Immunocytochemical localization of a calmodulin-like protein from Bacillus subtilis

Previous studies on the extractability of the calmodulin-like (BsCaM) protein from B. subtilis cells implied that the protein might be associated with the membrane fraction. To more precisely locate the protein in cells, immunocytochemistry was used. Bovine brain anti-calmodulin antibodies and gold-conjugated secondary antibodies were used to localize the protein in thin sections of late-logarithmic phase cells and spores. The BsCaM was found almost exclusively in the cell wall/membrane regions of fixed, sectioned cells. The association of the BsCaM with the membrane was confirmed by ELISA and Western blot assays of preparations of membrane vesicles.;The BsCaM was partially purified from the soluble intracellular extracts of B. subtilis 3036 cells, grown in a chemical defined sporulation medium. Purification involved urea lysis, ammonium sulfate and acid precipitations and affinity chromatography. The purified protein stimulated both bovine brain and bovine heart phosphodiesterase and crossreacted with anti-calmodulin antibodies from bovine brain.;Demonstration of calcium-binding activity was determined by several methods including: Ruthenium red, Stains-All and ;The BsCaM was synthesized in a constitutive manner from the end of logarithmic growth through eight hours of sporulation as determined by antibody crossreactivity immunoblot and enzyme-linked-immunoabsorbent-assay,;In an attempt to clone the B. subtilis calmodulin gene, two genomic libraries were constructed and screened with anti-calmodulin antibodies from bovine brain. Sequence analysis of two cloned inserts revealed a high homology with B. subtilis groEL and a penicillin binding protein pbpD genes.;The B. subtilis calmodulin-like protein appears to be localized in the cell membrane of the bacterial cell, it is synthesized in a constitutive manner from logarithmic growth through 12 h of sporulation. The protein activates cAMP phpsphodiesterase, binds calcium ions and crossreact with anti-calmodulin antibodies from bovine brain.