Parallel-up structure evidences the molecular directionality during biosynthesis of bacterial cellulose

Abstract

The "parallel-up" packing in cellulose Ialpha and Ibeta unit cells was experimentally demonstrated by a combination of direct-staining the reducing ends of cellulose chains and microdiffraction-tilting electron crystallographic analysis. Microdiffraction investigation of nascent bacterial cellulose microfibrils showed that the reducing end of the growing cellulose chains points away from the bacterium, and this provides direct evidence that polymerization by the cellulose synthase takes place at the nonreducing end of the growing cellulose chains. This mechanism is likely to be valid also for a number of processive glycosyltransferases such as chitin synthases, hyaluronan synthases, and proteins involved in the synthesis of nodulation factor backbones.

Figure 1

The definition of the directionality of the cellobiose repeat unit with respect to the crystallographic axes.

Figure 2

Evaluation of the chain directionality by microscopic visualization. (A) Schematic diagram of the mode of reaction used in this study. N.E., nonreducing end; R.E., reducing end. (B and C) Electron micrographs of typical microcrystals of cellulose from Cladophora. Arrowheads, see text.

Figure 3

Theoretical background to identify the directionality of c axis by diffraction. (Upper middle) The initial situation of the microcrystal without any tilt. (Lower middle) The corresponding diffraction pattern. The anti-clockwise/clockwise rotation around c axis gives the lower left/lower right diffraction diagrams, respectively.

Figure 4

Two sets of tilt-diffraction patterns from the labeled microcrystal. (A) A microcrystal with its top end labeled with silver grains. (B) Patterns obtained from initial Cladophora. (C) Patterns obtained from annealed Cladophora.

Figure 5

Schematic representation of mode of chain packing in the unit cell of cellulose. (A) Triclinic unit cell. (B) Monoclinic unit cell. Monoclinic angle γ is obtuse.

Figure 6

A sets of tilt-diffraction patterns from the nascent microfibril of Acetobacter. (A) A microfibril ribbon spun out from the cell. Circled areas indicate where a set of diffraction patterns were taken. (B) The corresponding diffraction patterns.

Figure 7

Model for the polymerization of cellulose chain by addition of sugar residues at the nonreducing end.