Dataset Information

Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

ABSTRACT: Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

SUBMITTER: Florea M

PROVIDER: S-EPMC4806288 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

Florea Michael M Reeve Benjamin B Abbott James J Freemont Paul S PS Ellis Tom T

Scientific reports 20160324

Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in add ...[more]

PMID: 27010592

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Project description:Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.

Project description:Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of ?-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

Dataset Information

Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

Publications

Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

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