Respiratory Pathways in the Mycoplasma

SMITH, S. L. (Cornell University, Ithaca, N.Y.), P. J. VAN DEMARK, AND J. FABRICANT. Respiratory pathways in the Mycoplasma. I. Lactate oxidation by Mycoplasma gallisepticum. J. Bacteriol. 86:893-897. 1963.-Resting cells of Mycoplasma gallisepticum 293 required the addition of nicotinamide adenine dinucleotide, thiamine pyrophosphate, and flavine mononucleotide for the maximal rate of sodium lactate oxidation. Inhibitor studies, as well as spectrophotometric and chemical assays, indicate that the pathway of electron transport to oxygen during lactate oxidation does not involve heme catalysts, and is mediated by flavin-linked enzyme systems. The presence of reduced nicotinamide adenine dinucleotide-specific lactic dehydrogenase, menadione reductase, ferricyanide reductase, and reduced nicotinamide adenine dinucleotide oxidase activities was detected in cell-free extracts. No cytochrome c reductase or reduced nicotinamide adenine dinucleotide peroxidase activity was detected in these extracts. It has been shown by various workers (Rodwell and Rodwell, 1954; Tourtellotte, 1960; Neimark and Pickett, 1960) that glycolysis represents the major metabolic pathway in the fermentative Mycoplasma with lactic acid being the major end product under anaerobic conditions. However, with the exception of these studies, little is known concerning the respiratory pathways and energyyielding mechanisms of the fermentative pleuropneumonia-like organisms (PPLO). Since lactate is a substrate readily used by most strains of Mycoplasma, a study of its oxidation by a number of strains of PPLO has been undertaken in our laboratory. The present paper is a report of the studies of lactate oxidation by M. gallisepticum (Edward and Kanarek, 1960) 293. MATERIALS AND METHODS Culture. M. gallisepticum 293 originally was isolated in 1956 from a field case of chronic respiratory disease in poultry (Calnek and Levine, 1957). Its serological characteristics were described by Fabricant (1960). Since isolation, the organism has been maintained on laboratory media in continuous subculture for over 250 transfers. For this study, the organism was grown from a 1% inoculum in a broth medium consisting of 2.5% beef heart infusion, 10% horse serum, 1% yeast hydrolysate, 0.05% thallium acetate, and 1 million units per liter of penicillin on a reciprocal shaker at 37 C. The cells were harvested after 48 hr by centrifugation at 12,000 X g for 20 min and washed twice in distilled water. Resting-cell suspensions were prepared by resuspending the cells in approximately ',Jo of the original growth volume of distilled water. Cell-free extracts were prepared by sonic oscillation of a 10% suspension of cells in a 0.5% glutathione solution in a 10-kc oscillator for 10 min. The cell debris was removed by centrifugation at 20,000 X g for 1 hr. Manometric procedures. The oxidation of sodium lactate was followed by conventional manometric methods with air as the gas phase at 37 C; each Warburg vessel contained 100 ,umoles of phosphate buffer (pH 7.0), 50 ,umoles of sodium lactate, and approximately 3 mg of cells, with a total volume of 3.0 ml per flask. The center well of each vessel contained .15 ml of 40% KOH to absorb carbon dioxide. Spectrophotometric and chemical assays. Nicotinamide adenine dinucleotide (NAD)-linked enzymes were assayed spectrophotometrically by measuring the change in absorbancy at 340 m, with a Beckman model DU spectrophotometer at 27 C. The reduction of ferricyanide was determined at 400 m,u and cytochrome c at 550 m,u. Catalase activity was determined by measuring 893 on O cber 7, 2017 by gest http/jb.asm .rg/ D ow nladed fom SMITH, VAN DEMARK, AND FABRICANT