The mechanism of Acetobacter xylinum cellulose biosynthesis: direction of chain elongation and the role of lipid pyrophosphate intermediates in the cell membrane

The biosynthesis of Acetobacter xylinum ATCC 10821 cellulose has been studied with resting cells and a membrane preparation using 14C-pulse and chase reactions, with d -glucose and UDPGlc, respectively. Cellulose was biosynthesized from UDPGlc, and it was found to be tightly associated with both the cells and the membrane. The cellulose chains could be released from the cells and the membrane preparation by treating at pH 2, 100 °C for 20 min. The cellulose chains that were released from the pulse and pulse-chase reactions were purified and separated from any low molecular weight substances by gel chromatography on Bio-Gel P4. They were then reduced with sodium borohydride and hydrolyzed with 4 M trifluoroacetic acid at 121 °C for 2 h. Labeled products from the acid hydrolyzates were separated by paper chromatography and found to be d -glucose and d -glucitol. The amount of radioactivity in the products was determined by liquid scintillation counting. It was found that the pulsed products from the resting cells gave a ratio of d -[14C]glucitol to d -[14C]glucose of 1:11, and after chasing, the ratio decreased to 1:36. The pulsed products from the membrane gave a ratio of d -[14C]glucitol to d -[14C]glucose of 1:12, and after chasing for 5 min the ratio decreased to 1:43, and after 10 min, the ratio decreased to 1:66. These results show that the labeled d -glucitol obtained from the reducing end of the cellulose chain is chased into the interior of the cellulose chain during synthesis, showing that the cellulose chain is elongated from the reducing end. An insertion mechanism for the synthesis of cellulose from UDPGlc is proposed that involves lipid pyrophosphate glycosyl intermediates and three membrane enzymes: lipid phosphate:UDPGlc phosphotransferase, cellulose synthase, and lipid pyrophosphate phosphohydrolase.