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Abstract
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 β-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.
Highlights
Cellulose is produced by plants and by microorganisms, such as algae, bacteria and fungi [1]
To identify G. hansenii genes that are involved in the synthesis, secretion, assembly, and crystallization of cellulose, we previously used Tn5 transposon random mutagenesis to generate mutants that might be defective in any of the steps
We screened the previously generated Tn5 transposon insertion mutants of G. hansenii ATCC23769 for those that produced cellulose with altered morphologies
Summary
Cellulose is produced by plants and by microorganisms, such as algae, bacteria and fungi [1]. BC, like plant cellulose, consists of ordered glucan chains, but exhibits differing higher order molecular and nanoscale assembly resulting in unique structural and mechanical properties. Gluconacetobacter is a Gram-negative bacterium, which is capable of synthesizing large amounts of highly ordered, or crystalline, cellulose organized as twisting ribbons of microfibrillar bundles [7]. Gluconacetobacter has long been used as a model system for studying cellulose synthesis and composite assembly in higher plants. Images of electron micrographs showed that the surface of the Gluconacetobacter cell has 50–80 pore-like sites located in a regular row along the long axis of the cell, each of which is thought to secrete a 1.5 nm sub-elementary fibril composed of 10–15 glucan chains [8, 9]. Fasciation of bundles forms the twisting cellulose ribbon (40–60 nm), which aggregates to form a cellulose pellicle produced at the top of the culture medium
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