Exchange of type II dockerin-containing subunits of the Clostridium thermocellum cellulosome as revealed by SNAP-tags.
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.
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
Benjamin H
BH
Waller
Daniel G
DG
Olson
Devin H
DH
Currie
Adam M
AM
Guss
Lee R
LR
Lynd
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D000097722:Cohesins
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D002480:Cellulase; D002482:Cellulose; D002868:Chromosomal Proteins, Non-Histone; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000097722:Cohesins; C080369:CipA protein, Clostridium; C473132:cellulosome, Clostridium