Function of limited sorbitol oxidation in Gluconobacter oxydans

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Virginia Polytechnic Institute and State University

Bacteria of the genus Gluconobacter have very active, membrane-bound, NAD(P)-independent, polyol dehydrogenases which stoichiometrically produce the singlestep oxidation product of polyols provided in the growth medium. These bacteria have a high respiratory quotient which is believed to result from oxidations by these dehydrogenases. These organisms grow and survive at pH values as low as 2.5 leading to speculation that their membrane-bound dehydrogenase activity provides the rapid electron flow necessary to purge cells of toxic levels of hydrogen ions. These dehydrogenases are also believed to be used for energy metabolism, and there is no clear understanding of their function in the cell metabolism. Oxidation of sorbitol in Gluconobacter oxydans ATTC 621 was studied to determine if the oxidations by the membrane bound sorbitol dehydrogenase (mSDH) were required for growth, and whether they functioned to protect the cells in low pH environments. G. oxydans required a high concentration of sorbitol in the medium, and a reduction in the concentration to 0.1% decreased the rate and extent of growth. Using mutants with decreased levels of mSDH, we found that growth rates decreased as a result of this mutation, indicating that mSDH activity was needed for growth. No changes in the specific activity of mSDH in strain ATCC 621 occurred when the cells were grown at pH 7.0, 6.0, and 4.5. However, cytochrome levels were doubled in cells grown at pH 4.5 compared to pH 6.0 and 7.0. The increased cytochrome levels did not increase the oxygen uptake of the pH 4.5 grown cells on sorbitol. Cells grown at all pH values respired more rapidly when tested at pH 4.5, and respiration decreased as the pH increased. The higher activity at lower pH values may result from increased efficiency of mSDH, which has an in vitro pH optimum of 5.2. Magnesium and calcium increased the respiration of pH 6.0 grown cells but not pH 4.5 grown cells. Less cell mass per mg of sorbitol oxidized was obtained when cells were grown at pH 4.5 compared to pH 6.0 and 7.0. However, no differences were detected in the specific activity of any of the sorbitol oxidizing enzymes. The activity of mSDH in G. oxydans is necessary for the growth of this bacterium. The mSDH specific activity is not regulated by the growth pH, but increased levels of cytochromes and decreased cell yields indicate a change in the cell's oxidative system resulting from lowered growth pH values.