This project is aimed at gaining further understanding of the glp and dha regulons specifying respectively the respiratory and fermentative pathways for glycerol utilization in enteric bacteria. Possible physical interaction of the glycerol facilitator (permease) protein with glycerol kinase, resulting in more stringent specificity for substrate uptake and allowing simultaneous effector con of the permeability and the phosphorylation rate will be tested in E. coli. The studies will entail the isolation of the permease protein and the constitution of liposome vesicles capable of phosphorylative substrate uptake. The respiratory regulation of expression of genes encoding enzymes and proteins that participate in electron transfer from sn-glycerol 3-phosphate (an intermediate in the glp pathways) to various terminal acceptors (fumarate, nitrate, and oxygen), including effects that extend to other respiratory chains or fermentative pathways in E. coli, will be characterized with the aid of hybrid operons with lac structural genes fused to the promoters under investigation. Attempts will be made to determine by mutant analysis the number and nature of protein components in electron transfer pathways that commence with aerobic or anaerobic G3P dehydrogenase and terminate in fumarate reductase, nitrate reductase, or the cytochrome oxidases in E. coli. The biochemical mechanism and physiological significance of aerobic inactivation of Klebsiella pneumoniae glycerol dehydrogenase encoded by the dha regulon will be evaluated by comparisons with other NAD-linked enzymes of anaerobic or aerobic function and by selecting mutants with a glycerol dehydrogenase resistant to aerobic inactivation. The genetic organization of the dha regulon will be characterized by gene cloning, and the antagonistic interactions between the respiratory glp regulon and the fermentative dha regulon which impede their simultaneous expression will be probed with mutants blocked in the formation of various intermediates or gene products and by cloning the K. pneumoniae dha regulon into E. coli. Finally, the nature of DHA entry and trimethylene glycol exit will be explored with mutants blocked in the permeation processes.
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