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Wen Luo

Advisor: Malcolm Winkler, Ph.D.

pdxJ and serC are two genes involved in pyridoxal 5-phosphate biosynthesis in Escherichia coli K-12. pdxj is arranged in a five-gene complex superoperon. Previous studies suggested that two promoters rncP and pdxP directed the transcription of the superoperon. In this study, we confirmed the existence of three species of transcripts from the rnc-pdx operon. About 70% of pdxJ-acpS transcripts came from pdxP. The remainder was directed by rncP. RNase T2 protection assay showed that only functional rnc-recO transcripts decreased 50% in the wild type strain compared with that in a rnc null mutant, indicating that rnc-recO transcripts was autoregulated by RNase III, but had no effect on pdxJ-acpS transcripts. The result from a rho-15 mutant suggested the putative terminator in the middle of recO might be Rho-dependent.

We also pursued exhaustive investigation on pdxJ regulation under a variety of physiological conditions. No carbon sources and long-chain fatty acids showed significant effect on the regulation of pdxJ expression. Mutations in pdxB and pdxH, two other genes in the same biosynthetic pathway caused no difference in the pdxJ expression either. Previous in vitro studies showed that the serC promoter required a supercoiling template for its transcription and activation by LRP. In this work, we performed in vivo studies on the effect of supercoiling level on serC expression. Salt-shock induced serC expression roughly 3-fold. >Novobiocin, a gyrase inhibitor antagonized the effect of salt shock. About a 5-fold induction of serC-lacZ fusion expression was observed in stationary phase bacteria. RNase T2 protection assays and quantitative Western immunoblotting confirmed that induction was at the transcription resulting in increased SerC protein levels, however, the induction detected by direct assays was lower that of fusion expression. The stationary phase induction required only the -10 and -35 region of serC promoter and was independent of the ss and Hfq factors, two global regulators of stationary phase gene expression. Our results suggested that supercoiling and pH may play a role in the stationary phase induction. Deletion fusion studies confirmed th negative role of crp in the serC expression but excluded the 26 nt dyad symmetry as the binding site for the putative repressor.

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Research Info

Regulation of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12