Date of Award

8-1-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Jerry Simecka

Second Advisor

Ming-Chi Wu

Third Advisor

Stephen R. Grant

Abstract

Wei, Bangdong L., Positive Regulation of Acetate Metabolism and Motility by the RNA-binding Protein CsrA in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), August, 2000, 118 pp., 5 tables, 19 illustrations, bibliography, 175 titles. The carbon storage regulatory (Csr) system consists of a small RNA-binding effector protein, CsrA, and non-coding RNA, CsrB. CsrA acts as a global regulator and modulates specific mRNA stability in Escherichia coli. It regulates central carbon metabolism, physiology, and cell surface properties on a broad scale. In this study, the regulatory roles of csrA in acetate metabolism and motility were examined. The csrA gene was demonstrated to positively regulate acetyl-CoA synthetase and isocitrate lyase, while it did not affect phosphotransacetylase, isocitrate dehydrogenase, or citrate synthase. As a result, growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media. Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. The TCA cycle intermediates or pyruvate, but not glucose, galactose or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. Apparently, central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism are combined to cause metabolic stress by depleting the TCA cycle. The csrA gene was essential for motility and flagellum biosynthesis. Further studies elucidated the molecular mechanism by which CsrA positively regulates flagellum synthesis. Purified recombinant CsrA protein, which was isolated as a ribonucleoprotein complex consisting of one single CsrB molecule and ~18 CsrA subunits, directly stimulated the coupled transcription-translation of flhDC::lacZ in S-30 extracts and bound specifically to the 5’ non-coding segment of flhDC mRNA in mobility shift assay. The steady state level of flhDC mRNA was higher and its half-life was ~3-fold greater in a csrA wild type versus a csrA::kanR mutant strain, as shown by RT-PCR. Thus, CsrA is able to stimulate flhDC gene expression by a post-transcriptional mechanism that resembles its function in repression.

Comments

Wei, Bangdong L., Positive Regulation of Acetate Metabolism and Motility by the RNA-binding Protein CsrA in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), August, 2000, 118 pp., 5 tables, 19 illustrations, bibliography, 175 titles. W 4 W415P 2000