The metabolism of lactulose by Clostridium perfringens in batch and continuous culture

Abstract

The aim of this project was to initiate studies on lactulose (4-O-[beta]-D-galactopyranosyl D-fructofuranose) metabolism by human intestinal bacteria. The organism of choice was Cl. perfringens, due to its rapid metabolism of the sugar. Work was conducted with cells grown anaerobically, in both batch and continuous culture. Use of the latter system permitted closer approximation of the in vivo situation.Initial work was concerned with the identification of the metabolic pathway(s) involved. Radiolabelled lactulose was not available, hence a variety of 'cold' procedures were employed; including fluorimetric determination of key metabolites, and assay of key enzymes.Growth in medium supplemented with lactulose resulted in the induction ofB-galactosidase, whereas phospho-B-galactosidase activity could not be detected. This suggested that lactulose was accumulated in the free form and that the first step in intracellular metabolism was hydrolysis to its constituent monosaccharides. This was confirmed by the detection of intracellular free galactose and fructose. The galactosyl moiety was found to be metabolised by the Leloir pathway: the enzymes galactokinase, galactose 1-phosphate uridylyl transferase and UDPgalactose 4-epimerase were present in lactulose-grown cells, and both galactose 1-phosphate and glucose 1-phosphate were detected. The fructosyl moiety was most likely metabolised by an ATP-dependent fructokinase activity, present in lactulose-grown cells.Measurement of lactulose utilisation in a buffered incubation system permitted the study of various transport system inhibitors. Utilisation was inhibited by both CCCP and DCCD, suggesting that transport requires a proton gradient; formed by the action of the membrane-bound ATPase activity. The presence of a proton gradient-dependent uptake system is consistent with the findings from the metabolic studies. A similar metabolic system was responsible for lactulose metabolism under conditions of carbon limitation in the chemostat.Galactokinase was further studied. Basal levels of this enzyme were repressed by inclusion of glucose in the culture medium. Glucose also prevented induction of galactokinase by lactulose or galactose. This latter 'glucose effect' could not be abolished by the addition of cyclic AMP, and appeared to be mediated via inducer exclusion.