Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

Saxena, Inder M.; Lin, Fong Chyr; Brown, R. Malcolm

doi:10.1007/bf00016118

Cited by 181 publications

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“…Of particular interest is the antibody localization of rosette TCs discovered in advanced algae such as M. denticulata , Nitella spp, and Coleochaete scutata , which are thought to be progenitors of vascular plants Brown, 1987, 1988;Hotchkiss et al, 1989). The antibody approach could help to dissect the evolution of cellulose biosynthesis and rosette TC architecture (see also Herth, 1985b;Brown, 1990). …”

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Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

et al. 1999

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The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli , and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants. INTRODUCTIONCellulose is the most abundant biopolymer on earth and is the major constituent of the plant cell wall (Franz and Blaschek, 1990;. Cellulose is a biopolymer consisting only of ␤ -1,4-glucans. Approximately 40 ␤ -glucan chains are synthesized from a multimeric enzyme complex, and these chains associate immediately upon synthesis to form the crystalline entity known as a microfibril . Crystalline cellulose is defined by its various allomorphs, with cellulose I denoting the most abundant native crystalline form .One of the great enigmas in plant biology is the biosynthesis of cellulose. During the past 50 years, the site of cellulose synthesis has been investigated intensively, but until now, there has been no direct proof for the existence of a multimeric enzyme complex located in the plasma membrane of vascular plant cells. Roelofsen (1958) first suggested that cellulose might be assembled by a large enzyme complex at the growing tip. As early as 1972, Dobberstein and Kiermayer (1972) had visualized ordered particle complexes within "f-vesicles" of the Golgi apparatus in the green alga Micrasterias denticulata. These particles were implicated in the biosynthesis of cellulose. This work is significant historically, because the first observation of what was later to be beautifully imaged by freeze fracture was from sectioned material. Thus, the ordered granule complex, first postulated by Preston (1964), was found only eight years later, but it was not until the freeze-fracture techniq...

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“…The antibody approach could help to dissect the evolution of cellulose biosynthesis and rosette TC architecture (see also Herth, 1985b;Brown, 1990).…”

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Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

et al. 1999

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“…The GTs that show structural likeness to Alg8 have been demonstrated or predicted to be multispanning membrane proteins (Barny et al, 1996;Saxena et al, 1990). Here we investigated the membrane topology of Alg8 using translational fusions with PhoA to predict periplasmic and cytoplasmic regions of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…HCA was used to demonstrate Alg8's structural homology to family 2 (inverting) b-GTs (Campbell et al, 1997), which is one of the largest families of GTs (Campbell et al, 1997). Family 2 GTs include those involved in bacterial capsule or exopolysaccharide synthesis such as chitin synthesis by NodC in Sinorhizobium species, cellulose production by AcsAB in Gluconacetobacter xylinus (formerly Acetobacter xylinum), and hyaluronate synthesis by HasA in Streptococcus pyogenes (Campbell et al, 1997;Saxena et al, 1990Saxena et al, , 1995.…”

Section: Introductionmentioning

confidence: 99%

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

2008

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Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for bGTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for cdi-GMP [bis-(39,59)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.

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“…The genes encoding CESAs were first discovered in Acetobacter xylinum (Saxena et al, 1990;Wong et al, 1990). After six years, the first plant CesAs, GhCesA1 and GhCesA2 were identified in cotton (Pear et al, 1996).…”

Section: Structural Features Of Cesa Proteinsmentioning

confidence: 99%

Structure, organization, and functions of cellulose synthase complexes in higher plants

Festucci-Buselli

Otoni

Joshi

2007

Braz. J. Plant Physiol.

113

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Annually, plants produce about 180 billion tons of cellulose making it the largest reservoir of organic carbon on Earth. Cellulose is a linear homopolymer of beta(1-4)-linked glucose residues. The coordinated synthesis of glucose chains is orchestrated by specific plasma membrane-bound cellulose synthase complexes (CelS). The CelS is postulated to be composed of approximately 36 cellulose synthase (CESA) subunits. The CelS synthesizes 36 glucose chains in close proximity before they are further organized into microfibrils that are further associated with other cell wall polymers. The 36 glucose chains in a microfibril are stabilized by intra- and inter-hydrogen bonding which confer great stability on microfibrils. Several elementary microfibrils come together to form macrofibrils. Many CESA isoforms appear to be involved in the cellulose biosynthetic process and at least three types of CESA isoforms appear to be necessary for the functional organization of CelS in higher plants.

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Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

Cited by 181 publications

References 20 publications

Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

Structure, organization, and functions of cellulose synthase complexes in higher plants

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