Gluconeogenesis by Veillonella parvula M4: evidence for the indirect conversion of pyruvate to P-enolpyruvate.

Abstract

Cells of Veillonella parvula M4 growing in complex medium with lactate-U-14C as the carbon source were shown to synthesize complex cellular components with lactate carbon. Of the original labeled substrate, 69% of the label appeared in the metabolic end products (acetate, propionate, and CO2) while 25% was distributed among the products of biosynthesis. Of this latter radioactivity, the lipopolysaccharide (LPS2) fraction contained 22% of the total, 6% of which was found in lipid material and 16% in a carbohydrate fraction; this latter material was analyzed and shown to contain labeled glucosamine, galactosamine, galactose, glucose, and ribose. The remaining 2.6% was distributed in DNA (0.6%), cellular amino acids (0.6%), and the extracellular polysaccharide (LPS1) fraction (1.4%). Further analysis showed that this latter fraction was composed of only glucose and galactose. Of the cellular amino acids, arginine, aspartate, glutamate, alanine, and serine were labeled.Since the above results indicated that gluconeogenesis in V. parvula M4 proceeds, at least in part, from lactate, enzyme studies were undertaken with dialyzed crude extracts of the organism to determine the possible route of lactate conversion to P-enolpyruvate. Evidence is presented for the presence of pyruvate kinase and PEP carboxykinase activity in the organism. Since extract preparations produced significant quantities of (32P)ADP, but negligible quantities of (32P)AMP, during assays containing (α-32P)ATP and pyruvate, it was concluded that PEP synthetase or pyruvate, phosphate dikinase are absent in the organism. Thus, from these and previous results, it was concluded that in V. parvula M4, lactate is converted to P-enolpyruvate via the intermediate, oxaloacetate.