Distinctions in Mollicutes Purine Metabolism: Pyrophosphate-Dependent Nucleoside Kinase and Dependence on Guanylate Salvage

Abstract

Cell preparations of Acholeplasma jlorum LIT, Acholeplasma axanthum S743T, Acholeplasma granularum BTS-39T, Spiroplasma jloricola 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 characterized by lack of a cell wall, complex nutritional requirements, and small genome of generally low guanine-pluscytosine 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][18][19][20][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 involved in purine salvage and interconversion been completely 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 PPidependent 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 METHODSOrganisms. 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 ). Organisms were incubated statically at 37°C until mid-log phase of grow...

Metabolism of Members of the Spiroplasmataceae

“…This reaction may be involved in maintaining oxaloacetate levels and hence may affect malate dehydrogenase activity, as well as the concentration of cellular nicotinamide adenine dinucleotide and NADH. There is no de novo synthesis of purines in members of the Mollicutes, so aspartate presumably does not contribute its nitrogen to the purine ring, but the nitrogen of aspartate has been reported to be transferred to the amino group of inosine monophosphate in the synthesis of adenosine monophosphate by extracts of a number of mollicute species (52).…”

“…Adenosine monophosphate, inosine monophosphate, and guanosine monophosphate are known to be synthesized in mollicutes in the following ways: (i) in a one-step reaction, from the respective nucleobase and phosphoribosyl pyrophosphate, and (ii) in a two-step reaction (in the first part, ribose 1-phosphate or deoxyribose 1-phosphate is used to form the ribo-or deoxyribonucleoside, and then in the second part, ATP or PP, is used as the orthophosphate donor to form the ribo-or deoxyribomononucleotide) (52). All spiroplasmas were able to synthesize these mononucleotides by the one-step reaction or by the two-step reaction in which PP, was used as the orthophosphate donor.…”

“…Other reports have described optimal growth responses to various additives in formulations of semidefined and defined media (2,4,5, 18, 22, 24,52). A number of studies have reported the chemical contents and the processing or appearance of radioactivity from either lipids or lipid precursors into membrane components of growing spiroplasmas (3,13,15, 23, 31, 32) or the uptake of compounds such as [14C]thymidine (50), [32P]phosphate, or 14C-labeled amino acids (1).…”

Purine and Pyrimidine Metabolism in Mollicutes Species

“…Disintegrations per minute were converted to picomoles of product by using the specific activity values supplied by the manufacturer. For purines, we estimated that we could detect 0.3 pmol of product synthesized per min per mg of protein, a value which is at least 10-fold greater than the value which we could previously detect (39).…”

“…In cytoplasmic fractions we studied 37 enzyme activities involved in the salvage and synthesis of purine and pyrimidine nucleobases, ribonucleosides, deoxyribonucleosides, ribomononucleotides , and deoxyribomononucleotides. Specific radioactive assays for the purine reactions were performed as previously described (19,38,39), but with modifications. For example, the incubation time for the assay for 5'-nucleotidase activity was reduced to 2 to 5 min from 4 to 8 min.…”