COMPLEX PHENOTYPES OF MUTIPLE MUTANTS OF E COLI

大肠杆菌多种突变体的复杂表型

• 批准号: 6520198
• 负责人: KEVIN D YOUNG
• 金额: $ 22.37万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520198
• 关键词: Escherichia coli; bacterial genetics; bacterial proteins; binding proteins; combinatorial chemistry; gene expression; gene interaction; gene mutation; genetic strain; microorganism culture; mutant; penicillins; peptidoglycan; phenotype

Some biological traits are mediated by combinations of genes or proteins whose interactions are so complicated that we can not predict an organism's phenotype even with an intimate knowledge of its genotype. The twelve penicillin binding proteins (PBPs) of Escherichia coli form a model system for the study of such "complex phenotypes." The PBPs synthesize, modify and maintain the rigid peptidoglycan layer of the bacterial cell wall and are the targets of our most important single class of antibiotics, the beta-lactams. Nonetheless, despite decades of work, we do not know the detailed biological functions of these enzymes nor can we describe the biochemical pathways by which they operate. This information is becoming increasingly important with the rise of antibiotic resistant organisms. Our long term objective is to explain the structure, synthesis, and function of bacterial peptidoglycan so that more rational antimicrobial strategies can be devised. Therefore, we constructed 192 E. coli strains from which were deleted every possible combination of eight different PBPs. This comprehensive set of mutants allowed us to show that such a combinatorial genetic strategy produces results impossible to classic genetic approaches. Preliminary screening of the mutants revealed unusual and unanticipated phenotypes, including: capsule production, morphological aberrations, phage resistance, resistance to antibiotic- induced lysis, and temperature sensitivity. In most cases, the traits did not appear in cells with fewer than three or four mutations, and these phenotypes depended in a complex way on the combinations of active PBPs. We propose to complete the screening of this set of mutants for traits likely to be affected by a alterations in the peptidoglycan-e.g., in antibiotic-or chemically-induced autolysis, phage resistance, protein secretion, and in the morphogenesis of extracellular structures. In addition, analytical techniques will be adapted so that phenotypic predictions can be made from knowledge of the genotype in complex situations. Three significant results can be anticipated. First, we will identify new phenotypes in basic cellular processes in which the PBPs and peptidoglycan play fundamental biological roles. Second, we will understand better how the PBPs maintain the bacterial cell wall and how beta-lactam antibiotics induce its destruction. And third, the compilation of extensive and defined datasets will allow us to develop appropriate tools to investigate complex relations between genotype and phenotype.

一些生物学特性是由基因或蛋白质的组合介导的，这些基因或蛋白质的相互作用是如此复杂，以至于即使我们对生物体的基因型有了深入的了解，我们也无法预测生物体的表型。大肠杆菌的12种青霉素结合蛋白（PBP）形成了研究这种“复杂表型”的模型系统。“PBPs合成，修饰和维持细菌细胞壁的刚性肽聚糖层，并且是我们最重要的一类抗生素β-内酰胺的目标。尽管如此，尽管几十年的工作，我们不知道这些酶的详细生物学功能，也不能描述它们运作的生化途径。随着抗生素耐药微生物的兴起，这一信息变得越来越重要。我们的长期目标是解释细菌肽聚糖的结构、合成和功能，以便设计出更合理的抗菌策略。因此，我们构建了192 E。大肠杆菌菌株中删除了8种不同PBPs的每种可能组合。这一组全面的突变体使我们能够表明，这种组合遗传策略产生的结果是经典遗传方法所不可能的。突变体的初步筛选揭示了不寻常的和未预料到的表型，包括：荚膜产生、形态畸变、噬菌体抗性、对抗生素诱导的裂解的抗性和温度敏感性。在大多数情况下，这些特征不会出现在少于三个或四个突变的细胞中，并且这些表型以复杂的方式依赖于活性PBPs的组合。我们建议完成这组突变体的筛选，以获得可能受肽聚糖改变影响的性状，例如，在抗生素或化学诱导的自溶、噬菌体抗性、蛋白质分泌和细胞外结构的形态发生中。此外，将调整分析技术，以便在复杂情况下根据基因型知识进行表型预测。可以预期三个重要结果。首先，我们将确定新的表型在基本的细胞过程中，PBPs和肽聚糖发挥基本的生物学作用。其次，我们将更好地了解PBPs如何维持细菌细胞壁以及β-内酰胺抗生素如何诱导其破坏。第三，广泛和定义的数据集的汇编将使我们能够开发适当的工具来研究基因型和表型之间的复杂关系。