In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

Bureau, Thomas E.; Brown, R. Malcolm

doi:10.1073/pnas.84.20.6985

Cited by 60 publications

(42 citation statements)

References 32 publications

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“…Our experiments provide the first direct evidence that the linear TCs contain the c-di-GMP-binding protein. The c-di-GMP-binding protein is associated with the cytoplasmic membrane, as suggested by biochemical and sequencing data (9,32).…”

Section: Discussionmentioning

confidence: 99%

“…Investigations of A. xylinum as a model organism for cellulose biosynthesis (for reviews, see references 28, 29, and 35) have led to the following: (i) in vitro cellulose biosynthesis (1,2,9,16,34), (ii) discovery of cyclic diguanylic acid (c-di-GMP) as a specific activator of cellulose biosynthesis in A. xylinum in vitro (30), (iii) purification and identification of cellulose synthase (23,24,25), and (iv) the first isolation of the cellulose synthase gene (31,36).…”

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confidence: 99%

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Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of Acetobacter xylinum

Kimura

et al. 2001

J Bacteriol

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Section: Discussionmentioning

confidence: 99%

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Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of Acetobacter xylinum

Kimura

et al. 2001

J Bacteriol

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“…The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils from membrane-localized cellulose-synthesizing complexes (10) that are organized in a linear row along the long axis of the bacterial cell (8,56). Among the organisms known to produce cellulose, the mechanism of cellulose synthesis at present is best understood in this bacterium.…”

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Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization

et al. 1994

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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...

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“…The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The most reliable methods to prove the presence of crystalline cellulose are x-ray and electron diffraction analyses (Lin et al, 1985;Bureau and Brown, 1987). Highly purified cellulase conjugated to colloidal gold has been used as a probe to identify cellulose by TEM (Lin et al, 1985).…”

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[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

Okuda

Kudlicka

et al. 1993

Plant Physiol.

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In vitro B-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Cossypium hirsutum) fiber cells. The reaction mixture favoring &1,4-glucan synthesis included the following effedors: MgZ+, Caz+, cellobiose, cyclic-3':5'-CMP, and digitonin. The ethanol insoluble fraction from this reaction contained &1,4-glucan and 8-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the &1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated produd favoring &l,4-glucan synthesis strongly resembled that of cellulose II. O n the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan produd was found to be exclusively 8-1,4-linked. Enzymic hydrolysis confirmed that the produd was hydrolyzed only by cellobiohydrolase 1. Autoradiography proved that the produd was synthesized in vitro. l h e degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the @-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble produd. O n the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.Cellulose is the most abundant macromolecule on earth (Brown, 1985). It serves a major structural role in the cell wall of plants, some algae, and certain fungi and is the primary component of economical products such as wood, cotton, and paper. Because of its tremendous abundance and its physiological and economical importance, many attempts at in vitro cellulose synthesis have been made with cell-free systems from various sources during the past three decades Brown, 1989b;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model (Ross et al., 1987;Brown, 1989a;Lin and Brown, 1989;Lin et al., 1990;Saxena et al., 1990;Wong et al., 1990;Mayer et al., 1991;Saxena et al., 1991). In higher plants, however, numerous failures to detect activity for in vitro cellulose synthase have continued because it has remained impossible to make preparations from higher plant cells capable of synthesizing true microfibrillar cellulose or even appreciable quantities of (3-1,4-glucan (Read and Delmer, 1991).Severa1 methods for the characterization of products synthesized in vitro, such as solubility properties, acid hydrolysis, acetolysis, specific enzymic digestion, and linkage analysis after periodate oxidation or methylation, have been used either alone or in various combinations (Fry, 1988). Insolubility in strong alkali (eg. 24% KOH) has been used for the detection of p-1,4-glucan, but (3-1,3-glucan synthesized in vitro may also be present in the alkali-insoluble fraction (Hayashi et al., 1987). The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The mo...

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In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

Cited by 60 publications

References 32 publications

Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of Acetobacter xylinum

Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of Acetobacter xylinum

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization

[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

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