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Total DNAs from nine strains of Acetobacter xylinum, two strains of Acetobacter aceti, and one Acetobacter pasteurianus strain were examined for the extent of digestion by various restriction endonucleases. The majority of the endonucleases cleaved the total DNAs with a frequency expected from the number of sites present in DNA sequences deposited in the GenBank data base. However, the restriction enzyme digestions identified two different genomic DNA modifications in Acetobacter. One sequence-specific modification protected total DNAs from seven of the A. xylinum strains against cleavage by EcoRI (GAATTC). Digestion of total DNAs from A. xylinum ATCC 10245 (DNA not cut by EcoRI) and the closely related A. xylinum NRCC 17005 (DNA cut by EcoRI) with Tsp509I (AATT) revealed differences in restriction frequencies that indicated methylation of the first or second adenine within GAATTC. Another sequence-specific modification rendered total DNAs from all the 12 strains recalcitrant to digestion by HinfI. The latter modification indicated that species of the genus Acetobacter contain a solitary DNA methyltransferase that probably methylates adenine in GANTC.Key words: Acetobacter, genomic DNA, modifications, EcoRI, HinfI.

Evidence from phase and polarizing microscopy as well as scanning electron microscopy indicates that the cellulose microfibrils in the spherulites of bacterial cellulose are oriented tangentially, not radially. Also, the orientation may be limited to only a fraction of the thickness of the pellicle. It is suggested that the tangential deposition may be caused by a gradient of concentration of a weakly soluble inhibitor of cellulose formation about a center.

Formation of cellulose microfibrils from precursors elaborated by Acetobacter xylinum takes place remote from the cell surface and without an extensive, amorphous, intermediate high polymer. The microfibrils increase in mass by growth only at one or both tips. The rate of microfibrillar growth per bacterial cell at 25 °C is constant at 0.1 μ per minute up to at least 7 minutes, incubation time. New microfibrils may be produced continuously during the incubation period.

The development of non-spherulitic, threadlike birefringence, observable in pellicles of bacterial cellulose from Acetobacter xylinum, is described. This birefringence is a consequence of the formation of bundles of parallel cellulose microfibrils within the pellicle. The reason for this parallel orientation is unknown and all attempts to influence it have failed. The development of the birefringent threads within the pellicle is often accompanied by formation of streaks of lipid-like material Boating on the surface of the medium. The distribution of these streaks is similar to the distribution of the non-spherulitic birefringence. This similarity in distribution may be a reflection of a common origin.

Lysis of about 90% of the cells of Acetobacter xylinum by lysozyme may be obtained after 2 hours at pH 8.1 in phosphate–citrate buffer. The cell population is heterogeneous since the remaining 10% of the cells do not lyse, even on repetition of the above treatment. The lysozyme does not cause the dissolution of all the bacterial wall, as in other lysozyme-sensitive species, but appears to attack only a limited, local area on the surface of the bacterium. Following the local weakening of the wall, the cell contents escape into the medium leaving a distinct "hull" with the same size and shape as the cell. The oxidative capacity of the lysed cells is the same as that of the whole cells. Reasons for the heterogeneity of the bacterial wall, both within and between cells, remain obscure.

Development of the morphology and microstructure of colonies of Acetobacter xylinum growing on agar was studied by optical microscopy, and transmission and scanning electron microscopy. The mass of rapidly dividing cells surrounded by a sheath of cellulose microfibrils passes from a smooth spheroid to a flattened aggregate with a characteristic "pillowed" surface. This morphology is the result of a repeated extrusion of cells from the confirming sheath, followed by regeneration of a new portion of the sheath on the extrusion of cells from the confirming sheath, followed by regeneration of a new portion of the sheath on the extruded cell mass. Relations of this mechanism to others which produce similar shapes are indicated.

The morphological aspects of biosynthesis of cellulose by Acetobacter xylinum and Acetobacter acetigenus were studied by transmission electron microscopy of both freeze-etch replicas and sections of cellulose-free cells in suspension culture before and subsequent to the induction of cellulose synthesis. Also examined were freshly synthesized, thoroughly washed, cellulose pellicles. Thin sections of rapidly dividing, glucose-metabolizing cells of both species showed irregular features on the cell surface including a small polar invagination which sometimes contained or was associated with fibrils as fine as 3 nm in diameter of a substance which stains with electron-microscopic counterstains. Cellulose microfibrils in thin sections of freshly synthesized pellicles were coated with a surface material which also stained with the same counterstains (uranyl ions and lead ions). The effect of air-drying on freshly synthesized cellulose was striking. When examined by freeze-etching, thoroughly washed, never air-dried pellicles of both species showed a nascent form of cellulose fibril which consisted of a central, dense core surrounded by a sheath of amorphous gel. This sheath may be up to 100 nm wide. When the pellicle was air-dried and rehydrated before freeze-etching, the amorphous sheath was rare and shrunken but ordinary microfibrils of classical dimensions were visible. The sheath and core are sometimes closely associated with the envelope of the cells of both species. These observations can be interpreted in the context of recent advances in cellulose synthesis by assuming that chains of an initial, highly hydrated, intermediate polyglucan are released from the cell and that such chains associate to form a nascent fibril external to the cell but associated with the cell envelope. Air-drying of nascent fibrils converts them to classical microfibrils and this conversion is considered here in molecular terms.

Some lipids, particularly diglycerides, cholesterol, or cholesterol derivatives, are able to induce visible birefringence in pellicles of extracellular bacterial cellulose produced by Acetobacter xylinum when they are added in small amounts to the surface of cultures. This birefringence is due to orientation of a fraction of the cellulose microfibrils, not the cells, in the pellicle. Orientation may begin with small microscopic domains which are present in pellicles of untreated cultures and some of which may overgrow others under the influence of a lipid. How the lipids induce alignment of the microfibrils in the bacterial culture is unknown but the process appears to involve an interaction between newly formed extracellular microfibrils and the microcrystalline surface of the lipid.

Two-dimensional analogues of spherulites are formed in the pellicle of bacterial cellulose produced by static cultures of Acetobacter xylinum. These spherulites are much larger (sometimes more than 1 cm diameter) than those usually observed in other natural or synthetic polymers. They are due to the radial orientation of bacterial cellulose microfibrils in limited regions of the plane of the pellicle. These spherulites interact to form characteristic, dendritic structures of great complexity which resemble leaves of higher plants.