Energy-dependent incorporation of sphingolipid precursors and fatty acids in Bacteriodes melaninogenicus

Abstract

Washed cells of Bacteroides melaninogenicus are unable to incorporate the sphingolipid precursor 3-ketodihydrosphingosine (3KDS) or dihydrosphingosine into the complete sphingolipids ceramide phosphorylethanolamine (CPE) and ceramide phosphorylglycerol (CPG), whereas growing cultures are able to do so. This result suggested that an energy source was required by washed cells to initiate the incorporation of 3KDS. Investigation of a number of energy sources for B. melaninogenicus showed that glutamine was active in driving the incorporation of 3KDS. This system shows saturation kinetics. Besides glutamine, only asparagine and reduced nicotinamide adenine dinucleotide (NADH) are effective; glutamate and other compounds are inactive. The glutaminedriven system is sensitive to 2,4-dinitrophenol, azide, N,N'-dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenylhydrazone. Asparagine plus NADH shows a synergistic effect in stimulating the incorporation of 3KDS into CPE and CPG in washed cells. However, glutamine plus NADH and glutamine plus asparagine show no such synergy. The cytochrome-free mutant ofB. melaninogenicus, strain S, incorporates 3KDS in a manner similar to the parent strain when glutamine is used to drive the reaction; NADH or asparagine, however, are ineffective when used with strain S. Vitamin K-depleted cells ofB. melaninogenicus are similar to vitamin K-grown cells, when glutamine or NADH is used to drive the 3KDS incorporation. Glutamine and NADH are also effective in stimulating the incorporation of palmitate and acetate by washed cells ofB. melaninogenicus. Increased incorporation of these fatty acids into CPE, CPG, 3KDS, and other phospholipids is significantly increased by the presence of glutamine or NADH. Thus, energization of the membrane of B. melaninogenicus by glutamine or the electron transport system by NADH or asparagine is required for sphingolipid and other phospholipid synthesis. The relationship of this energization to possible transport of sphingolipid precursors is discussed.

Glutamine-stimulated amino acid and peptide incorporation in Bacteroides melaninogenicus

“…Lev and Milford have shown that glutamine stimulates the incorporation of sphingolipid pret Present address: Department of Microbiology, Southern Mllinois University at Carbondale, Carbondale, IL 62901. cursors into complete sphingolipids in washed cells of B. melaninogenicus (10) and that the effect of glutamine is energy mediated. In the present study I show that glutamine stimulates incorporation of amino acids and peptides into the cell envelope.…”

“…B. melaninogenicus subsp. levii (ATCC 29147; isolated from bovine rumen [10]) was used in the following experiments. Cells were obtained from cultures grown in a Trypticase yeast extract basal medium supplemented with 0.025% hemolysed erythrocytes and 0.1 ,.g of vitamin K, per ml.…”

“…The supernatant was reduced in volume under a vacuum, and a sample was applied to a 0.1-mm cellulose thin-layer plate. This was developed with propan-2-ol-formic acid-water (10:2:10) (2), and the locations of the radioactive spots were determined by radioautography, as described previously (10).…”