Browsing by Subject "Escherichia coli"
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Item Effects of the Pleiotropic Gene csrA on Glycogen Metabolism in Escherichia Coli(1995-06-01) Yang, Honghui; Ming-Chi Wu; Wayne NicholsonYang, Honghui, Effects of the pleiotropic gene csrA on glycogen metabolism in Escherichia coli. Master of Science (Biomedical Sciences), June, 1995, 78 pp., 3 tables, 17 illustrations, bibliography, 59 titles. The csrA gene negatively regulates the expression of four genes glgB, glgC, glgA and glgS involved in glycogen synthesis. It also negatively regulates glgY, which encodes the enzyme glycogen phoshorylase involved in glycogen degradation, but no effect was observed on the glycogen debranching enzyme in this pathway. In addition, csrA exhibits a positive effect on the glycolytic enzyme triosephosphate isomerase. No significant effects were observed on the expression of two genes (zwf & gnd) participating in the pentose phosphate pathway. In vitro expression of glgB, glgC and glgA was specifically inhibited by cell extracts containing the csrA gene product (CsrA). This study provides evidence that csrA encodes an important regulator of intermediary carbon metabolism in Escherichia coli.Item Identification of Novel Genes Involved in Escherichia coli Biofilm Formation(2003-05-01) DesPlas, Rebecca L.; Jerry Simecka; Ming-Chi WuDesPlas, Rebecca L., Identification of Novel Genes Involved in Escherichia coli Biofilm Formation. Master of Science (Microbiology), May 2003, 77 pp., 6 tables, 11 figures, references, 55 titles. Transposon mutagenesis using a miniTn10::camR transposon generated 800 random insertion mutants displaying altered biofilm phenotypes as compared to the parent strain, TRMG F/M. Transduction of the resistance marker confirmed approximately 150 biofilm mutants. Amplifications of the insertion sites, nucleotide sequencing and BLAST searches against E. coli K-12 genomic databases, identified118 of these sites. Many of the interrupted genes are not known to be associated with biofilm formation. Four mutations were transduced into E. coli K-12 MG1655, creating altered biofilm phenotypes. A plasmid clone of the nhaAR operon complemented the corresponding mutations. Results indicate that the genes identified in this study influence biofilm formation. However, further studies are needed to determine the degree of impact in a wild type strain background.Item Positive Regulation of Acetate Metabolism and Motility by the RNA-Binding Protein CsrA in Escherichia coli(2000-08-01) Wei, Bangdong L.; Jerry Simecka; Ming-Chi Wu; Stephen R. GrantWei, 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.Item The Effect of CsrA on Biofilm Development in Escherichia coli(2001-05-01) Jackson, Debra White; Julian Borejdo; Richard Easom; Jerry SimeckaJackson, Debra W., The Effect of CsrA on Biofilm Development in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), May 2001, 127 pp., 2 tables, 15 illustrations, bibliography, 138 titles. CsrA, carbon storage regulator, is a small RNA-binding protein that acts as a global regulator and modulates specific mRNA stability in Escherichia coli. CsrA regulates central carbon metabolism in addition to flagella biogenesis. In this study, the phylogenetic distribution of csrA and its role in Escherichia coli biofilm development were examined. CsrA homologs were examined using Southern hybridization experiment and by analyzing existing sequencing data and was found to be widespread among eubacteria. CsrA was shown to be capable of acting as a genetic switch for biofilm formation and dispersal. A csrA mutant of E. coli was shown to increase biofilm formation and exhibit apparent pillars and channels characteristic of a mature biofilm. Over-expression of csrA completely inhibited biofilm formation in E. coli K-12 and decreased biofilm formation in related enteric pathogens. Induction of csrA expression from a multicopy plasmid caused dispersal of a pre-formed biofilm. Gene expression studies revealed that csrA expression is dynamically regulated during biofilm formation. Several outer-membrane factors and global regulators that have been implicated in biofilm formation were examined for effects on biofilm formation in a csrA mutant. Crystal violet adherence assays revealed that flagella and type I pili affect biofilm formation in a scrA mutant strain; however, colonic acid and curli fimbriae did not exhibit quantitative effects on biofilm formation in the csrA mutant, but the stationary phase sigma factor, RpoS, had no quantitative effect on csrA mutant biofilm formation. Therefore, a csrA mutant will form a biofilm in the absence of each of these outer-membrane factors and global regulatory factors of biofilm formation. The effects of csrA on biofilm formation were found to be mediated in part through its effects on intracellular glycogen metabolism. Thus the redirection of carbon flux, in response to environmental and/or physiological cues, is important for biofilm development.