Cellulose degradation system of Cytophaga hutchinsonii
In this project, Cytophaga hutchinsonii, an aerobic gliding bacterium with cellulose-degrading ability, was studied, since its cellulase system was unknown and might be very different from those of other cellulose-degrading species. Only ß-1,4- endoglucanases and non-specific ß-glucosidases were found in the C. hutchinsonii genome sequence, whereas specific exoglucanases were apparently absent. Almost all putative cellulases were composed of catalytic domains only, without carbohydrate-binding modules. Samples from C. hutchinsonii cultures were analyzed by using TLC and colorimetric assays. Glucose was detected in the cellobiose grown culture, but not in cellulose-grown cultures, suggesting that cellobiose is hydrolyzed extracellularly rather than being directly assimilated, and that cellulose may not be degraded via cellobiose. Also, cellobiose-based cultures caused greater acidification of the medium than glucose or cellulose grown cultures. Nine putative cellulases were expressed in four bacterial strains. In some cases, expression was toxic to host cells. The crude lysates were tested for endoglucanase, specific exoglucanase or nonspecific ß-glucosidase activity. CHU_1280 and CHU_1842 showed apparent endoglucanase activity when expressed in Citrobacter freundii. Four putative GH family 3 ß-glucosidases with similar conserved domains were expressed in Escherichia coli JM109 and E. coli BL21(DE3)pLysS. One of these, CHU_2268, was found to possess MUC-degrading ability. This suggests that CHU_2268 may be the 'missing' exoglucanase in C. hutchinsonii. Another two ß-glucosidases, CHU_2273 and CHU_3784, possessed only MUG-degrading activity.
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