The Nutrition and Metabolism of Mycoplasma: Progress and Problems

Abstract

Part A MetabolbmResults of earlier work on the carbohydrate metabolism have been summarized (Rodwell, 1960). The observation that glucose is not metabolized anaerobically by cell suspensions, although lactate is the major produce of glucose breakdown during growth in nonaerated cultures, was puzzling.It was suggested that glycerol oxidation might proceed by a flavoproteincatalyzed oxidation of L-glycerol-3-phosphate (GP) . Because glycerol is an essential nutrient and glucose is not fermented by cell suspensions, it was postulated that this pathway must be essentially irreversible, and that no other freely reversible NAD-linked pathway exists from hexose to GP. It has since been found that, when glycerol is the growth-limiting nutrient, glycerol carbon is diluted with carbon derived from glucose (Plackett, unpublished). The nature of the terminal respiratory system catalyzing the transfer of electrons from reduced nicotinamide adenine dinucleotide (NADH) to oxygen was not investigated, although no cytochrome system could be found (Rodwell and Rodwell, 1954a).The carbohydrate and oxidative metabolism was therefore reexamined. The aims were to explain why glucose is fermented during growth in nonaerated cultures but not by washed cell suspensions, how glucose carbon enters glycerol compounds and to determine the nature of the terminal respiratory system. METHODSOrganisms. Strain V5 of M. mycoides and a goat mycoplasma (strain Y, Laws, 1956) were used. Strain Y (referred to as strain G Y in earlier publications) is closely related serologically and biochemically to M. mycoides. It differs from strain V5 in that it grows almost as well in nonaerated cultures as in aerated ones, but no differences have been observed in its fermentative or respiratory activities under the two conditions. Cultural Conditions. The growth medium was devised by Buttery (unpublished). Its composition in amounts per 1 is: "Panmede" (Paines and Byrne Ltd., Greenford, England) 30 g; Na2HP04 (anh) 10 g; glucose 4 g; ox serum 100 ml; adenine 12.5 mg; guanine 12.5 mg; sodium oleate 0.1 m-mole; sodium palmitate 0.1 m-mole and glycerol 1.7 m-mole. The pH is adjusted to 7.9 with sodium hydroxide and the medium sterilized by Seitz filtration. Cultures were grown in 500 ml Florence flasks containing 100 ml medium and rotated at 100 rpm during incubation (rotated flask cultures) or in 150 ml Erlenmayer flasks containing 100 ml medium and incubated without rotation (static cultures). The inoculum was 1 .O ml and the flasks were incubated for 16-1 8 hr for strain Y and for 20-24 hr for the slower-growing strain V5. Glucose is oxidized to acetate and carbon dioxide in rotated flask cultures, lactate is the major product of glucose breakdown in static cultures.

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

“…Viable cell counts, W; turbidity, 0 ; and SH thymidine incorporation, A, were determined on replicate tubes during incubation at 37". Growth-promoting properties of serum albumin It was concluded (Rodwell, 1967) that in medium Cr, in which unesterified fatty acids are added in a total concentration of 0.1 mM, albumin is required to bind fatty acids. The concentration of BSA required for growth increased with increasing fatty acid concentrations, and albumin was not required when the concentration of fatty acids was decreased below a critical value as in medium D. Mycoplasmacells are rapidly lysed by long chain monoenoic fatty acids in concentrations lower than those required for optimum growth, and lysis is prevented by BSA (Fig.…”

“…Medium C contained BSA to bind fatty acids (Rodwell, 1967). It is likely that, in this medium, cholesterol is dispersed by an interaction with fatty acids.…”

“…Deficiencies of glycerol, cholesterol or fatty acids resulted in marked morphological changes, cell death and lysis (Rodwell & Abbot, 1961). Medium C (Rodwell, 1967) containing defatted bovine serum albumin (BSA) and a pronase digest of BSA as undefined components, gave good growth of strain Y, and was used to study the fatty acid growth requirements. The problem of supplying fatty acids in a non-toxic form, and of cholesterol in an available form, is considered further in this paper.…”

A Defined Medium for Mycoplasma Strain Y

“…Strain Y, isolated from a goat (Laws, I 956), resembles Mycoplasma mycoides in its nutrition and metabolism (Rodwell, 1960(Rodwell, , 1967, but differs in that its growth is not improved by aeration. The following strains were also used: v5, GLADYSDALE and KH,J of M .…”

“…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.…”