Zahid, Nageena: Osmotic stress response in the industrially important bacterium Gluconobacter oxydans. - Bonn, 2017. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Nageena Zahid}},
title = {Osmotic stress response in the industrially important bacterium Gluconobacter oxydans},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2017,
month = mar,

note = {G. oxydans is commercially used for the oxidative fermentation of various sugars, sugar acids, and alcohols to produce biotechnologically valuable products. The current study was conducted to explore the (so-far unknown) mechanisms of osmoprotection in the organism. One common strategy to cope with osmotic stress is the intracellular accumulation of compatible solutes. For the identification of the presence of compatible solute in G. oxydans, total cellular metabolites were extracted from control and osmotic stress cultures and analyzed by 13C NMR spectroscopy and HPLC. Both analytical methods revealed that G. oxydans accumulated mannitol as compatible solute in response to osmotic stress to a maximum concentration of 2.7 µmol mg-1 total cell protein. The intracellular mannitol concentration varied and depended upon the type of osmoticum and the carbon source. Additionally, the exogenous supply of mannitol assisted the stressed cells to alleviate osmotic stress, to maintain their proliferation rates, and to enhance their catalytic efficiency.
Furthermore, the role of two cytoplasmic fructose reductases (Gox0849 and Gox1432) was elucidated with respect to intracellular mannitol formation and osmotolerance of G. oxydans. Enzyme assays revealed that Gox1432 (EC catalyzed a NADPH-specific fructose reduction, while Gox0849 (EC preferred NADH as cofactor. The corresponding genes were deleted, and growth behavior of the mutants was analyzed under non-stress and osmotic-stress conditions. During cultivation in high-sugar media, the mutant lacking gox1432 showed retarded growth, while the growth of strain Δgox0849 was unaffected. The sensitivity of the cells towards osmotic stress was increased when both fructose reductases were simultaneously deleted. Likewise, the intracellular mannitol content was reduced in the mutants lacking gox1432 during growth under osmotic stress. These results indicated the importance of Gox1432 in intracellular mannitol formation and osmotolerance of G. oxydans. Another line of evidence came from the transcript abundance of gene gox1432 which was 30-fold higher than that of gox0849. Analogously, the activity of the NADPH-dependent enzyme Gox1432 was 10-fold higher in comparison to the NADH-dependent enzyme Gox0849 in the cytoplasmic fractions of osmotically stressed cells. Moreover, a plasmid-based overexpression of gox1432 in the Δgox1432 deletion mutant enabled the cells to recover from osmotic stress. Hence, the present findings provide detailed insights into the molecular mechanisms of mannitol-mediated osmoprotection in G. oxydans, and are helpful to optimize the processes of industrial products formation in highly concentrated sugar solutions.},

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