An ORF2gene located upstream of the cellulose synthase(hcs)operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and C-13 solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. Oil the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose 1. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose 1 microfibrils by this microorganism. (C) 2002 Elsevier Science (USA). All rights reserved.