Factors affecting hexose phosphorylation in Acetobacter xylinum

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Self-immobilized recombinant Acetobacter xylinum for biotransformation

Biofilms have been successfully applied for biotransformation for decades, especially in the area of bioremediation due to the feature of harsh reaction condition resistance. Acetobacter xylinum is known for its cellulose pellicle forming capability. Like biofilm, A. xylinum cells are immobilized by simultaneously produced cellulose nanofibers in the pellicle. A recombinant A. xylinum was constructed with the expectation that the cells could be self-immobilized and achieve a desired and stable biotransformation. d-Amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as the model enzyme to be expressed in the recombinant A. xylinum. The constructed recombinant A. xylinum not only successfully produced DAAO activity but also self-immobilized by cellulose nanofibers in both the static and shaken culture. Although self-immobilized cells demonstrated a DAAO activity approximately 10% of the cell crude extract activity, it provided the benefits of improved thermal stability, operational stability, and easy retrieval for repeated use.     

Occurrence of Free Ceramide in Acetobacter xylinum

Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) from rice seeds and probably the first well-defined cystatin superfamily member of plant origin, was immunologically investigated for its occurrence in rice seeds during maturation and germination. The enzyme-linked immunosorbent assay (ELISA) using anti-oryzacystatin Immunoglobulin G showed that all the investigated 23 cultivars of rice, Oryza sativa L. japónica, contained oryzacystatin at 1 ~ 4 mg % in their seeds. Particularly, oryzacystatin levels were high in precocious cultivars and low in sticky rice cultivars. The use of the ELISA method for the representative rice cultivar, Nipponbare, gave the result that in the seed maturation process, oryzacystatin was synthesized in precedence to total seed protein. In the germination process, oryzacystatin tended to decrease in accordance with degradation of total seed protein.     

Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

Regulation of hexose phosphate metabolism in Acetobacter xylinum

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.     

The structure of cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus.

The structure of the pellicles and cells of the cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus, was studied by transmission electron microscopy of thin sections and freeze-etch replicas of glucose-stimulated cell suspensions, quiescent cell suspensions, and discrete pellicles. These bacteria have a relatively thin cell wall in section, with several irregular features superimposed on an otherwise simple, Gram-negative morphology. There are no flagella or pili. Unfixed, unextracted cells, viewed as whole mounts, show spherical or ellipsoidal bodies of undetermined composition which disappear after extraction with water or ethanol and propylene oxide. For both species, there are several kinds of cell surface irregularities, some of which are localized protrusions of the cell envelope. A variety of irregularities is seen frequently on cells in the first minutes of glucose incubation, on cells in a discrete pellicle, on quiescent cells, and on starved cells. Immediately after the addition of glucose to cellulose-free cells in suspension culture, fine fibrils appear on and (or) near the cell envelope. The fine fibrils are frequently as small as 3 nm in diameter in both freeze-etch and thin-section preparations and are frequently associated with freshly synthesized cellulose fibrils. Starved cells in suspensions free of (classical) microfibrils sometimes reveal stubs of an extracellular structure whose morphology resembles that of a nascent cellulose fibril.     

Synthesis of mannosyl cellobiose diphosphate prenol in Acetobacter xylinum

The enzymatic synthesis of a β-mannosyl (1 → 3) β-glucosyl (1 → 4) α-glucose-1-pyrophosphate-prenol (allylic) by Acetobacter xylinum preparations is described. Glucose pyrophosphate lipid, already known to be formed from UDP-glucose and endogenous phosphate lipid, is demonstrated to accept another glucose from UDP-glucose to give a cellobiose pyrophosphate lipid. The latter in turn accepts mannose from GDP-mannose to form a mannosyl cellobiose pyrophosphate lipid. The structure of the trisaccharide and the way it is linked to the lipid moiety were established by enzymatic and chemical methods such as mild alkaline and acid hydrolysis, phenol treatment, partial acid hydrolysis and acetolysis, periodate oxidation, borohydride reduction, and treatments with glycosidases. The α-unsaturated, polyprenolic nature of the lipid was inferred from and confirmed by the reaction between UDP-glucose and ficaprenol monophosphate to give glucose pyrophosphate ficaprenol, which had the same properties as the glucose pyrophosphate lipid formed from the endogenous acceptor. The allylic structure proposed for the endogenous acceptor is suggested by the lability to phenol treatment and catalytic reduction of its glycosylated derivatives. The enzyme preparation also synthesizes a β-mannose phosphate prenol (allylic), which does not seem to participate in the trisaccharide synthesis. The possible role of these sugar prenols in the synthesis of exopolysaccharides is considered.     

Electron Microscopy of a Non-pellicle-forming Strain of Acetobacter xylinum

A non-pellicle-forming culture of Acetobacter xylinum, isolatedfrom a pellicle-forming culture by repeated transfer of thelatter through agitated medium, has been examined. The cellsof the non-pellicle-producing culture are often greatly elongatedand possess a profusion of intra-cytoplasmic membranes. Freeze-fracturedcells reveal extensive face views of both the outer and cytoplasmicmembranes and show a cell coat composed of a large number ofshort fibrils arising from the outer membrane. X-ray diffractionof the cells in some cases shows the cellulose-I pattern butshadow-contrasted preparations do not show long microfibrils.It is suggested that the non-pellicle formers produce celluloseas only very short microfibrils which are synthesised all overthe cell surface and that there are changes in the outer membranecompared to pellicle-producing cells. The possible significanceof these differences between pellicle-forming and non-pellicle-formingA. xylinum strains is discussed.