This laboratory has been studying the global responses of the model organism Escherichia coli to reducing agents, such as 1-thioglycerol or dithiothreitol. In the course of these investigations we discovered that cells protect themselves from thiols by methylating them, followed by secretion of the methylated thiol. Cells use their precious S-adenosyl methionine resource for this process, even if it means growth stoppage. Exposure to thiols result in global changes in hundreds of genes in E. coli. Among the notable responses are an up-regulation in porphyrin and riboflavin syntheses. We isolated a mutant, which produces an inordinate amount of porphyrin, when exposed to thiols. The biochemical basis for this turned out to be the dramatic increase in the enzyme glutamyl-tRNA reductase, the rate limiting enzyme of porphyrin biosynthesis in E. coli.

The mutated gene is yet to be identified!

In the meanwhile, we began looking for the locus of control for the reductive stress response. We reasoned, that should such a locus exist, if it were to be mutated, it would result in a thiol hypersensitive phenotype. Sure enough, we found a thiols hypersensitive mutant. The cause turned out to be an inability of the cell to make ubiquinone. Complementation of this mutant with a plasmid carrying the ubiX gene, removed thiol hypersensitivity and restored the ubiquinone making capacity. Connecting thiol hypersensitivity with missing ubiquinone lead us to postulate that the periplasmic disulfide reductase system is linked to the respiratory chain. Indeed, when we examined known mutants of the disulfide reductase system they turned out to be thiol hypersensitive also.

It was odd that ubiX genes should restore ubiquinone synthesis in our mutant because there is a second gene, ubiD, which is supposed to catalyze the same biosynthetic step, the decarboxylation of 3-hydroxy-octaprenyl benzoate. The only way this could happen is if this second gene, ubiD, was also mutated. When we attempted to sequence ubiD of E. coli we discovered that the location of this gene on the chromosome was not known. After considerable amount of work, we identified the open reading frame yigC as the ubiD gene. To our surprise we found no mutation in the ubiD gene of the ubiquinone deficient mutant. So why was this mutant ubiquinone deficient? The answer turned out to be a mutation in its ubiG gene, which codes for multiple methylating steps in ubiquinone synthesis. This finding only deepened the mystery! Now we are faced with having to explain how a mutation in the ubiquinone biosynthesis can be complemented by either the ubiX or the ubiG genes. To solve this riddle we are attempting to knock out the ubiX gene in E. coli and to study its physiological consequences. We have data that support the notion that the ubiquinone biosynthetic enzymes may form a tightly integrated complex. Our eventual goal is to identify the nature of protein-protein interactions among these proteins.