STUDIES ON THE METABOLISM OF ARGININE BY MYCOPLASMA

This report describes briefly some studies, done in part with the collaboration of Michael Barile, on the biochemical and physiological significance of arginine utilization for energy generation by mycoplasma. Historically, the first indication of the physiological importance of arginine for mycoplasma was the finding of Schlesinger and his group,' as well as that of Kenney and Pollock,2 indicating that the rapid depletion of arginine in mammalian cell culture medium was due to contamination by mycoplasma. Schimke and Bade3 showed that the breakdown of arginine to ornithine in contaminated cell cultures was an extensive process that occurred by way of the so-called arginine dihydrolase pathway. This pathway is well known in streptococci and certain other bacteria,4 and was first demonstrated in mycoplasma by Smith.s The arginine dihydrolase pathway is widespread among mycoplasma species, being present in 10 of 18 species of mycoplasma tested, including those of human, bovine, avian, murine, swine, goat, and cell culture origin.6 Human strains having this pathway include M . horninis types 1 and type 2 ( M . artliritidis), M . orale, and M . salivarium. M . fermentans and M . pneumoniae do not contain the enzyme pathway. The arginine dihydrolase pathway, shown in FIGURE 1, comprises three enzymes: (1) arginine deiminase, which hydrolyzes arginine to citrulline, (2) ornithine transcarbamylase, which forms carbamyl phosphate and ornithine, (3) carbamate kinase, which synthesizes ATP from ADP and carbamyl phosphate. In streptococci, the metabolism of arginine by this pathway results in the formation of energy (ATP) used for growth.' I should like to outline evidence supporting the concept that the utilization of arginine by this pathway, with the concomitant synthesis of ATP, can constitute a major energy source for mycoplasma species containing this pathway. Dependence of Growth on Arginine Content of Medium. FIGURE 2 shows a typical experiment with M. hominis 07, showing that in a brain-heart infusion broth containing 10% horse serum, growth ceases at a point at which arginine is totally depleted from the medium.8 If, initially or later as indicated by the arrow, additional arginine is added, growth proceeds to a new plateau where it is limited for unknown reasons. No arginine analogue, citrulline, ornithine, or a variety of other compounds, including glutamine, glycerol, pyruvate, succinate, acetate, or /3-hydroxybutyrate, was as effective in promoting growth as arginine. The fact that a compound does not stimulate growth could be due to a lack of the required enzymes, or to a lack of permeability. In the case of citrulline, the requisite enzymes are preesnt-i.e., ornithine transcarbamylase and carbamate kinase. Yet citrulline ,is ineffective in promoting growth because it does not appreciably enter the cell^.^^^ Similar results of a growth dependence on arginine have been obtained with other mycoplasma species containing the arginine dihydrolase pathway, including M. hominis type 1, cell culture strain ERKS, avian strain NTF, and M. orale. Relationship between Arginine Degraded and A TP Requirement for Growth. During growth of certain mycoplasma strains, there is an extensive conversion of arginine to ornithine.3 In the case of M. hominis 07, 90% of the arginine utilized