CLONING AND ANALYSIS OF THE VITAMIN K2 GENE CLUSTER

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In humans and other higher animals, vitamin K (as vitamin K2 or menaquinone) serves as an essential cofactor in the formation of several clotting factors. The menaquinone-dependent biochemical reaction in clotting factor synthesis is the Gamma-carboxylation of glutamic acid. Among prokaryotic cells, on the other hand, menaquinone functions as a lipophilic electron carrier in membrane-bound electron transfer chains. Most bacterial species containing menaquinone are capable of synthesizing it de novo, and there is some evidence that intestinal flora can supply their animal hosts with menaquinone in the absence of dietary sources of the vitamin. Knowledge of the biosynthetic steps in bacterial menaquinone formation and the regulation of this biosynthesis should enhance our understanding of this aspect of bacterial-human interaction. The long-term objectives of the applicant's research program are: (i) to discover the principles governing regulations of bacterial menaquinone biosynthesis, both at the metabolic and the genetic levels; ii) to relate these principles to the functioning of menaquinone in the biology of prokaryotic cells. The specific aims of this research proposal are: i) the use of molecular cloning techniques to isolate from the bacterium Bacillus subtilis a cluster of genes (the men operon) encoding information for the menaquinone biosynthetic enzymes and associated regulatory regions; ii) the physical analysis of this DNA fragment by restriction mapping, subcloning, nucleotide sequence determination, and transcript mapping; iii) establishment of the presence, order, and functionality of structural genes within the operon fragment by recombination and complementation measurements of subcloned sequences.