The Biosynthesis of Cell Wall Lipopolysaccharide in Escherichia coli: VI. ENZYMATIC TRANSFER OF GALACTOSE, GLUCOSE, N-ACETYLGLUCOSAMINE, AND COLITOSE INTO THE POLYMER

Abstract

Escherichia coli J-5, a uridine diphosphate galactose 4-epimeraseless mutant of Escherichia coli 0111-B4, produces a cell wall lipopolysaccharide which lacks galactose and colitose (3,6-dideoxy- l -xylohexose) and contains reduced quantities of glucose and N-acetylglucosamine. With the use of a particulate fraction of the cell envelope of this mutant (which contains glycosyl transferases and lipopolysaccharide acceptor), the transfer of galactose, glucose, N-acetylglucosamine, and colitose to the incomplete lipopolysaccharide was demonstrated. The addition of the various sugars to the acceptor apparently occurred in a sequential manner as indicated by the following observations: (a) galactose was transferred directly to the polymer from uridine diphosphate galactose; (b) transfer of glucose (from uridine diphosphate glucose) occurred only in the presence of uridine diphosphate galactose; the product of these reactions was released from the lipopolysaccharide by hydrolysis and shown to be a newly synthesized glucosylgalactose disaccharide; (c) in a similar manner, N-acetylglucosamine transfer from uridine diphosphate N-acetylglucosamine to the polymer required the prior transfer of both galactose and glucose; (d) colitose was transferred (from guanosine diphosphate colitose) to the polymer in small, but reproducible, amounts only when the nucleotide derivatives of galactose, glucose, and N-acetylglucosamine were included in the incubation mixture; and (e) no evidence was obtained for the formation of oligosaccharide intermediate in any of the transferase systems studied. It is suggested that the transfer of galactose, glucose, and N-acetylglucosamine to the mutant polymer pertains to the biosynthesis of a “core” polysaccharide that has a structure similar to that observed in Salmonella. In addition, small amounts of colitose were incorporated into lipopolysaccharide when GDP-colitose-14C was incubated with the particulate enzyme preparation in the presence of UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine.