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The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology MECHANISTIC STUDIES INTO BACTERIAL CELLULOSE SYNTHESIS A Dissertation in Biochemistry, Microbiology, and Molecular Biology by John B. McManus © 2017 John B. McManus Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2017 This dissertation of John B McManus was approved* by the following: Ming Tien Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee B. Tracy Nixon Professor of Biochemistry and Molecular Biology Hemant Yennawar Director, X-ray Crystallography Laboratory Frank L. Dorman Associate Professor of Biochemistry and Molecular Biology Squire Booker Professor of Chemistry Professor of Biochemistry and Molecular Biology Scott B. Selleck Department Head, Biochemistry and Molecular Biology *Signatures are on file in the Graduate School ii ABSTRACT Cellulose, a polymer of β-1,4-linked glucose units, is synthesized by plants, animals, fungi, and bacteria. It is the major component of the plant cell wall and, thus, is thought to be the most abundant biological molecule on earth. Cellulose synthase, a membrane-bound glycosyltransferase, synthesizes cellulose through the processive addition of glucose, from UDP-glucose, to the non-reducing end of the cellulose polymer. This enzyme is often found with other proteins in large cellulose synthase complexes. The primary focus of the present work is a greater understanding of the mechanisms for initiation, elongation, and termination of the cellulose polymer. Structural studies of cellulose synthase from Rhodobacter sphaeroides, called BcsA- BcsB, have revealed much regarding the mechanisms for polymer elongation, however the mechanisms for initiation and termination largely remain a mystery. Two tools were developed for the present work. First was the isolation of a core catalytic subunit of the cellulose synthase complex. The catalytic heterodimer, AcsA- AcsB, was isolated from Gluconacetobacter hansenii by two methods—product entrapment and affinity chromatography—and then subsequently kinetically characterized. Second was the development of a tool for the size analysis of in vitro- synthesized cellulose. Existing methods of cellulose solubilization and separation by gel permeation chromatography were adapted for this tool. Initiation of cellulose synthesis can follow either a primer-dependent or a primer- independent mechanism. Using size analysis of in vitro-synthesized cellulose, both AcsA-AcsB and BcsA-BcsB were shown to follow a primer-independent mechanism for iii initiation. Reducing end analysis of in vitro-synthesized cellulose from both enzymes indicated that glucose is sufficient to initiate synthesis. Finally, BcsA-BcsB demonstrated UDP-glucose hydrolase activity, allowing for the construction of a plausible mechanism for self-priming. Here, cellulose synthase hydrolyzes UDP-glucose and subsequently binds the liberated glucose to initiate new cellulose polymer synthesis. Size analysis also revealed processivity differences between the two enzymes. To investigate the factors underlying processivity, changes in selected residues in the BcsA transmembrane channel were made. In one variant, the processivity and kinetic rates were altered. Finally, size analysis indicated that the active elongation of the polymer by both AcsA-AcsB and BcsA-BcsB is faster than the overall turnover numbers. This suggested that the catalytic cycle minimally consists of a fast and a slow phase. With these data, a minimal kinetic mechanism for cellulose synthesis was constructed. To see if the overall turnover number was equally as slow in vivo, quantitative western blotting was employed to calculate the turnover number in whole G. hansenii cells. The turnover number in vivo was much faster than for purified AcsA-AcsB. Three cellulose synthase complex accessory proteins were investigated to test for their involvement in catalysis. Following this, the turnover number for AcsA-AcsB was measured at each step during purification by quantitative western blotting. This analysis showed a decrease in the turnover number immediately after cell lysis, suggesting that association into the cellulose synthase complex, in whole cells, positively impacts the cellulose synthesis rate of AcsA-AcsB. iv TABLE OF CONTENTS LIST OF ABBREVIATIONS ............................................................................................ ix LIST OF FIGURES .............................................................................................................x LIST OF TABLES ............................................................................................................ xii ACKNOWLEDGEMENTS ............................................................................................. xiii CHAPTER 1: CELLULOSE AND CELLULOSE SYNTHESIS .......................................1 Cellulose Morphology, Crystallinity, and Degree of Polymerization .....................3 Cellulose Synthesis in Bacteria ................................................................................6 Regulation of Cellulose Synthesis by cyclic-di-GMP ..................................8 Gluconacetobacter hansenii as a Model for Cellulose Synthesis ................9 The Molecular Biology of Cellulose Synthesis in G. hansenii ...................12 BcsA-BcsB: The Structural Model for the Bacterial CSC Catalytic Core ............................................................................................15 Initiation of Cellulose Synthesis ............................................................................20 Heparosan Synthase...................................................................................22 Cyclic Glucosyl Synthase ...........................................................................22 STATEMENT OF THE PROBLEM .................................................................................23 SUMMARY .......................................................................................................................24 CHAPTER 2: ACSA-ACSB: THE CATALYTIC CORE OF THE CELLULOSE SYNTHASE COMPLEX ...........................................................................28 Introduction ............................................................................................................28 Materials and Methods ...........................................................................................29 Culture Conditions .....................................................................................29 Cloning of AcsAB-his .................................................................................30 Protein Concentration Determination .......................................................30 AcsA-AcsB Purification by Product Entrapment .......................................31 AcsA-AcsB Purification by Affinity Chromatography ...............................32 Radiometric Measurement of Cellulose Synthase Activity ........................32 Spectrophotometric Measurement of Cellulose Synthase Activity.............33 SDS-PAGE and Western Blot Analysis ......................................................33 N-terminal Sequencing...............................................................................34 v Trichloroacetic Acid-Mediated Cell Lysis .................................................34 Results ....................................................................................................................34 Purification of AcsA-AcsB by Product Entrapment ..................................34 Processing of AcsAB ..................................................................................38 Purification of AcsA-AcsB by Affinity Chromatography ...........................38 Kinetic Characterization of AcsA-AcsB .....................................................42 Discussion ..............................................................................................................45 AcsA-AcsB is the Catalytic Core of Cellulose Synthase ............................45 Processing of AcsAB ..................................................................................45 Kinetic Characterization of AcsA-AcsB .....................................................46 CHAPTER 3: INITIATION OF CELLULOSE SYNTHESIS ..........................................48 Introduction ............................................................................................................48 Materials and Methods ...........................................................................................50 Preparation of Solvents..............................................................................50 Expression and Purification of BcsA-BcsB................................................51 Modification and Gel Permeation Chromatography of Cellulose in Tetrahydrofuran .........................................................................................51 Dissolution and Gel Permeation Chromatography of Cellulose in Dimethylacetamide / 8% Lithium Chloride ...............................................52 Reducing End Modification and Analysis ..................................................53 Glucose Quantification by Enzyme-Linked Assay .....................................54 Gel Permeation Chromatography of Glucose and Cellobiose ..................55 Reducing End Quantification Using Bicinchoninic Acid...........................55 Results ....................................................................................................................56