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Microbial cellulose has proven to be a remarkably versatile biomaterial and can be used in wide variety of applied scientific endeavors, such as paper products, electronics, acoustics, and biomedical devices. In fact, biomedical devices recently have gained a significant amount of attention because of an increased interest in tissue-engineered products for both wound care and the regeneration of damaged or diseased organs. Due to its unique nanostructure and properties, microbial cellulose is a natural candidate for numerous medical and tissue-engineered applications. For example, a microbial cellulose membrane has been successfully used as a wound-healing device for severely damaged skin and as a small-diameter blood vessel replacement. The nonwoven ribbons of microbial cellulose microfibrils closely resemble the structure of native extracellullar matrices, suggesting that it could function as a scaffold for the production of many tissue-engineered constructs. In addition, microbial cellulose membranes, having a unique nanostructure, could have many other uses in wound healing and regenerative medicine, such as guided tissue regeneration (GTR), periodontal treatments, or as a replacement for dura mater (a membrane that surrounds brain tissue). In effect, microbial cellulose could function as a scaffold material for the regeneration of a wide variety of tissues, showing that it could eventually become an excellent platform technology for medicine. If microbial cellulose can be successfully mass produced, it will eventually become a vital biomaterial and will be used in the creation of a wide variety of medical devices and consumer products.

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The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of 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 single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/ Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains.Cellulose is an extracellular polysaccharide, synthesized as long f-1,4 glucan chains that associate to form the microfibrils commonly observed in cellulose-synthesizing organisms. The synthesis of this biopolymer takes place via a single polymerization step, utilizing UDP-glucose as the substrate and-catalyzed by the enzyme cellulose synthase (UDP-glucose: 1,4-p-D-glucosyltransferase), without the apparent involvement of any intermediates (40). However, the crystalline structure of cellulose microfibrils and their association with organized intramembranous particles observed in most cellulose-synthesizing organisms suggest a more intricate mechanism of cellulose biogenesis than that deduced from the polymerization reaction alone. Because of their association with cellulose microfibrils, the intramembranous particles are believed to be sites of cellulose synthesis and extrusion. The nature of these particles is incompletely understood at present, and it is not known at the molecular level how they become organized into structures that are referred to as terminal synthesizing complexes (TCs) that are characteristic for most organisms (5). In higher plants, the TC is arranged as a rosette; in a large number of algal species, the TC is arranged as a linear row(s) of particles (7). Even though the cellulose produced by all organisms chemically is the same in its primary compositi...