title: Functional reconstitution of cellulose synthase in Escherichia coli.
abstract: Cellulose is a high molecular weight polysaccharide of β1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.
authors.affiliation: Research Institute for Sustainable Humanosphere (RISH), Kyoto University , Uji, Kyoto 611-0011, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Imai
authors.forename: Shi-Jing
authors.initials: SJ
authors.lastname: Sun
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Horikawa
authors.forename: Masahisa
authors.initials: M
authors.lastname: Wada
authors.forename: Junji
authors.initials: J
authors.lastname: Sugiyama
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D005964:Glucosyltransferases / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D014961:X-Ray Diffraction
chemical_list: D029968:Escherichia coli Proteins; D005964:Glucosyltransferases; C478648:cellulose synthase