A fungal homologue of neuronal calcium sensor-1, Bbcsa1, regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the DBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H + pump/ATPase was noted in the mutant. A slight growthdeficient phenotype was observed for the DBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the DBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached. INTRODUCTIONIntracellular Ca 2+ levels affect many signal transduction cascades that transduce stimuli to appropriate cellular responses. Cellular Ca 2+ levels are carefully controlled by a diverse set of Ca 2+ channels, pumps, transporters and calcium-binding proteins (Cunningham, 2011). Members of the last-named group are often divided into two classes: calcium buffers and calcium sensors. Neuronal calcium sensor-1 (NCS-1)/frequenin (the yeast homologue) represents a subfamily of the calcium sensors that contain three functional calcium-binding EF-hand motifs and show high-affinity calcium binding at concentrations below that which would activate other calcium transducers such as calmodulin (Burgoyne, 2004). Upon calcium binding, these proteins are thought to undergo a conformational change allowing for interactions with target proteins and/ or receptors. Many NCS proteins are N-terminally myristoylated, with the solvent accessibility of this lipid moiety dependent upon Ca 2+ binding. This 'switch' is considered to act as a reversible mechanism for association of NCS proteins with membranes and their components, although in some sy...