Bacterial spores maintain metabolic dormancy and have high resistance to external pressure. Germination requires degradation of the spore cortex and the participation of germination-specific cortex-lytic enzymes (GSLEs). Previously reported GSLEs have been identified in bacteria and facilitate germination. In this study, we have characterized a novel spore lytic enzyme, Ply67, from Bacillus pumilus phage vB_BpuM_BpSp. Ply67 had a similar cortex-lytic activity to GSLEs but disrupted the inner membranes (IMs) of spores, leading to spore killing rather than germination. The amino acid sequence of the complete protein, Ply67FL, exhibited 40% homology to the GSLE SleB. Domain prediction showed that Ply67FL was composed of three domains: a signal peptide, N-terminal domain protein and C-terminal domain protein. Ply67FL rapidly caused E. coli cells lysis when it was expressed in E. coli. The protein containing the C-terminal domain protein, Ply67C, could kill B. pumilus spores. The protein containing the N-terminal domain protein, Ply67N, could combine with the decoated B. pumilus spores, indicating that N-terminal was the binding domain and C-terminal was the hydrolase domain. The protein lacking the signal peptide but containing the N-terminal and C-terminal domain proteins, Ply67, had activity against spores of various Bacillus species. The surface of spores treated with Ply67 shrank and the permeability barrier was disrupted, and the inner contents leaked out. Immunoelectron microscopic observation showed that Ply67 was mainly acted on the spore cortex. Overall, Ply67 is a novel spore lytic enzyme that differs from other GSLEs not only in amino acid sequence but also in activity against spores, and Ply67 might have the potential to kill spores of pathogenic Bacillus species, e.g., B. cereus and B. anthracis.

中文翻译：

---

来自芽孢杆菌噬菌体的新型孢子裂解酶可杀死孢子

细菌孢子保持代谢休眠，对外界压力有很强的抵抗力。萌发需要孢子皮层的降解和萌发特异性皮质溶解酶 (GSLE) 的参与。先前报道的 GSLE 已在细菌中鉴定出来并促进发芽。在这项研究中，我们表征了一种来自短小芽孢杆菌噬菌体 vB_BpuM_BpSp 的新型孢子裂解酶 Ply67。Ply67 具有与 GSLE 相似的皮质溶解活性，但破坏了孢子的内膜 (IM)，导致孢子被杀死而不是萌发。完整蛋白质 Ply67FL 的氨基酸序列与 GSLE SleB 具有 40% 的同源性。域预测表明 Ply67FL 由三个域组成：信号肽、N 端域蛋白和 C 端域蛋白。Ply67FL 迅速引起大肠杆菌。大肠杆菌细胞在大肠杆菌中表达时裂解。含有 C 端结构域蛋白 Ply67C 的蛋白质可以杀死短小芽孢杆菌孢子。含有 N 端结构域蛋白 Ply67N 的蛋白质可以与脱壳的短小芽孢杆菌孢子结合，表明 N 端是结合域，C 端是水解酶域。缺乏信号肽但含有 N 端和 C 端结构域蛋白 Ply67 的蛋白质对各种芽孢杆菌属的孢子具有活性。Ply67处理的孢子表面收缩，渗透屏障被破坏，内部内容物泄漏。免疫电镜观察表明Ply67主要作用于孢子皮层。全面的，