Cellulose complementing factor (Ccp) is a new member of the cellulose synthase complex (terminal complex) in Acetobacter xylinum

Abstract

The cellulose complementing factor (Ccp) is known to be involved in cellulose production in the Acetobacter species. However, its precise functions remain unclear. In the current study, we identified the coding region of the ccpAx gene (ccp gene from Acetobacter xylinum) and the localization of the CcpAx in cells by generating fusion proteins tagged to an enhanced green fluorescent protein (EGFP). From the results of N-terminal sequencing of CcpAx-EGFP-fusion protein, which recovered 65% of cellulose-producing abilities of the wild-type to the ccpAx gene-knockout mutant, the ccpAx gene was determined to encode a protein with the molecular weight of 8 kDa. The amino acid sequence deduced had high similarities with the C-terminal regions of Ccp proteins from other Acetobacter species. Fluorescence microscopy analysis showed that CcpAx was longitudinally localized along with one side of the cell membrane. Additionally, the localization of AxCeSD, which is thought to be a member of the cellulose synthase complex [terminal complex (TC)] in A. xylinum, was determined in the same manner as CcpAx. Fluorescence microscopy analysis showed that AxCeSD had a localization pattern similar to that of CcpAx. Pulldown assays and isothermal titration calorimetry analysis clearly showed a significant interaction between CcpAx and AxCeSD. Taken together, these data strongly suggest that CcpAx functions as a member of the TC in A. xylinum.