MECHANISTIC AND PHYSIOLOGICAL INSIGHTS INTO POST- TRANSCRIPTIONAL REGULATION BY SMALL RNAS SGRS AND DICF BY DIVYA BALASUBRAMANIAN 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, 2014 Urbana, Illinois Doctoral Commitee: Associate Professor Carin K. Vanderpool, Chair Professor Jeffrey F. Gardner Professor William W. Metcalf Professor James M. Slauch Abstract Base pairing small regulatory RNAs (sRNAs) are important post-transcriptional regulators of gene expression in bacteria. These sRNAs deploy novel mechanisms to regulate mRNA targets leading to various physiological outcomes during stress conditions including, but not limited to, iron starvation, carbon flux and metabolism, virulence, and quorum sensing. In this study, we investigate the multitude of clever mechanisms that two sRNAs, SgrS and DicF, utilize to regulate gene expression, and the physiological consequences of such regulation. The SgrS sRNA participates in a response to a growth inhibitory stress condition called sugar-phosphate stress caused by the toxic accumulation of phosphorylated sugars. SgrS combats this stress with its RNA base pairing function by silencing translation of sugar transporters that import the stress molecules. SgrS was previously shown to negatively regulate the manXYZ broad sugar-substrate transporter. In this study we demonstrate that SgrS binds at manX and in the intergenic region of manXY to translationally silence this operon. We show that pairing at both these sites is critical for degradation of the manXYZ polycistron, and is also crucial for providing maximal relief from stress. SgrS is a dual-function sRNA in that in addition to its RNA function, it also produces a peptide called SgrT. Here, we investigate the mechanistic relationship between these two functions of SgrS. We demonstrate that while mutating the sgrT translation initiation sequences have little impact on SgrS base pairing properties, mutations in the sgrS base pairing region increase SgrT production. Further, the SgrS RNA function was the primary means of action against sugar-phosphate stress; SgrT production lags SgrS synthesis. We therefore propose a model in which the two ii independent functions of SgrS act at different stages in response to sugar-phosphate stress. The DicF sRNA is encoded on the dicBF operon on the cryptic Qin prophage in E. coli. The only known target of DicF is the ftsZ mRNA, whose gene product is essential for cell division in bacteria. Using reporter gene fusions to predicted target genes, we identified three genes in metabolism that are also targets of DicF. DicF also requires core E. coli proteins for its base pairing properties. We also demonstrate that in addition to inhibiting cell division, DicF leads to growth inhibition of cells. Lastly, the gene products of the dicBF operon are cumulatively toxic to E. coli cells. Characterizing mechanistic contributions of sRNAs to significant physiological outcomes will better our understanding of these novel regulatory RNA elements. iii Acknowledgements I would like to thank my advisor, Dr. Cari Vanderpool for giving me this incredible opportunity to undertake my Ph.D. journey in her laboratory. Cari fostered a very friendly and fun-loving laboratory environment that made my experience here nurturing and enjoyable. I am also extremely grateful to Cari for being a great mentor by providing me with the freedom to do the experiments that I wanted to pursue and greatly improve my scientific writing and communication abilities. I would also like to extend my thank you to all my committee members: Dr. Jeff Gardner, Dr. Bill Metcalf, Dr. Jim Slauch and the late Dr. Charles Miller for invaluable feedback on my projects throughout the years. I am especially thankful to Dr. Jim Slauch for his useful suggestions during our joint lab meetings and for having an open door to any of my questions. I would also like to thank my undergraduate mentor, Dr. Brad Goodner, who inspired me to pursue a career in research. I am truly thankful to all members of the Vanderpool laboratory, both past and present for providing me with a collaborative, supportive and fun atmosphere to work in. My wonderful experience in the Vanderpool lab came from the amazing co-workers in the lab. I would not have been able to come to a Ph.D. program or finish the program without the unequivocal support of my parents and sister, Madhu, throughout my many years here at UIUC. I am truly thankful to my family. Last, but certainly not the least, I would like to thank my family in Champaign, consisting of my fiancé Shriram and our pet bunny for seeing me through hard times of my Ph.D. career, for believing in me, and for their unwavering support. iv Table of Contents Chapter 1: Introduction ....................................................................................................... 1 1.1. RNA regulators ............................................................................................................ 1 1.1.1. Regulatory RNAs in eukaryotes ............................................................................ 1 1.1.2. Bacterial regulatory RNAs (small RNAs) ............................................................. 3 1.2. The carbohydrate phosphoenolpyruvate phosphotransferase system ........................ 14 1.2.1. The major glucose transporters EIIAGlc and EIIBCGlc ........................................ 15 1.2.2. The broad sugar-substrate transporters EIIABMan and EIICDMan........................ 16 1.3. The small RNA SgrS-mediated regulation of sugar transporters .............................. 17 1.3.1. The sugar-phosphate stress.................................................................................. 17 1.3.2. The small RNA SgrS ........................................................................................... 18 1.3.3. SgrT ..................................................................................................................... 21 1.4. The small RNA DicF ................................................................................................. 22 1.4.1. Discovery ............................................................................................................ 22 1.4.2. Distribution of DicF in bacteria .......................................................................... 24 1.5. Aim of this study ........................................................................................................ 25 1.6. Figures........................................................................................................................ 27 Chapter 2: Materials and methods .................................................................................... 30 2.1. Strains and plasmid construction ............................................................................... 30 2.2. RNA extraction, Northern blot analysis and Quantitative Real-Time PCR .............. 35 2.3. β-galactosidase assays ................................................................................................ 36 2.4. Kinetic fluorescence assays ....................................................................................... 37 2.5. Protein extraction and Western blot analyses ............................................................ 37 2.6. Growth assays ............................................................................................................ 38 2.7. RNA-Seq conditions and data analyses ..................................................................... 39 2.8. Tables ......................................................................................................................... 40 Chapter 3: Deciphering the interplay between two independent functions of the small RNA regulator SgrS in Salmonella ................................................................... 47 3.1. Introduction ................................................................................................................ 47 3.2. Results ........................................................................................................................ 49 3.2.1. Ectopic expression of SgrS base pairing and SgrT in Salmonella ...................... 49 3.2.2. Translation of sgrT has minimal effects on SgrS riboregulation ........................ 50 3.2.3. Mutations in the sgrS base pairing lead to increased SgrT production ............... 51 3.2.4. SgrT production increases when base pairing activity is impaired without Hfq ................................................................................................................... 53 3.2.5. Riboregulation of SgrS is necessary for growth rescue of stressed Salmonella cells............................................................................................................. 56 3.2.6. Production of SgrT lags synthesis of SgrS RNA ................................................ 57 3.3. Discussion .................................................................................................................. 58 3.4. Figures........................................................................................................................ 64 v Chapter 4: Post-transcriptional regulation of central metabolism by the small RNA DicF ............................................................................................................... 75 4.1. Introduction