Functional analysis of the acsD gene for understanding cellulose biosynthesis in Gluconacetobacter xylinus
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The acsD gene is a unique gene present in the cellulose biosynthesis operon in G. xylinus. With the use of homologous recombination, the acsD gene disruption mutation was created in the G. xylinus genome. Phenotypic characterization of the acsD gene mutant was investigated with the assistance of light and electron microscopy observations, carboxymethyl cellulose alterations, and lower temperature incubation. The microscopic analysis of the cellulose ribbons secreted from the acsD gene mutant shows that the polymerization and the crystallization components in mutant cells were functional. Observations of the mutant cells after incubation with carboxymethyl cellulose and temperature changes indicate that the arrangements of the pores on the cell surface have been altered. These arrangements led to decreased cellulose secretion capacity of the mutant cells. Successful complementation was achieved by using gene expression plasmids with green fluorescence protein tag in the acsD mutant background. Anti-GFP antibodies were used to determine the in vitro localization of the protein. Using subcellular fractionation and western blotting, the AcsD protein was found to be localized in the periplasm of the cells. Taking all these results together, a new model for bacterial cellulose biosynthesis has been suggested and discussed.