Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PPi-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PPi-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P2). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P2), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PPi-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.
Cytoplasmic fractions from species of the Mollicutes genera Entomoplusma, Mesoplasma, Mycoplusma, and Acholeplusma were assayed for NADH oxidase (NADH ox), ATP-and PP,-dependent phosphofructokinase (PFK), ATP-and PP,-dependent deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (MPde), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase, hypoxanthine-guanine phosphoribosyl transferase, dUTPase, and uracil-DNA glycosylase (UNG) activities. Membrane fractions were also examined for NADH ox activity. These activities were used as indicators of the presence and relative activities of major MoZZicutes metabolic and DNA repair pathways. This was the first study to determine the presence of these enzymes in members of the genera Entomoplusma and Mesoplusma. Using the data obtained, we constructed a preliminary scheme for distinguishing genera of the class Mollicutes on the basis of the results of signature functional enzyme assays. This scheme includes phylogenetic relationships deduced from rRNA analyses, but is more informative with respect to metabolic potential. The criteria used include the presence of PP,-dependent PFK, urease, dUTPase, and dGUOK activities. Entomoplasma ellychniae ELCN-lT (T = type strain), Entomoplasma meluleucae M-lT, Mesoplasma segertii F7T, Mesoplusma entomophilum TACT, Mesoplusma jlorum LIT, Mycoplusma fermentans PGIST, and AchoZeplasma multibcale PN525T were similar in most respects. NADH ox activity was localized in the cytoplasm of these organisms. These strains had ATP-dependent PFK, MDH, LDH, ATP-and PP,-dependent dGUOK, and UNG activities, but not dUTPase or G6Pde activities. In contrast, Acholeplusma equifetale C112T, Acholeplasma oculi 19LT, Acholeplasma hippikon CIT, Acholeplusma modicum PG49T, and Acholeplasma momm 72-043T had membrane-localized NADH ox activity, PP,-dependent PFK, G6Pde, and dUTPase activities, and significantly lower MDH and LDH activities and exhibited a faster rate with PP, than with ATP in the dGUOK reaction. All of the members of the MoZZicutes tested had hypoxanthine-guanine phosphoribosyl transferase, phosphoenolpyruvate carboxylase, and (except for Mesoplasma entomophilum TACT) UNG activities. All of the Acholeplusma strains except Acholeplusmu multilocale PN52ST had TK, TMPK, and UNG activities. Mesoplusma entomophilum TACT was distinguished by having no detectable dUTPase, UNG, TK, and TMPK activities, indicating that there is a severe restriction in or an absence of a synthetic route to dTTP. Our data also suggest that A. multiZocale PN52ST is a member of an unrecognized metabolic subgroup of the genus Acholeplusm or is not an Acholeplusma strain.Distinguishing members of the class Mollicutes by their metabolic characteristics has been of very limited phylogenetic and taxonomic usefulness. Standards for describing Mollicutes taxa have been published previously (7). Characteristics such as gross cellular morphology, colonial appearance, genome size, and serologic...
Cultures of the Mollicutes (mycoplasma) Acholeplasma laidlawii B, Acholeplasma morum, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma fermentans and Mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in pH and glucose concentration, and concentrations of ATP, ADP, AMP, lactate and pyruvate. The adenylate energy charge (ECA), the mean generation time, and the number of nmol of ATP (mg dry weight)-1 were calculated for cultures in the mid-exponential growth phase. The maximum cell concentrations ranged from 0.2 X 10(10) to 5.0 X 10(10) c.f.u. ml-1. Doubling times ranged from 0.34 to 3.29 h. The fermentative, nonarginine-requiring A. laidlawii B, A. morum, and M. gallisepticum, as well as the fermentative, arginine-requiring M. fermentans, utilized glucose and produced lactate and pyruvate. The non-fermentative, non-arginine-requiring M. bovis neither utilized glucose nor produced lactate or pyruvate. The non-fermentative, arginine-requiring M. arginini utilized glucose, but did not produce lactate or pyruvate. At mid-exponential growth phase, the average ECA of A. laidlawii B was 0.90, a value similar to that reported for Spiroplasma citri and other bacteria. In contrast, the average ECA of A. morum and the four Mycoplasma species was 0.70. In A. laidlawii B at mid-exponential growth phase, ATP accounted for 97% of the total adenylate nucleotide pool.(ABSTRACT TRUNCATED AT 250 WORDS)
Immunofluorescence has proved useful in the identification of mycoplasmas and detection of antibodies against them (1-3). For Mycoplasma pfieumoniae, the indirect fluorescent antibody test has been found to be highly specific and high fluorescent antibody titers to M . pneumoniae have been correlated with resistance to clinical illness (4). The purpose of this study was to develop antigenic preparations for a variety of mycoplasma species which would be both reliable and convenient to use in fluorescent antibody tests. Two methods of preparing mycoplasma antigens for fluorescence tests have thus far been reported. One, as described by Liu ( S ) , employs frozen sections of chick embryo lung infected with M . ifmeumoniae. In the second, and most commonly used precedure (6), mycoplasma colonies growing on agar are transferred to microscope slides and fixed in a hot water bath. The Liu technique employing egg-grown antigens is technically difficult and time consuming; the procedure utilizing hot water fixation gives nonspecific fluorescence primarily because of the presence of contaminating agar.Broth-grown mycoplasmas have been shown to adhere to glass surfaces (7). We have exploited this property by using glass cov-
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