Chemical and biological properties of an extracellular lipopolysaccharide from Escherichia coli grown under lysine-limiting conditions

Abstract

Lipopolysaccharide was prepared from the extracellular lipoglycopeptide produced by the lysine-requiring mutant Escherichia coli A.T.C.C. 12408 grown under lysine-limiting conditions. The lipid moiety, containing glucosamine phosphate and four fatty acids (lauric acid, myristic acid, beta-hydroxymyristic acid and palmitic acid) corresponded in composition to lipid A of known bacterial lipopolysaccharides. The components of the polysaccharide moiety were d-glucose, d-galactose, l-glycero-d-manno-heptose, 3-deoxy-2-oxo-octonic acid, ethanolamine and phosphate. These are the constituents of the polysaccharide of the cell-wall antigens from rough strains of E. coli. Lipopolysaccharides were also prepared from whole cells of E. coli 12408 grown with excess or limited amounts of lysine; they were identical in carbohydrate composition with the extracellular lipopolysaccharide. The biological properties of this material also resembled those of known lipopolysaccharides; it was antigenic, pyrogenic, toxic and had adjuvant activity.

Biological Studies on Free Endotoxin and a Non-toxic Material from Culture Supernatant fluids of Escherichia coli 078 K 80

“…The origin of these substances in culture fluids is uncertain, but it seems unlikely that they result from leaching or lysis of the bacteria during growth. Taylor et al (1966) showed that their lipopolysaccharide was excreted by a. rough lysine-dependent mutant when lysine was deficient in the medium, whereas the E. coli 078 K80 (used in the present work) was a smooth ovine enteropathogen without any unusual requirements. As Taylor et al (1966) found, the endotoxin in the culture fluid appeared to be derived by excretion from the bacteria rather than b y lysis or leaching.…”

“…Taylor et al (1966) showed that their lipopolysaccharide was excreted by a. rough lysine-dependent mutant when lysine was deficient in the medium, whereas the E. coli 078 K80 (used in the present work) was a smooth ovine enteropathogen without any unusual requirements. As Taylor et al (1966) found, the endotoxin in the culture fluid appeared to be derived by excretion from the bacteria rather than b y lysis or leaching. The non-toxic fraction probably arose similarly, although its rdation to endotoxin is not yet established (Marsh & Crutchley, 1967).…”

“…Shear & Turner (I 943) studied the tumour-necrotizing ability of their cell-free endotoxic lipopolysaccharide. Todd, Shaw, Blain & Boyle (1958), showed that bacteria grown in inorganic media liberated endotoxin into the culture fluid and, Taylor, Knox & Work (1966) showed that a lipopolysaccharide endotoxin was released into cultures by a lysine-requiring mutant of Escherichia coli. Our own results showed that in aerated Endotoxin of E. coli 419 liquid cultures of E. coli 078 K80, large amounts of endotoxin were liberated into the culture fluid, together with a non-toxic !entity which could be separated on DEAE cellulose and purified using Sephadex G-IOO (Marsh & Crutchley, 1967).…”

Purification and Physico-Chemical Analysis of Fractions from the Culture Supernatant of Escherichia coli O78K80: Free Endotoxin and a Non-Toxic Fraction

“…Shear & Turner, 1943) have isolated tumour-necrotizing substances (endotoxins) from supernatant fluids of cultures of Gram-negative bacteria, most other workers have preferred to extract endotoxins from the bacteria themselves. An exception has been Work and her collaborators (Bishop & Work, 1965, Taylor, Knox & Work, 1966' who found endotoxic lipopolysaccharides in culture supernatant fluids of lysinerequiring mutants of Escherichia coli grown under lysine-limited conditions.…”

“…Shear & Turner, 1943) have isolated tumour-necrotizing substances (endotoxins) from supernatant fluids of cultures of Gram-negative bacteria, most other workers have preferred to extract endotoxins from the bacteria themselves. An exception has been Work and her collaborators (Bishop & Work, 1965, Taylor, Knox & Work, 1966' who found endotoxic lipopolysaccharides in culture supernatant fluids of lysinerequiring mutants of Escherichia coli grown under lysine-limited conditions.The release of large quantities of free endotoxin into the culture supernatant fluids of Gram-negative bacteria, under favourable growth conditions, has recently been described (Crutchley, Marsh & Cameron, 1967a). The present paper will deal with the fractionation of supernatant fluids of cultures of Escherichia coli serotype 078K80 and the subsequent physical and chemical characterization of the fractions ; the accompanying paper (Crutchley et al 1967 b) will discuss the biological properties of these fractions.…”