Complex polysaccharide degrading enzyme arrays synthesized by a marine bacterium

Periphytic bacterium strain designated 2-40 (2-40) was isolated from the salt marsh grass, Spartina, in the Chesapeake Bay. 2-40 can degrade numerous insoluble complex polysaccharides (ICP) to utilize recalcitrant ICP of plant, fungal, and animal origin. This research set out to: (1) better understand how 2-40 regulates the production of these enzyme systems in single and multiple carbon source minimal media (MM), (2) study novel cell surface structures, which we term degradosomes, (3) document cell morphogenesis during batch growth in agar and chitin.; Regulation of 2-40 ICP degradative enzyme systems allows it to survive in echoniches where ICP concentrations are constantly fluctuating. Each of the 2-40 carbohydrase systems were elicited at highest levels in supernatant fractions by the homologous substrate. Glucose repressed all of the carbohydrase systems, excluding amylase. Sole carbon source agarose supported the synthesis of the most heterologous carbohydrase systems.; 2-40 produced a {dollar}beta{dollar}-agarase system, which was induced by agar, agarose, and agaro-oligosaccharides. Four predominant agarases were synthesized, with molecular weights of 98, 90, 60, and 42 kDa. Similarly, 2-40 synthesized four extracellular chitinases, with molecular weights of 200, 98, 66, and 52.5 kDa. The chitinase system was induced by chitin and chito-oligosaccharides.; Both scanning and transmission electron microscopy revealed cell surface protuberances, degradosomes, that correlated with the degradation of either agarose or chitin. Immunoelectron microscopy revealed that these degradosomes contained agarase or chitinase. Cells elaborated both agarase(s) and chitinase(s) containing degradosomes, packaging each enzyme system in a separate degradosome. 2-40 degradosome proteins cross-reacted in Western blots with antisera against Clostridium thermocellum cellulosome enzyme or scaffolding protein. This is the first report of these structures in a gram-negative marine bacterium and also in any organism with multiple ICP degradative systems.; Fine structure analysis revealed progressive changes in cell topology and morphogenesis during progression of cells to late stages of growth. These changes were consistent with the formation of a survival cell type (ultramicrocells). 2-40, grown in ICP or during starvation, produced membranous tubules containing agarases or chitinases and bleb-like membranous vesicles. Thus, 2-40 is both enzymatically and developmentally highly regulated to survive the dynamic constraints of the estuarine ecosystem.