PEPTIDOGLYCAN SYNTHESIS IN BACILLUS SUBTILIS

描述（由申请人提供）：肽聚糖细胞壁的合成是大多数细菌的基本过程，也是抗生素的有效靶点。参与肽聚糖聚合的主要酶（青霉素结合蛋白（PBP））的活性是已知的，但该家族中的多种蛋白质控制细胞形状和分裂的具体机制尚不清楚。拟议研究的长期目标是表征枯草芽孢杆菌主要PBP的功能结构域，并鉴定这些蛋白质与其他蛋白质之间的相互作用。将进行营养生长和孢子形成过程中肽聚糖合成的研究。对参与聚合特定细胞壁结构的蛋白质网络的理解将揭示抗生素开发的新的潜在靶点。孢子肽聚糖形成过程和孢子萌发过程中肽聚糖降解过程的知识将有助于孢子杀灭方法的发展。这些方法将普遍适用于防御孢子生物武器。 将产生变体A类PBP，包括活性位点突变体、截短蛋白和含有来自相关蛋白的结构域的嵌合蛋白，以及它们执行主要B的各种功能的能力。将评估枯草杆菌A类PBP，PBP 1。将测量酶活性和对表型特性（细胞长度、细胞直径、生长速率、肽聚糖结构和PBP 1定位）的影响。类似的研究将检查PBP 2c在孢子壁合成中的作用。免疫沉淀，PBP和自溶素活性测定，确定的突变株，蛋白质印迹法和质谱法将用于鉴定在细胞生长，分裂和孢子形成过程中与A类PBP相互作用的蛋白质。蛋白质-蛋白质相互作用的破坏和表型特性的变化之间的相关性。将使用免疫荧光显微镜和免疫沉淀法确定细胞形状确定、细胞分裂和孢子形成所需的B类PBP的细胞定位和蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): Synthesis of the peptidoglycan cell wall is an essential process in most bacteria and is an effective target for antibiotics. The activities of the major enzymes, the penicillin-binding proteins (PBPs), involved in peptidoglycan polymerization are known, but the specific mechanisms by which the multiple proteins in this family control cell shape and division are not clear. The long-term objectives of the proposed studies are characterization of the functional domains of the major PBPs of Bacillus subtilis and identification of interactions between these and other proteins. Studies of peptidoglycan synthesis during both vegetative growth and spore formation will be undertaken. An understanding of the network of proteins involved in polymerizing particular cell wall structures will reveal new potential targets for antibiotic development. Knowledge of the processes of spore peptidoglycan formation and of spore peptidoglycan degradation during germination will contribute to the development of methods for spore killing. Such methods will be generally applicable to defense against spore-based biological weapons. Variant class A PBPs will be produced, including active site mutants, truncated proteins, and chimeric proteins containing domains from related proteins, and their abilities to carry out the various functions of the major B. subtilis class A PBP, PBP1, will be assessed. Enzymatic activities and effects on phenotypic properties (cell length, cell diameter, growth rate, peptidoglycan structure, and localization of PBP1) will be measured. Similar studies will examine the role of PBP2c in spore wall synthesis. Immunoprecipitation, assays for PBP and autolysin activities, defined mutant strains, western blotting, and mass spectrometry will be used to identify proteins that interact with class A PBPs during cell growth, division, and sporulation. Correlations will be made between disruptions of protein-protein interactions and changes in phenotypic properties. Cellular localization and protein-protein interactions of class B PBPs required for cell shape determination, cell division, and spore formation will be determined using immunofluorescence microscopy and immunoprecipitation.