Membrane-associated sialyltransferase complexes of Escherichia coli K-235 catalyze the synthesis of sialyl polymers which remain associated with the cell envelope. Sialyl monophosphorylundecaprenol is an intermediate in the formation of these unique surface structures, and fluidity of the lipid phase is required for the proper function of the enzyme complex (Troy, F.A., Vijay, I.K., and Tesche, N. (1975) J. Biol. Chem. 250, 156-163, 164-170). In membranes containing an increased unsaturated fatty acid content of the phospholipids, obtained by growing cells at 15 degrees C, synthesis of polysialic acid was uncoupled from synthesis of the sialyl lipid-linked intermediate. Using reconstruction experiments, the importance of the role of an endogenous acceptor in polymer formation was suggested by the unexpected finding that polysialic acid synthesis could be reactivated in inactive membranes by the addition of an exogenous acceptor which contained sialic acid. Concomitant with polymer synthesis was a rapid loss of labeled sialic acid from the lipid phase. The activated sialic acid was shown to be transferred directly to the exogenous acceptor. These results establish: 1) that the temperature-induced alteration in polymer synthesis resulted from the inability of cells grown at 15 degrees C to either synthesize or assemble a functional endogenous acceptor and not from a defect in the synthesis of the sialyltransferase; 2) the intermediate precursor role of lipid-soluble sialic acid in sialyl polymer synthesis; and 3) that the exogenous acceptor served directly as an "acceptor" and not as a catalytic "effector" which stimulated an inactive membrane-enzyme complex. These results are in accord with the possibility that the low temperature-induced derangement in polymer formation is a consequence of the altered lipid structure resulting from the greater unsaturated fatty acid content in the membrane phospholipids. U-14C-labeled exogenous acceptor was isolated from the culture filtrate of cells grown at 37 degrees C and purified to homogeneity by preparative polyacrylamide gel electrophoresis. The pure acceptor was characterized structurally as a homopolymer of sialic acid with a degree of polymerization of approximately 12. Potassium borohydride reduction of the acceptor prior to complete hydrolysis with neuraminidase established that the polymer possessed a free reducing terminus of sialic acid. Subsequent structural studies showed that these oligomers of sialic acid appeared in the culture filtrate as a result of acid-catalyzed hydrolysis from membrane-associated polysialic acids of about 150 to 200 sialyl residues. Marked diminution of several membrane proteins was observed for cells grown at 15 degrees C. The possible relationship of these alterations to the upward shift in unsaturated lipids and to the loss of a functional endogenous acceptor is currently under study.