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Investigations of the early stages of transport by the transenvelope lipopolysaccharide transporter in E. coli Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Blake Robert Bertani Graduate Program in Microbiology The Ohio State University 2019 Dissertation Committee Natividad Ruiz, Ph.D., Advisor Irina Artsimovitch, Ph.D. Ross Dalbey, Ph.D. Patrice Hamel, Ph.D. 1 Copyrighted by Blake Robert Bertani 2019 2 Abstract The cell envelope of bacteria mediates their interaction with the outside world and determines what can enter the cell. Gram-negative bacteria have a cell envelope defined by two membranes: an inner membrane, which surrounds the cytoplasm, and an outer membrane, which together with the inner membrane delimits an additional cellular compartment termed the periplasm. The inner membrane of Gram-negative bacteria is primarily composed of a phospholipid bilayer. The outer membrane, in contrast, contains phospholipids in its inner leaflet, and the essential glycolipid lipopolysaccharide (LPS) in the outer leaflet. The presence of LPS in the outer leaflet renders the outer membrane relatively impermeable, and therefore grants the cell resistance to noxious compounds in the environment, such as antibiotics. LPS is synthesized in the cytoplasmic face of the inner membrane, though it can also undergo non-stoichiometric modifications in the periplasmic face, and must thereafter be transported across the rest of the cell envelope. Transversal of the inner membrane by LPS is mediated by the ATP-binding cassette transporter MsbA. LPS extraction from the inner membrane, and subsequent transport across the rest of the cell envelope, is mediated by the LPS transport, or Lpt, complex. The Lpt complex is composed of eight proteins: a dimer of LptB in the cytoplasm that binds and hydrolyzes ATP ii to drive LPS transport; two transmembrane domains, LptF and LptG, which form a cavity in the inner membrane that accepts LPS and extracts it; LptC, LptA, and LptD, which form a bridge across the periplasm to allow the hydrophobic portion of LPS to traverse the aqueous periplasm; and LptE, which in conjunction with LptD facilitates the transport of LPS across the outer membrane. Here, we describe work in which we dissect the molecular mechanisms by which the Lpt system’s inner membrane complex, LptB2FGC, interacts with LPS. In chapter two, we describe the identification of a residue within LptG, K34, which is critical for LPS transport. Through structure-function and suppressor analyses, we show K34 of LptG mediates early contact with unmodified LPS as it enters the cavity formed by LptF and LptG. Further, our results imply that modified LPS interacts with the transporter distinctly from unmodified LPS. Chapter three describes the structure of the LptB2FGC inner membrane complex, as determined by X-ray crystallography, and defines the path LPS takes to the periplasmic bridge of the Lpt system through site-specific crosslinking. We show that the transmembrane anchor of LptC intercalates between transmembrane domains LptF and LptG and is ergo likely to be functionally involved in transport. Further, the periplasmic bridge is preferentially coupled to LptF, rather than LptG. In chapter four, we utilized a mutant producing LptG altered at residue K34 to perform suppressor analysis and further elucidate how the LptB2FGC complex interacts with LPS. We found that increased LPS production could iii suppress the defects produced by this variant, but not those produced by other altered lpt alleles. We therefore postulate that this suppression could be used as a probe of what stages of LPS transport are altered by a given lpt allele, or Lpt system inhibitor. Finally, we found that partial-loss-of-function lpt alleles suppressed otherwise lethal overproduction of LPS, which we believe supports a model in which LPS is essential to balance the rate at which material is added to the leaflets of the outer membrane. iv Acknowledgments I’ve heard it said that no man is an island, and that’s certainly been true for me during my graduate school career. A large number of people have supported me throughout this endeavor and made it possible. Thus, I’d like to take this opportunity to express to them my gratitude. In no particular order, I’d like to thank my mentor Natividad Ruiz, for her exceptional patience, insight, and when called for, constructive criticism. My advisory committee, for feedback and helpful suggestions, and for bearing with me when I tried to articulate to them my latest ideas. The members of the Ruiz lab, past and present: Emily Butler, for helping me get started in the lab alongside Brent Simpson, who has been both a good co-worker and good friend to me in the last several years; Emily Lundstedt, or “Emily II,” as she has affectionately been dubbed, for being there to bounce ideas off or just listen to me gripe; Sujeet Kumar, for incessant trolling and being trolled in kind (and occasionally actually talking science); Andrew Wilson, for talking shop, and keeping the office stocked with coffee; and finally, Becky Davis, for keeping the ship afloat. I would also like to thank Ao Mei and Giancarlo Casillas- Vargas, undergraduates mentees of mine who both contributed data and strains to my projects, and, I hope, taught me to be a better mentor. My entering class cohort, for helping get me through that first year, and being around to v commiserate in the years after. Last, I would like to thank my friends and family, for helping me cling to sanity during the most stressful portions of my graduate career. I never could have made it this far without the support of everyone mentioned here, and likely at least a few who weren’t (sorry to you neglected folks), and so for helping me get this far, you have my thanks. -Blake R. Bertani vi Vita 2014 ...................................................... B.S. Microbiology,The Ohio State University 2014-2015 .............................................. Graduate Fellow, Department of Microbiology, The Ohio State University 2015-Present .......................................... Graduate Research and Teaching Associate, Department of Microbiology, The Ohio State Unversity Publications Tomsic J, He HL, Akagi K, Liyanarachchi S, Pan Q, Bertani B, Nagy R, Symer DE, Blencowe BJ, de la Chapelle A. A germline mutation in SRRM2, a splicing factor gene, is implicated in papillary thyroid carcinoma predisposition. Scientific Reports. 2015;5. doi: ARTN 1056610.1038/srep10566. PubMed PMID: WOS:000357264800001. Bertani BR, Taylor RJ, Nagy E, Kahne D, Ruiz N. A cluster of residues in the lipopolysaccharide exporter that selects substrate variants for transport to the outer membrane. Molecular Microbiology. 2018;109(4):541-54. doi: 10.1111/mmi.14059. PubMed PMID: WOS:000446522100010. Bertani B, Ruiz N. Function and Biogenesis of Lipopolysaccharides. EcoSal Plus. 2018;8(1). doi: 10.1128/ecosalplus.ESP-0001-2018. PubMed PMID: 30066669; PMCID: PMC6091223. vii Owens TW, Taylor RJ, Pahil KS, Bertani BR, Ruiz N, Kruse AC, Kahne D. Structural basis of unidirectional export of lipopolysaccharide to the cell surface. Nature. 2019;567(7749):550-3. doi: 10.1038/s41586-019-1039-0. PubMed PMID: 30894747. Fields of Study Major Field: Microbiology viii Table of Contents Abstract .............................................................................................................................. ii Acknowledgments ............................................................................................................ v Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xii List of Figures ................................................................................................................ xiii Chapter 1: Background ................................................................................................... 1 Foreword ........................................................................................................................ 1 1.1 Introduction to the Gram-negative cell envelope and lipopolysaccharides .. 1 1.2 Structure and function of lipopolysaccharides .................................................. 3 1.2.1 Main features of the structure of LPS ......................................................... 3 1.2.2 The function of LPS ....................................................................................... 5 1.3 The lipopolysaccharide biosynthesis pathway .................................................. 8 1.3.1 Kdo2-lipid A biosynthesis: the Raetz pathway ........................................... 8 1.3.2 Biosynthesis of the core oligosaccharide ................................................. 12 1.3.3 Biosynthesis of the O antigen .................................................................... 16 1.4 Regulation of lipopolysaccharide biosynthesis ............................................... 20 1.5 Modification of lipopolysaccharide structure ................................................... 21 1.5.1 Modifications of lipid A ................................................................................. 22 1.5.2 Modifications to the core oligosaccharide ................................................ 26 1.5.3 O-antigen modification ...............................................................................

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