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Open Iyer PR__Thesis.Pdf The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology BIOCHEMICAL CHARACTERIZATION OF THE PROTEINS INVOLVED IN GLUCONACETOBACTER HANSENII CELLULOSE SYNTHESIS A Dissertation in Integrative Biosciences by Radhakrishnan Iyer Prashanti 2012 Radhakrishnan Iyer Prashanti Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2012 The dissertation of Radhakrishnan Iyer Prashanti was reviewed and 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 Nicole R. Brown Associate Professor of Wood Chemistry Charles T. Anderson Assistant Professor of Biology Peter J Hudson Program Chair, Integrative Biosciences Director, The Huck Institutes of the Life Sciences * Signatures are on file in the Graduate School ii ABSTRACT Gluconacetobacter hansenii is a Gram-negative bacterium, considered as the model organism for studying the process of cellulose biogenesis. This is due to its unique ability to synthesize and secrete copious amounts of cellulose as an extracellular polysaccharide, in its growth medium. G. hansenii is therefore an ideal bacterium to study cellulose as a material as well as cellulose synthesis as a biological process. We have therefore employed this bacterium as our subject for our inquiry into the biochemistry of the process of cellulose synthesis. In this work, the main area of focus is towards understanding the bacterial cellulose synthesis and secretion complex, in terms of its component proteins, their structure, organization and their interactions. Two parallel approaches were used to understand and gain insights into the cellulose synthesis complex. One was to heterologously express and purify the proteins that are known to be involved in cellulose synthesis for structural studies or for generation of antibodies. Another method was to isolate the cellulose synthase complex from the G. hansenii cells and dissect its component proteins to reveal as-yet-unknown constituents of this complex. The cellulose synthase operon encodes for three proteins, AcsAB, AcsC and AcsD. AcsD protein was heterologously expressed and purified. The pure protein was employed for structural characterization as well as for antibody generation. Using the specific anti-AcsD antibody, the subcellular localization of this protein was identified to be the periplasmic space. Studies of AcsD using gel filtration, analytical ultracentrifugation and dynamic light scattering revealed that it exists as an octamer in solution. Structural characterization of AcsD using small angle-X-ray scattering reveals that, in accordance with its crystal-structure, the protein forms a complex of a tetramer of dimers that assumes a cylindrical conformation with a central pore. The predicted non-membrane regions of the cytoplasmic membrane-bound cellulose synthase (AcsAB) protein were heterologously overexpressed and purified using affinity methods. Specific antibodies generated against these regions revealed that the protein, though encoded by a single gene, is actually processed into three polypeptides AcsA (45 kDa), AcsB1 (34 kDa) and AcsB2 (95 kDa). Western blot of the fractions from a sucrose density centrifugation combined with sequence-based analysis revealed that the AcsB2 protein is localized in the periplasmic region of the bacterial cell. The genome of G. hansenii 23769 was sequenced to provide a database for mass- spectrometry based-proteomic studies of cellulose synthesis. The completed genome is now a public database in NCBI. These studies involved Multidimensional Protein Identification Tool (MudPIT) analysis of the total membrane (TM), outer membrane (OM) and the cytoplasmic membrane (CM) fractions of the cells for the comparison of the proteomic profile of the three compartments. This revealed that the AcsB, AcsC and the AcsD were largely concentrated in the OM whereas the CM compartment contains lower abundance of AcsAB protein. Using blue native polyacrylamide gel electrophoresis (BN-PAGE), the protein complexes in solubilized G. hansenii TM were isolated and the complex containing the proteins involved in cellulose was located using the specific antibodies against AcsD and AcsA. As revealed by LC-MS analysis of the antibody cross-reacting gel-band, this complex also contains phosphoglucomutase, glucose-6-phosphate isomerase and UDP- glucose pyrophosphorylase. These proteins were known to be involved in cellulose iv biosynthesis pathway, but this work presents evidence for the existence of these proteins in association with the proteins involved in the cellulose synthesis and secretion complex. In addition to using gel-based methods for identifying the components of the cellulose synthesis complex, zymography was used to demonstrate in-vitro cellulose synthesis using detergent solubilized membranes. This study directs towards a greater efficiency of the detergent dodecyl maltoside compared to Triton-X 100, in solubilization of the TM, while retaining the enzyme-activity. In summary, we have used a combination of traditional and modern biochemical approaches to study the protein components of the cellulose synthesis machinery. Our work has resulted in a sequenced genome, structural analysis and localization of AcsD, and identification of processing of the AcsAB. We have also presented evidence that the proteins involved in the cellulose biosynthetic process, indeed exist as a complex and have identified other proteins relevant to the process of cellulose synthesis, to be components of this complex. Based on our findings, we have proposed our model for the bacterial cellulose synthesis and extrusion complex. v TABLE OF CONTENTS LIST OF ABBREVIATIONS ............................................................................. x LIST OF FIGURES…………………………..................................................... xi LIST OF TABLES ............................................................................................. xiii ACKNOWLEDGEMENTS ............................................................................... xiv CHAPTER I BIOCHEMISTRY OF CELLULOSE SYNTHESIS ...................... 1 Cellulose and its impact on our lives .................................................................... 1 Chemical structure of cellulose ........................................................................... 4 Physical properties of cellulose ........................................................................... 6 G. HANSENII AS THE MODEL ORGANISM FOR CELLULOSE SYNTHESIS ………………… ...... .................................................................... 9 Bacterial cellulose: properties and uses ……...................................................... 11 Visualization of cellulose-synthesizing complexes ............................................ 13 Uridine diphosphate glucose (UDP-glucose) ..................................................... 15 Cyclic diguanylate (c-di-GMP) .......................................................................... 16 In vitro cellulose synthesis in presence of the regulator ….…........................... 19 Identification of the genes involved in cellulose synthesis ............................... 20 BACTERIAL CELLULOSE SYNTHASE OPERON……………................... 22 acsAB ……….................................................................................................... 23 AcsC .................................................................................................................. 25 AcsD................................................................................................................... 26 acs operon is flanked by genes that modulate cellulose synthesis .................... 27 dgc and pdeA genes ......................................................................................... 28 Cellulose biosynthetic pathway and mechanism of cellulose synthesis ..….… 30 STATEMENT OF THE PROBLEM ................................................................ 33 SUMMARY ...................................................................................................... 34 CHAPTER II WHOLE GENOME SEQUENCING OF GLUCONACETOBACTER HANSENII 23769.................................................. 38 INTRODUCTION ............................................................................................. 38 STEPS IN GENOME SEQUENCING……..…................................................. 40 General outline of sequencing protocol ............................................................. 40 reparation of single stranded DNA library ..................................................... 41 Amplification of the library by emulsion PCR .............................................. 41 Sequencing by synthesis (Pyrosequencing) ......................................................41 Paired-end library generation ......................................................................... 43 SOLid sequencing ........................................................................................... 45 ANALYSIS OF THE SEQUENCE USING SOFTWARE TOOLS ............... 46 Assembly ......................................................................................................... 46 Generation of contigs ...................................................................................... 46 Scaffolds .........................................................................................................
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