| Chitin is an important carbon and nitrogen source for marine bacteria, supporting up to 30% of bacterial production in the Delaware Bay. I chose to study chitin degradation by Vibrio harveyi because vibrios are commonly associated with chitin in the marine environment and because previous work showed that V. harveyi produces both chitin-binding proteins that facilitate attachment to chitin and chitinases that degrade chitin.; I cloned a chitinase gene, chiB, from V. harveyi . This gene encodes a 109 kDa endochitinase which is modified posttranslationally to produce two lower molecular weight chitinases. ChiB is a family 18 glycosyl hydrolase and has a similar domain structure to other bacterial chitinases with distinct catalytic and chitin-binding domains. Evolutionary trees for the catalytic and binding domains of ChiB and other bacterial chitinases suggest that they have evolved separately. Sequence analysis also revealed a mosaic pattern of domain arrangement, lending support to the role of modular evolution or domain shuffling in generating the diversity of bacterial chitinases.; To better understand attachment to chitin I made mutants of V. harveyi by insertion mutagenesis. Both attachment to and degradation of chitin by the mutants were higher than the wild type. The mutated region in one mutant (mutant B) was identified. The suicide vector integrated immediately downstream of luxO and luxU, two genes involved in quorum sensing regulation of luminescence in V. harveyi. Chitinase activity of mutant B was higher than the wild type at low cell densities but the same as the wild type at high cell density, suggesting that some density dependent regulatory component had been damaged.; Finally, in order to better understand the role a bacterium's surface composition plays in adhesion, I examined the adhesion of V. harveyi and two mutants, B and M, to three different surfaces: glass, aminated polyurethane, and sulphonated polyurethane. The mutants overexpress a suite of membrane proteins, are more hydrophobic, and attach differently than the wild type. It appears that the overexpressed proteins are mediating the increased attachment by disrupting the repulsive forces between the bacterium and the test surfaces and allowing the bacterium to get close enough to the surface for specific interactions to occur. |
