Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

Abstract

Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium iondependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose-and Ca2+-dependent manner and stimulated NAD kinase from peas in a dosedependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzymelinked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pl of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.

Allosteric Regulation of Bacillus subtilis NAD Kinase by Quinolinic Acid

“…Scheme of de novo NAD(P) biosynthesis in bacteria. The pathway has been described previously (7,8). The enzymes (genes) involved in each step are abbreviated as follows: LASPO (nadB), L-aspartate oxidase; QS (nadA), quinolinate synthase; QAPRT (nadC), QA phosphoribosyltransferase; NMNAT (nadD), NaMN adenylyltransferase; NADS (nadE), NAD synthetase.…”

Calcium signalling in bacteria

“…When sodium dodecyl sulfate-polyacrylamide gel electrophoresis of calmodulin is performed in the presence of ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), calmodulin migrates with a significantly different mobility (39). These distinctive properties have assisted in the identification of calmodulin-like proteins in a wide range of bacteria including Myxococcus xanthus (32), Saccharopolyspora erythraea (6), and B. subtilis (22). In addition, calmodulin-like proteins have been discovered in several cyanobacteria (for reviews, see references 59 and 69), for example, in Nostoc sp.…”

“…In B. subtilis, for example, spores are enriched twofold in a heat-stable calmodulin-like activity that stimulates PDE and sporulation is inhibited by the calmodulin inhibitor trifluoperazine at levels at which the endogenous calmodulin-like protein is inhibited (22). Proteins are turned over two or three times during sporulation, and again in B. subtilis, lowering calcium levels to 2 M inhibits sporulation and reduces proteolysis by 60% (56).…”

Prokaryotic calcium‐binding protein of the calmodulin superfamily Calcium binding to a Saccharopolyspora erythraea 20 kDa protein

“…Recently, a 23 kDa calcium binding protein from Bacillus subtilis has been purified and characterized [6]. The protein was found to stimulate bovine brain phosphodiesterase and pea NAD kinase and reacted with anti-bovine brain calmodulin antibodies.…”

“…•However, the amino acid composition shows marked differences from that of eukaryotic calmodulins -for one thing, the B. subtilis protein is reported to possess no less than 63 Ser, 48 Gly and 11 His, while bovine calmodulin contains 4, 11 and 1 of these residues, respectively [6].…”