Groupe de Recherche sur les Antimicrobiens et les Micro-organismes, UPRES EA 2656, IFR 23, Université de Rouen, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France.
J Bacteriol. 2010 May;192(9):2373-84. doi: 10.1128/JB.01546-09. Epub 2010 Feb 26.
This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.
这项工作报道了梭菌属中第一种已知的肽聚糖水解酶（Acp）的特性，该酶主要在营养生长期间产生。Acp 具有模块化结构，包含三个结构域：信号肽结构域、具有重复序列的 N 端结构域和 C 端催化结构域。纯化的重组 Acp 催化结构域对几种革兰氏阳性细菌的细胞壁表现出溶菌活性。通过将反向相高效液相色谱（RP-HPLC）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析相结合，分析枯草芽孢杆菌肽聚糖的消化产物，确定了其水解特异性，结果显示具有 N-乙酰葡萄糖胺酶活性。acp 表达的研究表明，在生长过程中持续表达，这表明 Acp 在梭菌属的生长中具有重要作用。此外，使用抗 Acp 抗体进行细胞分级分离和间接免疫荧光染色，发现 Acp 位于对数生长期营养细胞的隔膜肽聚糖中，这表明 Acp 在细胞分离或分裂中发挥作用。通过使用移动组 II 内含子策略（ClosTron）获得了 acp 缺失突变株。显微镜检查表明，acp 突变株缺乏营养细胞分离，以及用抗 Acp 抗体孵育的野生型菌株，这表明 Acp 在细胞分离中起关键作用。野生型和 acp 突变株对 Triton X-100、胆汁盐和万古霉素诱导的应激的比较反应表明，Acp 参与了这些应激诱导的自溶。总的来说，Acp 似乎是一种主要的细胞壁 N-乙酰葡萄糖胺酶，参与营养生长和应激诱导的自溶。
