Methionine Recycling Pathways in Klebsiella

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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.