 inhibits the fungal life cycle by antagonizing CaM action.

Calmidazolium in the protozoan (fungal-like protist) Dictyostelium discoideum induces both a rapid release of Ca²⁺ from intracellular storage compartments and an influx of Ca²⁺ across the plasma membrane, which leads to an instant global transient increase in calcium concentration. W7 also induces transient elevations of calcium concentration which are slow and seen in some cells (43). It is not known if the same phenomena occur in B. emersonii, but the efflux of calcium is an essential event to the germination process in this fungus (18). Thus, it is possible that calmidazolium and W7 inhibit B. emersonii germination by affecting calcium homeostasis and not only by their CaM-antagonizing properties.

In conclusion, the data presented in this study indicate that external calcium is only necessary during B. emersonii sporulation, whereas CaM and CaMKII are essential for growth and sporulation of the fungus.