COMPLEX PHENOTYPES OF MUTIPLE MUTANTS OF E COLI

一些生物特征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如此复杂，以至于即使我们对一个生物的基因型式有深入的了解，我们也无法预测它的表型。大肠埃希氏菌的十二种青霉素结合蛋白(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多糖层，是我们最重要的单一类抗生素--β-内酰胺类抗生素的靶标。然而，尽管进行了几十年的工作，我们仍然不知道这些酶的详细生物学功能，也不能描述它们发挥作用的生化途径。随着抗药性生物体的兴起，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个大肠杆菌菌株，从这些菌株中删除了八种不同PBP的每种可能的组合。这组全面的突变使我们能够证明，这样的组合遗传策略产生了经典遗传方法不可能产生的结果。对突变体的初步筛选发现了不同寻常的和意想不到的表型，包括：胶囊生产、形态异常、噬菌体抗性、对抗生素诱导的裂解的抗性以及温度敏感性。在大多数情况下，这些特征不会出现在少于三到四个突变的细胞中，这些表型以一种复杂的方式依赖于活性多氯联苯的组合。我们建议完成这组突变体的筛选，寻找可能受到肽聚糖变化-例如抗生素-或化学诱导的自溶、噬菌体抗性、蛋白质分泌和细胞外结构形态发生影响的特征。此外，还将采用分析技术，以便在复杂情况下根据对基因的了解进行表型预测。可以预期有三个重要的结果。首先，我们将确定基本细胞过程中的新表型，在这些过程中，PBPs和多肽可以发挥基本的生物学作用。其次，我们将更好地了解多氯联苯如何维持细菌细胞壁，以及β-内酰胺类抗生素如何导致细菌细胞壁的破坏。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.