Methionine Recycling Pathways in Klebsiella

描述（由申请人提供）：硫是许多化学品的关键成分， 普遍分布的不可或缺的生物分子。在大多数细菌中，硫 被有机地同化成半胱氨酸，半胱氨酸作为硫供体， 细胞中所有其他含硫分子。蛋氨酸是一个单独控制的过程，使用半胱氨酸作为硫供体。Klebsiella 产气菌与相关模式生物大肠杆菌不同， 采用至少2种不同的途径将结合甲硫氨酸的硫再循环回 进入半胱氨酸池。其中一条通路似乎特异性地引导逆转录酶的产生， 转硫作用（从蛋氨酸中产生半胱氨酸）， 由甲硫醇生成无机硫中间体。因此，硫 E.大肠杆菌-作为一种模式生物的理解 许多生物体的生理学是同源的--代表着 代谢，并提供了一个不完整的看法，这种普遍分布 physiology.这些循环途径可能在克雷伯氏菌的利用中发挥作用 一种致病的生活方式，硫可能是限制， 在循环利用全球丰富的有机硫方面发挥着重要作用 化合物.利用遗传和分子方法，这些循环途径将 接受检查。(1)已鉴定的半胱氨酸和蛋氨酸生物合成基因 将通过插入诱变在物理上和遗传上表征， 基因敲除和基因融合。(2)插入诱变将定义每个 甲硫氨酸再循环途径遗传。酶分析和营养分析 测试将阐明每一个回收途径的步骤。(3)融合到 lacZ报告基因将用于鉴定全局调节蛋白， 推断它们协同作用模式。通过这种方式， 途径揭示了复杂代谢的演变， 导致基因获得、基因丢失和基因组改变的选择力。

DESCRIPTION (provided by applicant): Sulfur is a critical component of numerous universally-distributed, indispensable biomolecules. In most bacteria, sulfur is assimilated organically into cysteine, which serves as the sulfur donor for all other sulfurbearing molecules in the cell. Methionine is made is a separately controlled process, using cysteine as a sulfur donor. Klebsiella aerogenes is distinct from the related model organism Escherichia coli in employing at least 2 distinct pathways to recycle methionine-bound sulfur back into the cysteine pool. One pathway appears specific in directing reverse transsulfurylation (creating cysteine from methionine), and the likely generates an inorganic sulfur intermediate from methanethiol. Therefore, sulfur amino-acid metabolism in E. coli - used as a model organism for understanding the physiology of numerous organisms by homology - represents remnant metabolism and provides an incomplete view of this universally distributed physiology. These recycling pathways may play a role in Klebsiella exploitation of a pathogenic lifestyle, where sulfur may be limiting, and may play significant roles in recycling abundant global pools of organic sulfur compounds. Using genetic and molecular methods, these recycling pathways will be examined. (1) The identified genes for cysteine and methionine biosyntheses will be characterized physically and genetically via insertion mutagenesis, gene knock-out and gene fusion. (2) Insertion mutagenesis will define each methionine recycling pathway genetically. Both enzyme assays and nutritional testing will elucidate the steps of each recycling pathway. (3) Fusions to the lacZ reporter gene will be used to identify global regulatory proteins, and to infer their synergistic modes of action. In this way, characterization of these pathways sheds light on the evolution of complex metabolism, and on the selective forces that lead to gene acquisition, gene loss, and genome change.