Pathway for oxidative dissimilation of glycerol in Bacillur subtilis

It has been suggested that the main pathway for oxidative glycerol dissimilation in Bacillus subtilis involves a nicotinamide adenine dinucleotide (NAD)-dependent glycerol dehydrogenase (8). In this paper, evidence is presented that supports the notion that glycerol dissimilation in this organism proceeds primarily via an NAD-independent glycerol phosphate dehydrogenase. In strains 168 and W23 of B. subtilis, activity corresponding to the dihydroxyacetone kinase and NAD-dependent glycerol dehydrogenase reported by Wiame et al. (8) was found; however, glycerol kinase and an inducible NAD-independent glycerophosphate dehydrogenase of higher specific activity were also found in the same strains. It was also observed that approximately 10% of mutants unable to grow on glycerol as the major source ofcarbon and energy are lacking the NADindependent glycerophosphate dehydrogenase. The mutations were induced with ethyl methane sulfonate, and the mutants were isolated by replica plating after penicillin selection in minimal medium supplemented with tryptophan, histidine, methionine, proline, threonine, serine, and valine at 10 ,ug/ml (containing 0.3% glycerol as the carbon source). The mutant character is transformable at a frequency characteristic of single site mutations. The mutants grow on all carbon sources, except glycerol, that support the growth of the parent strain, e.g., glucose, mannitol, succinate, fumarate, galactose, and amino acids. These mutants have unchanged activity for glycerol dehydrogenase, indicating that it is the NADindependent enzyme that is involved in the dissimilation of glycerol. The NAD-dependent glycerol dehydrogenase apparently is the means by which the cell synthesizes glycerol from dihydroxyacetone or glyceraldehyde and 2,3-butanediol from acetoin (1). Although the cells contain the enzymes (glycerol dehydrogenase and dihydroxyacetone kinase) that might lead to dihydroxyacetone phosphate through an NAD-linked system, they probably do not use this pathway for the dissimilation of glycerol. The NAD-independent glycerophosphate dehydrogenase activity is induced by growth on glycerol and is repressed by growth on glucose (Table 1). A mutant of strain 168, designated B24, which is unable to grow on glycerol as a carbon source, does not form the NAD-independent enzyme. Glycerol gives no reaction in the assay system. The reaction with a-glycerophosphate does not require and is not stimulated by either NAD or nicotinamide adenine dinucleotide phosphate. Neither the oxidation of reduced nicotinamide adenine dinucleotide (NADH) nor the reduction of NAD is stimulated by dihydroxyacetone phosphate or a-glycerophosphate. Although the activities are measured in fairly crude extracts and may, therefore, be somewhat inaccurate, it seems clear that the NAD-independent enzyme has a much greater activity than the glycerol dehydrogenase. Extracts of cells of strain 168 grown in minimal medium show an activity of 1.2 x 10-2 MAmoles per min per mg of protein when assayed by the reduction ofNAD in the presence of glycerol. This may be compared to the activity of 6.2 X 10-2 ktmoles per min per mg of protein shown by extracts of the same strain grown in the same medium when assayed for NADindependent a-glycerophosphate dehydrogenase activity. The activity of the NAD-dependent glycerol dehydrogenase, although variable in extracts of cells grown on different media, does not respond to changes in the glycerol concentration of the growth medium. The finding that the NADindependent enzyme activity is inducible adds considerable support to the notion that it is on the pathway of oxidative glycerol dissimilation. Activities for glycerol kinase and dihydroxyacetone kinase were found to be 0.04 and 0.08 ,umoles per min per mg of protein, respectively, when measured by NAD reduction or NADH oxidation coupled with a-glycerophosphate dehydrogenase. The reaction mixture for glycerokinase activity at 25 C contained 0.3 ml of 0.1 M glycerol, 0.3 ml of 3 N hydrazine, 0.5 ml of 1 M carbonate, pH 9.5, 0.6 ml of 0.1 M adenosine