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Regulation of the Mannose Pts Operon by the Small Rna Sgrs

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Regulation of the Mannose Pts Operon by the Small Rna Sgrs REGULATION OF THE MANNOSE PTS OPERON BY THE SMALL RNA SGRS BY JENNIFER B. RICE DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2011 Urbana, Illinois Doctoral Committee: Assistant Professor Carin K. Vanderpool, Chair Professor John E. Cronan Professor Stephen K. Farrand Professor Charles G. Miller ABSTRACT A number of bacterial small RNAs (sRNAs) act as global regulators of stress responses by controlling expression of multiple genes. The sRNA SgrS is expressed in response to glucose-phosphate stress, a condition associated with disruption of glycolytic flux and accumulation of sugar-phosphates. SgrS has been shown to stimulate degradation of the ptsG mRNA, encoding the major glucose transporter. This study demonstrates that SgrS regulates the genes encoding the mannose and secondary glucose transporter, manXYZ. Analysis of manXYZ mRNA stability and translation in the presence and absence of SgrS indicate that manXYZ is regulated by SgrS under stress conditions and when SgrS is ectopically expressed. In vitro footprinting and in vivo mutational analyses showed that SgrS base pairs with manXYZ within the manX coding sequence to prevent manX translation. Regulation of manX did not require the RNase E degradosome complex, suggesting that the primary mechanism of regulation is translational. An Escherichia coli ptsG mutant strain that is manXYZ(+) experiences stress when exposed to the glucose analogs α- methyl glucoside or 2-deoxyglucose. A ptsG manXYZ double mutant is resistant to the stress, indicating that PTS transporters encoded by both SgrS targets are involved in taking up substrates that cause stress. We further demonstrate that SgrS binds to two sites on the manXYZ mRNA to coordinately down-regulate translation of all the cistrons, a mechanism reminiscent of that used by the eukaryotic miRNA, lin-4 for regulation of the lin-14 mRNA. We tested the hypothesis that regulation of manY and manZ is dependent on SgrS:manX base pairing and subsequent manXYZ mRNA degradation. Contrary to the hypothesis, we show that SgrS repression of ii manY and manZ translation can be decoupled from SgrS-dependent manXYZ mRNA degradation. Furthermore, SgrS regulates manY translation by a mechanism that is independent of SgrS:manX mRNA interactions. Instead, translational regulation of manY depends on SgrS pairing with sequences in the manX-manY intergenic region, upstream of the manY ribosome binding site. Mutational analysis suggests that while the SgrS-manY interaction is sufficient to stimulate some degradation of the manXYZ mRNA, pairing with both sites is required for maximal degradation of the manXYZ mRNA. Additional SgrS candidate-mRNA targets are also investigated. iii ACKNOWLEDGEMENTS I’d like to thank my advisor, Dr. Cari Vanderpool, for taking a chance on a fourth rotation student. I am truly grateful for the training, guidance, and overwhelming support I received during my time in her lab. I’d also like to thank her for fostering a fun, educational, and open atmosphere. I’d like to thank every single member of the Vanderpool Lab, past and present, who contributed to the fabulous, clown-rich environment. I’d like to extend many thanks to my committee members, Dr. Charles Miller, Dr. Stephen Farrand, Dr. John Cronan, and Dr. Jim Slauch for their very thoughtful and useful suggestions, and comments on my project. I’d also like to thank the Department of Microbiology - a collaborative, positive, and inviting atmosphere that heavily contributed to my graduate training. I’d like to extend a thank you to my high school biology teacher, Chris Luther, for evoking my passion for biology and showing me how fun this subject could be. I thank my dad, Bill Han, for sparking my scientific curiosity and being committed to my education and future endeavors. I’d like to thank my mom, Bonnie Han, for her unwavering support and trust in the choices I have made. I thank my second parents, Tom and Linda Rice for providing me the same support and encouragement they provide their very own children. Lastly, I would like to thank my husband, Grant Rice, for taking this journey with me. I am extremely grateful that we could be there for each other during our undergraduate and graduate studies. Most of all, I thank him for believing I had the ability to pass Organic Chemistry, get into a Ph.D. program and spend as many hours at the bench as he could :). iv TABLE OF CONTENTS CHAPTER 1: Introduction .................................................................................................................... 1 1.1. Escherichia coli ............................................................................................................................... 1 1.1.1. History and background ........................................................................................... 1 1.1.2. Genome ............................................................................................................................ 2 1.1.3. Model organism ............................................................................................................ 2 1.2. Cellular roles of RNA .................................................................................................................... 3 1.2.1. History of regulatory RNAs ...................................................................................... 3 1.2.2. Eukaryotic regulatory RNAs .................................................................................... 4 1.2.3. Bacterial regulatory RNAs ........................................................................................ 6 1.3. Characteristics of trans-encoded sRNAs in bacteria ........................................................ 9 1.3.1. Proteins involved in sRNA-dependent regulation ........................................10 1.3.1.1. Hfq .................................................................................................................10 1.3.1.2. RNase E and the degradosome ...........................................................11 1.3.2. Mechanisms of positive regulation .....................................................................12 1.3.3. Mechanisms of negative regulation ....................................................................13 1.4. The carbohydrate phosphoenolypyruvate phosphotransferase system .......................................................................................................................................................15 1.4.1. Example transporters ..............................................................................................17 1.4.1.1. Crr (EIIAGlc) and PtsG (EIICBGlc) .........................................................17 1.4.1.2. ManXYZ (EIIABMan, EIICDMan) ..............................................................18 1.5. The sugar-phosphate stress response.................................................................................19 1.5.1. Glucose-phosphate stress.......................................................................................19 1.5.2. SgrS .................................................................................................................................20 1.5.3. SgrR .................................................................................................................................22 1.5.4. SgrT .................................................................................................................................24 1.6. sRNAs in pathogenesis ..............................................................................................................25 1.7. Aim of this study ..........................................................................................................................26 CHAPTER 2: Materials and methods .............................................................................................28 2.1. Strain and plasmid construction ...........................................................................................28 2.2. Media and reagents.....................................................................................................................41 2.3. β-galactosidase assays ...............................................................................................................41 2.4. RNA extraction and northern blot analysis .......................................................................42 2.5. 5 RACE ............................................................................................................................................42 2.6. SgrS random mutagenesis .......................................................................................................42 2.7. In vitro RNA footprinting ..........................................................................................................43 2.8. RNA structure analysis ..............................................................................................................43 v CHAPTER 3: The small RNA SgrS controls sugar-phosphate accumulation by regulating multiple PTS genes ...................................................................................................44 3.1. Introduction...................................................................................................................................44 3.2. Results .............................................................................................................................................45
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