The Supply of Cholesterol and Fatty Acids for the Growth of Mycoplasmas

Abstract

S U M M A R YMycoplasma strain Y, serologically and biochemically related to Mycoplasma mycoides, grew well in a medium containing unesterified fatty acids, cholesterol and some samples of fatty acid-poor bovine serum albumin. With other samples of serum albumin, growth was accompanied by cellular lysis. Heating albumin solutions, treating them with charcoal, or varying the ratio of fatty acids to albumin, the concentration of cholesterol, or the order in which the lipid components were added to the medium, all influenced the amount of lysis. These effects may be due to varying interaction between fatty acids and cholesterol and between fatty acids and albumin. When fatty acids are firmly bound to albumin, their interaction with cholesterol is prevented, resulting in a cholesterol deficiency. Lysis was prevented by the addition of a heat-stable defatted serum protein fraction (fraction C), or a pronase digest of fraction C, both of which dispersed cholesterol.

A Defined Medium for Mycoplasma Strain Y

“…Growth was measured by turbidity at 660 mp; by the incorporation of 3H thymidine (Rodwell, 1969), or by protein determination, by the method of Lowry, Rosebrough, Farr & Randall (1951) with crystalline bovine serum albumin dried to constant weight as standard, in cells washed in 0.25 M-sodium chloride+0.02 M-sodium phosphate (pH 7.4) + 0.01 M-magnesium sulphate.…”

“…Media C, Cz I and C2 were as described (Rodwell, 1967(Rodwell, , 1969. Medium C3 had the same composition as medium C except that the concentrations of glycerol and of 'fatty acid poor' BSA were increased to 0.02 M and 2-7 g./l.…”

“…Cultures of strain Y were incubated vertically without agitation at 37'; cultures of the other strains were incubated in the same way and also in an inclined rack which was rotated at 10 rev./min. (rotated tube cultures).Growth was measured by turbidity at 660 mp; by the incorporation of 3H thymidine (Rodwell, 1969), or by protein determination, by the method of Lowry, Rosebrough, Farr & Randall (1951) with crystalline bovine serum albumin dried to constant weight as standard, in cells washed in 0.25 M-sodium chloride+0.02 M-sodium phosphate (pH 7.4) + 0.01 M-magnesium sulphate.In some cases, an accumulation of pyruvate during growth suggested a defect in the pyruvate oxidase system, and cells were then examined manometrically for pyru- …”

“…Growth assays. These were performed as described previously (Rodwell, 1969). Cultures of strain Y were incubated vertically without agitation at 37'; cultures of the other strains were incubated in the same way and also in an inclined rack which was rotated at 10 rev./min.…”

The Effect of Straight-chain Saturated, Monoenoic and Branched-chain Fatty Acids on Growth and Fatty Acid Composition of Mycoplasma Strain Y

“…Growth in medium C was, in some cases, limited by the availability of cholesterol. Better growth was obtained in a modified medium (medium C 2 ) containing in addition a pronase-treated defatted serum protein fraction with cholesterol-dispersing activity (Rodwell, 1969). While it was possible to alter and control the fatty acid composition of the membrane lipids to a large extent by altering the fatty acids supplied for growth in medium C 2, the lipids always contained fatty acids other than those added, in amounts varying from 4 to 8 % of the total.…”