The Agarase System of Saccharophagus Degradans Strain 2-40: Analysis of the Agarase System and Protein Localization

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 Gramnegative 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.