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Analysis of the Agarase System and Protein Localization ABSTRACT Title of Document: THE AGARASE SYSTEM OF SACCHAROPHAGUS DEGRADANS STRAIN 2-40: ANALYSIS OF THE AGARASE SYSTEM AND PROTEIN LOCALIZATION Nathan A. Ekborg, Doctor of Philosophy, 2005 Directed By: Professor Emeritus Ronald M. Weiner, Department of Cell Biology and Molecular Genetics and Professor Steven W. Hutcheson, Department of Cell Biology and Molecular Genetics Saccharophagus degradans (formerly “Microbulbifer degradans”) strain 2-40 is a Gram- negative marine bacterium isolated from the Chesapeake Bay. Analysis of 16s rDNA sequence indicated that S. degradans is related to a group of marine proteobacteria adept at degrading complex polysaccharides (CPs). S. degradans can depolymerize at least ten CPs including agarose. Agarose, an algal galactan, is degraded by few organisms. The agarase system of S. degradans was shown to be composed of five enzymes AgaA, AgaB, AgaC, AgaD and AgaE. These proteins contain glycoside hydrolase domains GH16, GH50 and GH86. S. degradans is the only organism known to collectively encode agarases with at least one of these domains. Unusual for agarases, AgaB and AgaE also contain multiple type-six carbohydrate binding modules. Furthermore, AgaE contains four thrombospondin type-three repeats whose function in prokaryotic proteins were unknown. The predicted agarases were characterized using a variety of methods including genomics, biochemical assays, proteomics and a newly described mutagenic technique. Agar degradation by S. degradans includes two depolymerases, AgaB and AgaC, a β-agarase II (AgaE) and a possible α-neoagarobiose hydrolase (AgaA). AgaB was found to be freely secreted while AgaC and AgaE were surface associated. AgaC is a predicted lipoprotein while AgaE did not have domains characteristic of surface localization. The Tsp-3 repeats, which are similar to repeats found on other cell surface enzymes, are the proposed cell surface anchoring sequences of AgaE. THE AGARASE SYSTEM OF SACCHAROPHAGUS DEGRADANS STRAIN 2-40: ANALYSIS OF THE AGARASE SYSTEM AND PROTEIN LOCALIZATION. By Nathan A. Ekborg Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2005 Advisory Committee: Professor Emeritus Ronald M. Weiner Co-Chair Professor Steven W. Hutcheson Co-Chair Assistant Professor Jocelyn DiRuggiero Associate Professor Richard C. Stewart Professor Emeritus J. Norman Hansen (Dean’s Representative) © Copyright by Nathan A. Ekborg 2005 Dedication This work is dedicated to my wife Kristin. Her love and constant patience is my guide. ii Table of Contents Dedication……………………………………………………………………………..ii Table of Contents.........................................................................................................iii List of Tables ................................................................................................................ v List of Figures.............................................................................................................. vi Chapter 1: Introduction................................................................................................. 1 Marine strain 2-40..................................................................................................... 1 Complex Polysaccharide Degradation...................................................................... 2 Strategies of Complex Polysaccharide Degradation................................................. 4 Cell Surface Enzyme Attachment in Bacteria .......................................................... 5 Agars and Agarose.................................................................................................... 8 Agarolytic Organisms ............................................................................................. 10 Agarases.................................................................................................................. 11 Modular Structure of Agarases ............................................................................... 16 Uses of Agar and Agarases..................................................................................... 17 The 2-40 Agarase System ....................................................................................... 19 Cell Surface Agarase Activity in strain 2-40 .......................................................... 20 Statement of Purpose .............................................................................................. 22 Chapter 2: Saccharophagus degradans gen. nov., sp. nov., a Versatile, Marine Degrader of Complex Polysaccharides....................................................................................... 23 Introduction............................................................................................................. 23 Materials and Methods............................................................................................ 25 Results..................................................................................................................... 26 Discussion............................................................................................................... 34 Chapter 3: Characterization of the Agarase System of S. degradans......................... 36 Materials and Methods............................................................................................ 38 Results..................................................................................................................... 47 Development of an S. degradans Genomic Library ............................................... 47 Identification of agaA and agaB............................................................................. 48 Genomic Sequence of S. degradans ....................................................................... 49 Identification of candidate agarases in the S. degradans genomic sequence ......... 52 Genomic arrangement of agarase genes encoded by S. degradans ........................ 53 Identification of domains present in the agarases encoded by S. degradans .......... 59 Biochemical Activity of the Predicted Agarases .................................................... 65 Detection AgaB, AgaC, and AgaE by mass spectrometry...................................... 76 Construction of null mutations in S. degradans...................................................... 79 Discussion............................................................................................................... 87 Chapter 4: Surface Localization of a β-agarase II in Saccharophagus degradans..... 93 Introduction............................................................................................................. 93 Materials and Methods............................................................................................ 96 iii Results................................................................................................................... 101 Biochemical Evidence of Membrane Associated Agarase Activity................. 101 Antibody tools to probe AgaE and AgaB ......................................................... 102 Expression and localization of AgaE and AgaB during the growth cycle........ 106 Immuno-epifluorescence indicates AgaE is cell associated ............................. 114 Cellular localization of AgaE and AgaB by immunogold electron microscopy114 Immunogold electron microscopy of thin sections shows exocellular arrangements of AgaE ............................................................................................................. 118 Discussion............................................................................................................. 127 Chapter 5: Future Directions and Proposed Experiments..................................... 135 Background........................................................................................................... 135 Proposed Research................................................................................................ 138 Conclusions........................................................................................................... 147 Appendices................................................................................................................ 149 Appendix I ............................................................................................................ 149 Phenotypic Instability of E. coli EPI300 (pNE10)................................................ 149 Appendix II ........................................................................................................... 155 Variability of the molecular weight of AgaB ....................................................... 155 iv List of Tables Table 1.1 Characterized agarases………………………………….15 Table 2.1 Pair-wise alignments of related 16s rDNA sequences….31 Table 2.2 Distinguishing characteristics of strain 2-40 as compared to Microbulbifer spp. and T. turnerae………...33 Table 3.1 Strains and plasmids used……………………………….44 Table 3.2 Oligonucleotides primers used…………………………..45 Table 3.3 Predicted agarases identified in S. degradans……………54 Table 3.4 Proteins identified in culture supernatants of S. degradans………………………………………………78 Table 3.5 Frequency of recombination………………………..……86 Table 4.1 Agarases identified in culture and cell fractions of S. degradans………………………………105 Table 4.2 Frequency and density of labeling observed in immuno-electron
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