Biosynthesis of bacterial lipopolysaccharide, I. Enzymatic incorporation of galactose in a mutant strain of Salmonella

MJ Osborn, SM Rosen, L Rothfield… - Proceedings of the …, 1962 - National Acad Sciences
MJ Osborn, SM Rosen, L Rothfield, BL Horecker
Proceedings of the National Academy of Sciences, 1962National Acad Sciences
Although the mechanism of biosynthesis of homopolysaccharides, such as
glycogen,'dextran, 2 and starch, 3 has been studied in some detail, relatively little attention
has been given to the biosynthesis of the heteropolymeric mucopolysac-charides and
lipopolysaccharides elaborated by animals and microorganisms. A promising approach to
this problem has arisen out of recent work in several labora-tories4'I on the in vitro
biosynthesis of the complex capsular polysaccharides of gram-positive cocci. In addition …
Although the mechanism of biosynthesis of homopolysaccharides, such as glycogen,'dextran, 2 and starch, 3 has been studied in some detail, relatively little attention has been given to the biosynthesis of the heteropolymeric mucopolysac-charides and lipopolysaccharides elaborated by animals and microorganisms. A promising approach to this problem has arisen out of recent work in several labora-tories4'I on the in vitro biosynthesis of the complex capsular polysaccharides of gram-positive cocci. In addition, advances6-8 in structural analysis of the lipo-polysaccharides of gram-negative bacilli have stimulated considerable interest in the mechanism of biosynthesis of these very complex heteropolymers. The cellwall lipopolysaccharides of the enteric bacteria determine the specific somatic (0) antigenicity of these organisms, and form the basis of their serological classification; The lipopolysaccharides of Salmonella may be composed of as many as seven dif-ferent sugars, including such rare sugars as aldoheptose and 3, 6-dideoxyhexoses, 6 and may account for as much as ten per cent of the dry weight of the cell. The present communication describes an enzyme system, derived from a mutant strain of S. typhimurium, which appears to be involved in the biosynthesis of the cellwall lipopolysaccharide.
Mutants of Escherichia and Salmonella lacking the enzyme UDP-galactose-4-epimerase characteristically produce an abnormal cell-walllipopolysaccharide, which is distinguished from the wild type by the absence of galactose as well as certain other components of the normal polysaccharide. 9'10 Fukasawa and Nikaido'0 have shown that the defect in cell-wall biosynthesis is primarilythe result of the inability of the mutant to synthesize the precursor of cell-wall galactose, UDP-galactose, in the absence of an exogenous supply of the sugar. The block in UDP-galac-tose synthesis can be bypassed bygrowthin the presence of galactose, and under these conditions the composition of the lipopolysaccharide is similar to that of the wild type. We have confirmed these observations with an epimeraseless mutant of Salmonella typhimurium. The lipopolysaccharide of the wild type has been shown6 to containglucose, galactose, mannose, rhamnose, abequose (3, 6-dideoxy-D-galactose), and an unidentified aldoheptose. In contrast, only glucose and the aldoheptose can bedetected in hydrolysates of the mutant cell wall. Glucosamine is also present in both wild type and mutant but appears to be associated exclusively with the lipid component of the lipopolysaccharide. A particulate enzyme system isolated from extracts ofthe mutant strain catalyzes the transfer of galactose from UDP-galactose into a material which appears to be identical with the cell-wall lipopolysaccharide. Some properties of this UDP-galactose-lipopolysaccharide transferase and partial characterization of the product of the reaction are reported here. The system resembles that recently isolated from S. enteriditis by Nikaido. 11 Materials and Methods.-1. Chemicals: Galactose-C'4, uniformly labeled, was obtained from
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