Abstract Title of Dissertation: COMPLEX POLYSACCHARIDE DEGRADATION BY MICROBULBIFER DEGRADANS STRAIN 2-40: STUDIES OF THE CHITINOLYTIC SYSTEM AND CARBOHYDRASE ARCHITECTURE

Title of Dissertation: COMPLEX POLYSACCHARIDE DEGRADATION BY MICROBULBIFER DEGRADANS STRAIN 2-40: STUDIES OF THE CHITINOLYTIC SYSTEM AND CARBOHYDRASE ARCHITECTURE Michael B. Howard, Doctor of Philosophy, 2004 Dissertation Directed By: Professor Steven W. Hutcheson, Department of Cell Biology and Molecular Genetics Microbulbifer degradans strain 2-40 is a Gram negative marine bacterium that is able to depolymerize and metabolize a wide variety of complex polysaccharides, including chitin. Chitin is the second most abundant biopolymer in nature and is a widely available nutrient in many environments. The chitinolytic system of M. degradans consists of three chitin depolymerases (ChiA, ChiB, and ChiC), three Nacetylglucosaminidases (HexA, HexB, and HexC), a chitodextrinase (CdxA), a chitin-binding protein (CbpA), and a suite of enzymes involved in the transport and metabolism of GlcNAc and GlcNAc2. ChiB, the largest eubacterial chitinase described, includes two complete Glycoside Hydrolase family 18 catalytic domains; one is exolytic and the other is endolytic. These catalytic domains share similar reaction optima, temperature, pH, and metal ion sensitivities, and are shown to function synergistically in the depolymerization of crystalline chitin. ChiA, ChiB, and 44 other M. degradans carbohydrases contain polyserine linker regions. These linkers are composed predominantly of serine (79%), have an average length of 39 residues, and are encoded by all six serine codons without any obvious bias or pattern. Polyserine domains are found only between functional groups (e.g., catalytic, binding, or anchoring domains) and are only found in putative secreted, carbohydrate depolymerases. The sequence of the M. degradans genome was determined during the course of this work and permitted the first genomic analysis of a chitinolytic organism to be performed. Further, these experiments and subsequent analyses have lead to a greater understanding of how M. degradans is able to metabolize such a diverse collection of biopolymers. COMPLEX POLYSACCHARIDE DEGRADATION BY MICROBULBIFER DEGRADANS STRAIN 2-40: STUDIES OF THE CHITINOLYTIC SYSTEM AND CARBOHYDRASE ARCHITECTURE.