Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 11 Dec 2018 16:06:36 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1985, p. 497-501 0020-7713/85/040497-05$02 .OO/O Copyright zyxwvutsrq 0 1985, International Union of Microbiological Societies Vol. 35, No. 4 Distinctions in Mollicutes Purine Metabolism: Pyrophosphate-Dependent Nucleoside Kinase and Dependence on Guanylate Salvage VICTOR V. TRYON? AND J. DENNIS POLLACK* Department zyxwvutsr of Medical Microbiology and Immunology, The Ohio State University, Columbus, Ohio 43210 Cell preparations of Acholeplasma jlorum LIT, Acholeplasma axanthum S743T, Acholeplasma granularum BTS-39T, Spiroplasma jloricola zyxwvut 23-6T, Mycoplasma gallisepticum S6, and Mycoplasma arginini G230T were examined for 13 cytoplasmic enzyme activities involved in the salvage and interconversion of nucleobases, nucleosides, and 5’-mononucleotides.The unique pyrophosphate-dependent nucleoside kinase activity known only in Acholeplasma laidlawii B-PG9 was found in A . axanthum and A . granularum. All the organisms could be divided into three groups based upon their patterns of purine salvage and interconversion activities. All the tested organisms, A. laidlawii, and apparently Mycoplasma mycoides subsp. mycoides lack the ability to synthesize guanylates from other purine mononucleotides, indicating that these members of the class Mollicutes can only salvage guanylates. The plant epiphytes A. florum and S. jloricola had an identical purine enzyme pattern which was different from those of all other members of the Mollicutes studied. Mollicutes (mycoplasmas) is a class of procaryotes char- acterized by lack of a cell wall, complex nutritional require- ments, and small genome of generally low guanine-plus- cytosine ratio. Nutritional studies on representatives from each of the three families within the class demonstrate the absolute requirement for nucleobases or nucleosides for growth (3, 5, 8, 11, 17-21, 23). The nutritional requirement for purine precursors suggests the lack of a functional pathway for the de novo synthesis of purines and the dependence on salvage and interconversion enzymes for the biosynthesis of purine nucleotides (11, 24). Hamet et al. (6) examined purine salvage but not nucleotide interconversion activity in a number of Mycoplasma species. Only in Mycoplasma mycoides subsp. mycoides (11, 12), a species not generally available for study in the United States, and Acholeplasma laidlawii B-PG9 (24) have the pathways in- volved in purine salvage and interconversion been com- pletely described. Here we describe our examination of the class Mollicutes for the unique pyrophosphate (PPJ-dependent nucleoside kinase activity which is only known in A. laidlawii B-PG9 (24). When first reported, we speculated that the PPi- dependent enzyme activity might prove useful in the study of the phylogenetic relationships of the Mollicutes. We now have studied six other representative Mollicutes for this PPi-dependent activity and for 12 other cytoplasmic enzyme activities involved in the salvage and interconversion of purine bases, ribonucleosides, and 5‘-monophosphates. MATERIALS AND METHODS Organisms. Acholeplasma florum LIT, Acholeplasma axanthum S743T, Acholeplasma granularum BTS-39T, and Spiroplasma joricola 23-6T were obtained from J. G. Tully, National Institutes of Health, Frederick, Md. Mycoplasma gallisepticum S6 and Mycoplasma arginini G230T were obtained from our stock collection. Media and growth conditions. All organisms were grown in * Corresponding author. t Present address,: Department of Microbiology, University of Texas Health Sciences Center at San Antonio, San Antonio, TX 78284. our modification of Edward medium (1). Media were supple- mented with 2% (vol/vol) (acholeplasmas) or 5% (vol/vol) (mycoplasmas and spiroplasma) heat-inactivated horse se- rum (lot number 200031; KC Biologicals, Lenexa, Kans.). Media for the growth of M . arginini were supplemented with 0.3% (wtlvol) L-arginine hydrochloride (Sigma Chemical Co., St. Louis, Mo.). Organisms were incubated statically at 37°C until mid-log phase of growth (1 to 3 days). Cells were harvested as previously described (1, 14). Cells in dilute (1:20) buffer (13) were lysed osmotically or sonicated for 1- to 30-s pulses (Sonifier Cell Disruptor; Heat Systems, Melville, N.Y.) while on wet ice. Cell extracts were frac- tionated by centrifugation, dialyzed, and prepared as we described for A. laidlawii B-PG9 (14, 24). Purity of the fractions was monitored by localization of reduced nicotinamide adenine dinucleotide oxidase (13,14) and aden- osine triphosphatase (EC 3.6.1.3) (24) activities. Assay. The activities of 13 cytoplasmic enzymes involved in the salvage and interconversion of nucleobases, nucleo- sides, and 5’-mononucleotides were assayed as described for A. laidlawii B-PG9 (24). Briefly, l4C-1abeled purine sub- strates were mixed with cofactors at pH 7.5. Dialyzed cell extract containing 20 to 40 pg of protein (2) was added to start the reaction. The reaction mixtures were then incu- bated at 37°C with shaking for 4 to 60 min. Incubation times were selected such that greater than 50% of the starting labeled substrate remained at the end of the reaction period. In preliminary trials, the velocity of each of the reactions was shown to be linear over the prescribed time period and conditions (24). Reactions were terminated by heating at 95°C for 2 min. Samples (20 pl) of the heated reaction mixtures were spotted onto polyethyleneimine-cellulose plates (Analtech, Inc., Newark, Del.) with 10 pg each of nonradioactive standards (24). 14C-labeled substrate and product were separated by thin-layer chromatography in polar solvents (17, 24). Resolved purines were visualized by ultraviolet light, scraped into counting fluid, and assayed for radioactivity. In all cases, greater than 90% of the applied label was recovered from substrate and product spots. During initial experiments, all plates were autoradiographed after chromatography to help exclude the presence of com- peting reactions. Radioisotope data were corrected for 497