CHARACTERIZATION OF GENES IN THE CELLULOSE-SYNTHESIZING OPERON (ACS OPERON) OF ACETOBACTER-XYLINUM - IMPLICATIONS FOR CELLULOSE CRYSTALLIZATION

The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-loca lized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the d ifferent components involved in this process, we isolated an Acetobact er cellulose-synthesizing (acs) operon from this bacterium. Analysis o f DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A si ngle base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is descr ibed in the present paper, along with the sequences of the other two g enes (acsC and acsD). The requirement of the acs operon genes for cell ulose production was determined using site-determined TnphoA/Kan(r) Ge nBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the ac sD mutant produced reduced amounts of two cellulose allomorphs (cellul ose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the hcs operon genes in determi ning the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the differen t mutants; however, this arrangement was observed only in cells that a ctively produced cellulose microfibrils, suggesting that it may be inf luenced by the crystallization of the nascent glucan chains.