The Role of Cell-Wall Lipid in the Biosynthesis of Bacterial Lipopolysaccharide

Abstract

In gram-negative enteric bacteria, the cell envelope is composed largely of protein glycopeptide, lipopolysaccharide, and in addition, substantial amounts of lipid.1 The lipopolysaccharide contains the somatic 0-antigens as well as the structures responsible for endotoxin activity,2 and is distinct from the lipid fractions obtained by usual lipid extraction procedures. Until now, no biologic activity has been established for these lipid fractions, and their relation to the other components of the intact cell envelope has been unknown.In the present communication we present evidence that a specific interaction occurs between the lipopolysaccharide and the phospholipid component of the cell envelope, and that this interaction is essential for the enzymatic reactions leading to the synthesis of the polysaccharide portion of the lipopolysaccharide.Biosynthesis of the lipopolysaccharide has been studied in mutant strains which are unable to synthesize specific nucleotide sugars and which, therefore, produce incomplete lipopolysaccharides.3' 4 In Salmonella typhimurium, these studies have revealed the existence of several soluble enzymes which catalyze the sequential transfer of specific sugars into the growing lipopolysaccharides.6 Two of these reactions can be described as follows: Glucose-deficient lipopolysaccharide + UDP-glucose Glucosyl-lipopolysaccharide (+UDP) (1) Glucosyl-lipopolysaccharide + UDP-galactose -* Galactosyl-glucosyl-lipopolysaccharide (+UDP). (2) In reaction (1), the incorporation of glucose into the lipopolysaccharide is catalyzed by the enzyme UDP-glucose lipopolysaccharide glucosyl transferase, and the acceptor is obtained from a mutant deficient in UDP-glucose. In reaction (2), the incorporation of galactose is catalyzed by the enzyme UDP-galactose lipopolysaccharide galactosyl transferase, and the acceptor is derived from a mutant deficient in UDP-galactose.The major portion of these enzyme activities is found in the particulate cell envelope fraction, which also contains the acceptor lipopolysaccharide, but significant enzyme activity is also present in the soluble fraction. The soluble enzymes show an absolute requirement for the addition of the appropriate acceptor. In previous studies we have used the crude cell envelope fractions, heated to destroy their endogenous enzyme activity, as acceptors. It has been demonstrated that the monosaccharide units are transferred into the deficient lipopolysaccharides,5 but the lipopolysaccharides themselves, separated from the other components of the cell envelope by phenol extraction and purified by Mg++ precipitation and dialysis, do not act as acceptors in the transferase reactions. This unexpected finding suggested a requirement for an additional component of the cell envelope. 939

Genetic and physiological regulation of intrinsic proteins of the outer membrane of Salmonella typhimurium

“…Strain SA536 (Hfr K6, serA13) was obtained from K. Sanderson. The enriched medium was proteose peptone-beef extract (15); minimal medium (M9, reference 3) contained 22 mM Na.2HPO4, 22 mM KH.,PO4, 18 mM NH4Cl, 8.5 mM NaCl, 1.9 mM MgSO4, and 0.1 mM CaCl2, supplemented with either 0.4% D-glucose, Dgalactose, or glycerol; solid media contained 1.5% agar. Conditioned medium was prepared by growth of strain G30 in M9-glucose until the optical density at 600 nm reached 0.8 to 1.0 units.…”

Identification by mass spectrometry of glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative, produced by Pseudomonas aeruginosa

“…From the late 1960s to the 1970s, bacterial lipid composition and metabolism were highly studied, as shown by the numerous published reviews during this period . These works eventually led to an attempt to classify bacteria from their lipid composition and particularly the fatty acid chains involved in this composition .…”

Morphogenesis of the bacterial division septum: a new class of septation-defective mutants.

“…Strain R5312R was a spontaneous revertant of R53 that had regained the wild-type phenotype. Bacteria were grown at 370 in glucose-supplemented minimal medium containing 2.5% proteose peptone-beef extract (2) England Nuclear) to 20 ml of culture. After two to three generations of exponential growth, during which steady-state labeling was achieved, 10 mM sodium azide was added, the cells were collected by centrifugation at 1000 X g for 10 min, and immediately frozen.…”