Studies on central carbon metabolism and respiration of Gluconobacter oxydans 621H

Gluconobacter oxydans shows a number of exceptional characteristics, like the biphasic growth on glucose and the incomplete oxidation of glucose to gluconate (phase I, exponential growth,) and ketogluconates (phase II, linear growth), leading to an acidification of the medium down to pH values less than 4. Furthermore, growth and metabolism of G. oxydans is strongly dependent on the availability of oxygen. In the respiratory chain, two terminal end acceptors are present. The ubiquinol bd oxidase, preferably used under acidic pH, is less efficient in contribution to the proton motive force than the bo3 oxidase. An open question was the function of a cytochrome bc1 complex as well as soluble cytochrome c552, in the absence of a cytochrome c oxidase. For elucidation of the function of these respiratory chain components, a deletion mutant lacking the genes encoding the cytochrome bc1 complex was constructed and characterised. When cultivated on mannitol at pH 4 the deletion mutant showed retarded growth and substrate consumption. Therefore, the cytochrome bc1 complex is involved in energy supply of the cells under acidic pH when the more inefficient ubiquinol bd oxidase is upregulated. Interestingly, under oxygen limitation the deletion mutant released heme into the culture medium in the late stationary phase. Since hemes b and c are the prosthetic groups of the cytochrome bc1 complex heme excretion of the mutant is a consequence of absence of the corresponding apoenzymes, which is formed under oxygen limitation. The membrane-bound and respiratory chain-linked alcohol dehydrogenase (ADH) was reported to be a component of the respiratory chain and not merely an oxidoreductase. A connection to the cytochrome bc1 complex was investigated in this work. The oxidation velocity of the ADH of the mutant was significantly lower than that of the wild type was. Furthermore, the cytochrome bc1 complex was shown to be involved in the energy-dependent activation of the ADH in cells grown at pH 4, but a direct interaction between the cytochrome bc1 complex and the ADH was not demonstrated yet. In order to throw light on the regulation of the respiratory chain in conjunction with the overall metabolism, genome-wide DNA microarray analyses were carried out with G. oxydans 621H. Three conditions were investigated: I) oxygen limitation vs. oxygen excess, II) cultivation at decreased pH of 4 vs. cultivation at standard pH of 6 and III) growth phase II vs. growth phase I during growth on glucose pH 6, since the cytochrome bc1 complex deletion mutant showed growth retardation in growth phase II. Transcriptional analyses of oxygen-limited cells displayed an upregulation of genes encoding the cytochrome bc1 complex and both terminal oxidases. In cells grown at pH 4, an enhanced transcription of the genes encoding the more inefficient ubiquinol bd oxidase occurred. Since no direct connection between the glucose metabolism and the cytochrome bc1 complex was evident, glucose metabolism was further characterised in the wild type. C-Metabolome analysis and metabolic flux analysis (MFA) were applied to solve the question of the quantity and oxidation state of the substrate entering the cell for catabolism. MFA of phase I glucose cultures showed that 97% of the initial glucose was oxidised in the periplasm by the highly active and respiratory chain-linked glucose dehydrogenase, whereas only 3% of glucose proceeded into the cytoplasm. According to the model, intracellular glucose was predominantly oxidised to gluconate, subsequently phosphorylated by gluconate kinase and further metabolised via the pentose phosphate pathway. In addition, genome-wide transcriptional analysis of G. oxydans approved the reported assumption of a highly active pentose phosphate pathway, which is enhanced in growth phase II. In contrast, the Entner-Doudoroff pathway was almost inactive in growth phase I.