COMPLEX PHENOTYPES OF MUTIPLE MUTANTS OF E COLI

一些生物学性状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，以至于即使我们对一个生物体的基因型有了深入的了解，我们也无法预测它的表型。大肠杆菌的12种青霉素结合蛋白（PBPs）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，是我们最重要的一类抗生素-内酰胺类的靶标。然而，尽管经过了几十年的研究，我们仍然不知道这些酶的详细生物学功能，也无法描述它们运作的生化途径。随着抗生素耐药生物的增多，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，以便设计更合理的抗菌策略。因此，我们构建了192株大肠杆菌，从中删除了8种不同PBPs的所有可能组合。这种全面的突变体使我们能够证明，这种组合遗传策略产生的结果是经典遗传方法无法实现的。突变体的初步筛选显示了不寻常的和意想不到的表型，包括：胶囊生产，形态畸变，噬菌体抗性，对抗生素诱导裂解的抗性和温度敏感性。在大多数情况下，这些性状不会出现在少于3或4个突变的细胞中，这些表型以一种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，以寻找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，分析技术也将进行调整，以便在复杂情况下根据基因型的知识进行表型预测。可以预期三个重要结果。首先，我们将在PBPs和肽聚糖发挥基本生物学作用的基本细胞过程中确定新的表型。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，广泛和明确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。

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.