Cell extracts of Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, and Mycoplasma gallisepticum S6 were examined for 37 cytoplasmic enzyme activities involved in the salvage and biosynthesis of purines. All of these organisms had adenine phosphoribosyltransferase activity (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8). All of these organisms had purine-nucleoside phosphorylase activity (EC 2.4.2.1) in the synthetic direction using ribose-1-phosphate (R-1-P) or deoxyribose-1-phosphate (dR-1-P); this activity generated ribonucleosides or deoxyribonucleosides, respectively. The pyrimidine nucleobase uracil could also be ribosylated by using either R-1-P or dR-i-P as a donor. The synthesis of deoxyribonucleosides from nucleobases and dR-1-P has been reported from only one other procaryote, Eschenichia coli (L. A. Mason and J. 0. Lampen, J. Biol. Chem. 193:539-547, 1951

Cell-free extracts from 10 strains of Spiroplasma species were examined for 67 enzyme activities of the Embden-Meyerhof-Parnas pathway, pentose phosphate shunt, tricarboxylic acid cycle, and purine and pyrimidine pathways. The spiroplasmas were fermentative, possessing enzyme activities that converted glucose 6-phosphate to pyruvate and lactate by the Embden-Meyerhof-Parnas pathway. Substrate phosphorylation was found in all strains. A modified pentose phosphate shunt was present, which was characterized by a lack of detectable glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities. Spiroplasmas could synthesize purine mononucleotides by using pyrophosphate (PP,) as the orthophosphate donor. All spiroplasmas except Spiroplusmu floricolu used adenosine triphosphate (ATP) to phosphorylate deoxyguanosine; no other nucleoside could be phosphorylated with ATP by any spiroplasma tested. These results contrast with those reported for other mollicutes, in which PP, serves as the orthophosphate donor in the nucleoside kinase reaction. The participation of ATP rather than PPi in this reaction is unknown in other mollicutes regardless of the nucleoside reactant. Deoxypyrimidine enzyme activities were similar but varied in the reactions involving deamination of deoxycytidine triphosphate and deoxycytidine. All Spiroplasma spp. strains had deoxyuridine triphosphatase activity. Uridine phosphorylase activity varied among strains and is possibly group dependent. As in all other mollicutes, a tricarboxylic acid cycle is apparently absent in Spiroplasma spp. Reduced nicotinamide adenine dinucleotide oxidase activity was localized in the cytoplasmic fraction of all Spiroplusmu species tested. Our assays indicate that the members of the Spiroplasrnataceae are essentially metabolically homogeneous in the highly conserved pathways which we studied, but differ from other mollicutes in several important respects. These differences are of probable phylogenetic significance and may provide tools for recognition of higher taxonomic levels of mollicutes.The metabolism of the wall-less, helical, sterol-requiring members of the Spiroplasmataceae has been little studied. The insect and plant habitats of the spiroplasmas, as their unique phylogenetic position indicates (59), suggested to us that the metabolism of these organisms might differ from that described for other members of the class Mollicutes (10, 17,39,45,55). Identification of such metabolic differences would aid in the characterization, classification, and study of the phylogeny of the spiroplasmas. This information can also identify metabolic steps or loci that are susceptible to chemical modulations that could inhibit the spiroplasmal diseases of corn, citrus, or other plants.Most reports relating to the metabolism of spiroplasmas have concerned nutrition, noting, for example, the presence or absence of acid produced during growth with various sugars (8, 20,43,44,58). Other reports have described optimal growth responses to various additives in formulations of semi...

We studied the purine enzyme activities in dialyzed cytoplasmic extracts from the following eight species, representing four genera, of Mollicutes: Mycoplasma pneumoniae FHT (T = type strain) and M129, Mycoplasma bovigenitalium PG-1lT, Mycoplasma hominis PG-21T and 1620, Mycoplasma genitalium G-37T, Mycoplasma hyopneumoniae JT, Ureaplasma urealyticum T960T, Spiroplasma citri Mar0c-R8A2~, and Anaeroplasma intermedium 5LA. In an investigation of purine nucleoside kinase activity we also included M . hominis 13408, 10144, 13428, 1612, 1184, and Botte. All of these Mollicutes species except U . urealyticum had purine phosphoribosyltransferase activity for adenine, hypoxanthine, and guanine; U . urealyticum had only adenine phosphoribosyltransferase activity. All of the organisms had nucleoside phosphorylase activity which used either ribose 1-phosphate or deoxyribose 1-phosphate and adenine, hypoxanthine, or guanine for the synthesis of nucleosides and adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, or deoxyinosine in the reverse direction. All had 5'-nucleotidase activity for adenosine monophosphate, deoxyadenosine monophosphate, inosine monophosphate, or guanosine monophosphate. Only M. hominis 1620,13408,10144, and 13428, A. intermedium, and S . citri had pyrophosphate-dependent nucleoside kinase activity. Only S. citri had nucleoside kinase activity with adenosine triphosphate and deoxyguanosine. We studied pyrimidine enzyme activities in all of the Mollicutes species except M . hominis and M . bovigenitalium. All of the Mollicutes species assayed had thymidine, thymidylate, and deoxycytidine kinase and thymidine and uridine phosphorylase activities. All of the Mycoplasma spp. had deoxycytotidine monophosphate and cytidine-deoxycytidine deaminase activities. All of the Mycoplasma spp. and U . urealyticum lacked deoxyuridine triphosphatase activity. U . urealyticum lacked deoxycytidine monophosphate deaminase activity, but otherwise it resembled all of the Mycoplasma spp. A. intermedium and S . citri differed from each other and from Mycoplasma spp. and U . urealyticum in the patterns of pyrimidine enzyme activities. Pyrophosphate-dependent nucleoside kinase activity was the most variably detected activity. None of the Mycoplasma spp. except four of eight strains of M . hominis had this kinase activity. Likewise, U . urealyticum did not have the pyrophosphate-dependent nucleoside kinase activity; however, A. intermedium and S . citri did have this enzyme activity. The absence of deoxyuridine triphosphatase activity in all Mycoplasma spp. may be related to their proposed rapid evolution and the relative lack of conserved sequences in their 5s ribosomal ribonucleic acids.

No abstract

An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-l-phosphate, deoxyribose-1-phosphate, or xylose-l-phosphate as the pentose donor. Mg2e and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (R.P 0.57) corresponded to all enzyme activity. The Kms for the partially pwrified PNP with ribose-l-phosphate, deoxyribose-l-phosphate, and xylose-l-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding V11,,s were 12.5, 14.3, and 12.0 FIM min-', respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2