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 chytridi omycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRN A species encoding a predicted protein 91% identical to human CaM. B. emers onii CaM has been expressed in Escherichia coil as a fusion protein with gl uthatione S-transferase (GST) and purified by affinity chromatography and c leavage from the GST portion using a site-specific protease, In the presenc e of Ca2+, B. emersonii CaM exhibited a shift in apparent molecular mass si milar to that observed with bovine CaM and was able to activate the autopho sphorylation of CaM dependent protein kinase II (CaMKII) from rat brain. Ca M expression is developmentally regulated in IZ. emersonii, with CaM mRNA a nd protein concentrations increasing during sporulation to maximum levels o bserved just prior to the release of the zoospores into the medium. Both Ca M protein and mRNA levels decrease drastically at the zoospore stage, incre asing again during germination. The CaM antagonists compound 48/80, calmida zolium, and W7 were shown to completely inhibit B. emersonii sporulation wh en added to the cultures at least 120, 150, and 180 min after induction, re spectively. All these drugs also inhibited growth and zoospore production i n this fungus, The Ca2+ 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 sugges t that the Ca2+-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii.