Grass Gregor, Schierhorn Angelika, Sorkau Eduard, Müller Helmut, Rücknagel Peter, Nies Dietrich H, Fricke Beate
Institute for Microbiology, Faculty of Life Sciences, Martin Luther University, D-06097 Halle, Germany.
Infect Immun. 2004 Jan;72(1):219-28. doi: 10.1128/IAI.72.1.219-228.2004.
Bacillus cereus frequently causes food poisoning or nosocomial diseases. Vegetative cells express the novel surface metalloproteinase camelysin (casein-cleaving metalloproteinase) during exponential growth on complex, peptide-rich media. Camelysin is strongly bound to the cell surface and can be solubilized only by detergents or butanol. Camelysin spontaneously migrates from the surface of intact bacterial cells to preformed liposomes. The complete sequence of the camelysin-encoding gene, calY, was determined by reverse PCR on the basis of the N-terminal sequence and some internal tryptic cleavage peptides. The calY gene codes for a polypeptide of 21.569 kDa with a putative signal peptide of 27 amino acids (2.513 kDa) preceding the mature protein (19.056 kDa). Although the predicted amino acid sequence of CalY does not exhibit a typical metalloprotease consensus sequence, high-pressure liquid chromatography-purified camelysin contains one zinc ion per protein molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tryptic peptide mass fingerprinting confirmed the identity of this zinc-binding protein as CalY. Disruption of the calY gene results in a strong decrease in the cell-bound proteolytic activity on various substrates.
蜡样芽孢杆菌经常引起食物中毒或医院感染性疾病。在富含肽的复杂培养基上指数生长期间,营养细胞表达新型表面金属蛋白酶camelysin(酪蛋白裂解金属蛋白酶)。Camelysin与细胞表面紧密结合,只有通过洗涤剂或丁醇才能溶解。Camelysin会从完整细菌细胞表面自发迁移至预先形成的脂质体。基于N端序列和一些内部胰蛋白酶裂解肽段,通过反向PCR确定了camelysin编码基因calY的完整序列。calY基因编码一种21.569 kDa的多肽,在成熟蛋白(19.056 kDa)之前有一个由27个氨基酸组成的推定信号肽(2.513 kDa)。尽管预测的CalY氨基酸序列未显示典型的金属蛋白酶共有序列,但高压液相色谱纯化的camelysin每个蛋白质分子含有一个锌离子。基质辅助激光解吸电离飞行时间质谱和胰蛋白酶肽质量指纹图谱证实了这种锌结合蛋白就是CalY。calY基因的破坏导致细胞对各种底物的蛋白水解活性大幅下降。