SPORE PEPTIDOGLYCAN SYNTHESIS IN BACILLUS SUBTILIS

建议研究的长远目标如下： 所涉及的酶活性和基因产物的表征 在细菌内生孢子的肽聚糖细胞壁的合成中， 2)检查每种类型的肽聚糖结构的作用 在测定内生孢子抗性特性方面的改进。 的 具体目的是：1）确定内生孢子的结构 肽聚糖在其合成的第一阶段，并跟踪 2）确定休眠孢子的最终形态; 由于青霉素的损失而产生的这种结构的合成变化- 结合蛋白、自溶素和孢子形成相关基因产物; 和3）在体外纯化和表征参与 肽聚糖侧链裂解和胞壁内酰胺生产。 了解内生孢子抗性特性、休眠和 寿命可有助于更好的净化方法和方法 用于药物和疫苗的储存和运输。 肽聚糖 合成通常是抗生素作用有吸引力的靶点， 对这一过程的进一步研究将有助于确定 鉴定新的抗生素。 肽聚糖将从 枯草芽孢杆菌化学和酶促产孢培养物 处理，用溶菌酶消化，并通过高压液相色谱法进行分析。 液相色谱法（HPLC），使用先前开发的方法， 休眠运动肽聚糖的分析。 新的尿肽将是 通过氨基酸分析和质谱鉴定。外观 以及肽侧链丙氨酸和甘氨酸残基的损失，胞壁- 内酰胺生产和肽交联的变化将是 在整个孢子形成过程中进行定量。 分析将 使用缺乏单个青霉素结合蛋白的菌株重复， 自溶素和孢子形成相关基因产物。 cwlD基因 将对产物进行纯化，并在体外测定胞壁酰-L-丙氨酸 活性和用于胞壁内酰胺合成。 未成熟肽聚糖 来自突变菌株和纯化的胞肽的样品将用作 在这些试验中的底物。 反应进程将使用 HPLC法和通过将该方法适用于毛细管 电泳

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.