1. Exchange of type II dockerin-containing subunits of the Clostridium thermocellum cellulosome as revealed by SNAP-tags.
- Author
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Waller BH, Olson DG, Currie DH, Guss AM, and Lynd LR
- Subjects
- Bacterial Proteins genetics, Cell Cycle Proteins metabolism, Cellulose metabolism, Chromosomal Proteins, Non-Histone metabolism, Clostridium thermocellum genetics, Membrane Proteins genetics, Microscopy, Fluorescence, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Deletion, Cohesins, Bacterial Proteins metabolism, Cellulase metabolism, Clostridium thermocellum metabolism, Fluorescent Dyes metabolism, Membrane Proteins metabolism, Multienzyme Complexes metabolism
- Abstract
Clostridium thermocellum is a thermophilic anaerobic bacterium which efficiently hydrolyzes and metabolizes cellulose to ethanol through the action of its cellulosome, a multiprotein enzymatic complex. A fluorescent protein probe, consisting of a type II dockerin module fused to a SNAP-tag, was developed in order to gain insight into the quaternary configuration of the cellulosome and to investigate the effect of deleting cipA, the protein scaffold on which the cellulosome is built. Fluorescence microscopy suggested that the probe had localized to polycellulosomal protuberances on the cell surface. Surprisingly, fluorescence intensity did not substantially change in the cipA deletion mutants. Sequential labeling experiments suggested that this was a result of bound type II dockerins from CipA being replaced by unbound type II dockerins from the fluorophore-SNAP-XDocII probe. This mechanism of dockerin exchange could represent an efficient means for modifying cellulosome composition., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)
- Published
- 2013
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