SPORE PEPTIDOGLYCAN SYNTHESIS IN BACILLUS SUBTILIS

拟议研究的长远目标如下：1) 酶活性和涉及的基因产物的表征 在细菌内孢子和细菌内孢子的肽聚糖细胞壁的合成 2)检测每种类型的肽聚糖结构的作用 在测定内孢子抗性特性方面的改进。这个 具体目标是：1)确定内孢子的结构 肽聚糖在其合成的第一阶段，并跟踪 结构在休眠孢子中的最终形式；2)鉴定 由于失去青霉素而产生的这种结构的合成变化- 结合蛋白、自溶素和产孢子相关基因产物； 3)体外纯化和鉴定一种参与酶 肽聚糖侧链断裂与内酰胺类化合物的生产。 了解内生孢子抗性特性、休眠和 长寿可能有助于更好的去污方法和方法 用于储存和运输药品和疫苗。肽多糖 一般来说，合成是抗生素作用的一个有吸引力的目标， 对这一过程的进一步研究将有助于研究 新抗生素的鉴定。多肽多糖将从 枯草芽孢杆菌的化学和酶促芽胞培养 处理，用溶菌酶消化，并用高压分析 使用先前开发的用于 休眠运动肽多糖的分析。新型多肽将被开发 用氨基酸分析和质谱仪进行鉴定。外观 和多肽侧链丙氨酸和甘氨酸残基的损失， 内酰胺类物质的产生，以及多肽交联度的变化 在产孢子的整个过程中进行了量化。分析将是 重复使用缺乏个别青霉素结合蛋白的菌株， 自溶素和产孢子相关基因产物。CwlD基因 产品将被提纯，并在体外进行溶菌酰丙氨酸的检测 活性和内酰胺合成。未成熟的肽聚糖 来自突变菌株和纯化的鼠多肽的样本将作为 这些检测中的底物。将使用以下工具监控反应进度 高效液相色谱法及其在毛细管法中的应用 电泳法。

The long-term objectives of the proposed studies are: 1) characterization of the enzymatic activities and gene products involved in synthesis of the peptidoglycan cell wall of bacterial endospores and 2) examination of the role of each type of peptidoglycan structural modification in determining endospore resistance properties. The specific aims are: 1) to determine the structure of the endospore peptidoglycan in the first stages of its synthesis and to track the structure to its final form in the dormant spore; 2) to identify the changes in synthesis of this structure produced by loss of penicillin- binding proteins, autolysins, and sporulation-associated gene products; and 3) to purify and characterize in vitro an enzyme involved in peptidoglycan side chain cleavage and muramic-lactam production. Knowledge of principles of endospore resistance properties, dormancy,and longevity may contribute to better decontamination methods and methods for storage and transport of drugs and vaccines. Peptidoglycan synthesis in general is an attractive target for antibiotic action, further studies of this process will contribute to methods for identification of new antibiotics. Peptidoglycan will be purified from sporulating cultures of Bacillus subtilis by chemical and enzymatic treatments, digested with muramidase, and analyzed by high-pressure liquid chromatography (HPLC) using methods previously developed for analysis of dormant sport peptidoglycan. Novel muropeptides will be identified using amino acid analysis and mass spectrometry. Appearance and loss of peptide side chain alanine and glycine residues, muramic- lactam production, and changes in peptide cross-linking will be quantified throughout the sporulation process. The analysis will be repeated using strains lacking individual penicillin-binding proteins, autolysins, and sporulation-associated gene products. The cwlD gene product will be purified and assayed in vitro for muramoyl-L-alanine activity and for muramic-lactam synthesis. Immature peptidoglycan samples from mutant strains and purified muropeptides will serve as substrates in these assays. Reaction progress will be monitored using the HPLC method and by adaptation of the method to capillary electrophoresis.