Structure, Expression, and Functional Analysis of the Gene Coding for Calmodulin in the Chytridiomycete Blastocladiella emersonii

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S -transferase (GST) and puriﬁed by afﬁnity chromatography and cleavage from the GST portion using a site-speciﬁc protease. In the presence of Ca 2 1 , B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii , with CaM mRNA and protein concentra-tions increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca 2 1 channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca 2 1 -CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii . 3 and Approximately 14,000 recombinant phages were genomic clone containing an insert of 11 kb A complete cDNA encoding the B. CaM protein was obtained by screening a cDNA library constructed in the l gt11 vector, as previously described (24). was the 1.8-kb Pst I/ Sal I fragment from the B. CaM gene. of recombinant phages high-stringency of mM potassium 3 of eight identical clones an insert of kb. DNA sequence genomic to endonuclease restriction by Southern blot a 1.8-kb Pst I/ Sal I fragment hybridized to the 240-bp PCR-ampliﬁed fragment. This Pst I/ Sal I fragment was further digested with restriction endonucle- ases, 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 complete nucleotide sequence of 0.7-kb clone was determined subcloning into the pUCBM21 universal forward and reverse prim- and Delta Taq cycle-sequencing kit Analysis sequence data and sequence comparisons were performed with programs Blast-X PileUp extension An 18-nucleotide 15 to 1 hybridized to m B. annealing reaction carried out in 25 m of 100 mM piperazine- N , N 9 -bis(2-ethanesul-fonic acid) (PIPES) buffer M NaCl–5 mM EDTA at 50°C for 16 h. nucleic acids ethanol precipitated and resuspended in 100 m a solution containing 50 mM Tris-HCl buffer 3 mM MgCl ; dithiothreitol; dATP, dGTP; and 40 U RNase inhibitor (Boehringer annealed extended (pH 7.5), 10 mM MgCl 2 , 0.5 mM dithiothreitol, 0.1 mM Na 2 EDTA, and 100 m Ci of [ g - 32 P]ATP/ m mol, either in the presence of 2 mM CaCl 2 plus 2.5 m g of CaM from B. emersonii or bovine brain or 5 mM EGTA. Reactions were started by the addition of 50 ng of the a -subunit of CaM-dependent protein kinase II (CaMKII) puriﬁed from rat forebrain (kindly provided by R. E. Larson, Uni- versidade de Sa˜o and terminated by the addition of 4 m l 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 3 10 5 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 CaCl 2 ). 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. Afte r 7 h ofinduction 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 3 10 5 zoospores in 1.6 ml of germination solution (1 mM Tris-maleate buffer [pH 6.8], 1 mM CaCl 2 , 10 mM MgCl 2 , 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 Ca 2 1 -CaM incubated zoospores had differentiated germinating inhibitors separately dishes, incubation induced sporulate absence of of the the dishes three times incubation