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Comparative metabolism of Mesoplasma, Entomoplasma, Mycoplasma, and Acholeplasma
J D Pollack1, M V Williams, J Banzon
1Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210, USA. pollack.1@osu.edu
Abstract:
Cytoplasmic fractions from species of the Mollicutes genera Entomoplasma, Mesoplasma, Mycoplasma, and Acholeplasma were assayed for NADH oxidase (NADH ox), ATP- and PPi-dependent phosphofructokinase (PFK), ATP- and PPi-dependent deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (G6Pde), 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 Mollicutes metabolic and DNA repair pathways. This was the first study to determine the presence of these enzymes in members of the genera Entomoplasma and Mesoplasma. 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 PPi-dependent PFK, urease, dUTPase, and dGUOK activities. Entomoplasma ellychniae ELCN-1T (T = type strain), Entomoplasma melaleucae M-1T, Mesoplasma seiffertii F7T, Mesoplasma entomophilum TACT, Mesoplasma florum L1T, Mycoplasma fermentans PG18T, and Acholeplasma multilocale 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 PPi-dependent dGUOK, and UNG activities, but not dUTPase or G6Pde activities. In contrast, Acholeplasma equifetale C112T, Acholeplasma oculi 19LT, Acholeplasma hippikon C1T, Acholeplasma modicum PG49T, and Acholeplasma morum 72-043T had membrane-localized NADH ox activity, PPi-dependent PFK, G6Pde, and dUTPase activities, and significantly lower MDH and LDH activities and exhibited a faster rate with PPi than with ATP in the dGUOK reaction. All of the members of the Mollicutes tested had hypoxanthine-guanine phosphoribosyl transferase, phosphoenolpyruvate carboxylase, and (except for Mesoplasma entomophilum TAC(T)) UNG activities. All of the Acholeplasma strains except Acholeplasma multilocale PN525T had TK, TMPK, and UNG activities. Mesoplasma entomophilum TAC(T) 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. multilocale PN525T is a member of an unrecognized metabolic subgroup of the genus Acholeplasma or is not an Acholeplasma strain.
Insights
This study analyzed key enzymes in Mollicutes bacteria, revealing distinct metabolic pathways. The findings establish a new classification scheme for Mollicutes genera based on enzyme activities, aiding in understanding their metabolic potential.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mollicutes, a class of bacteria, lack cell walls and possess unique metabolic characteristics.
- Understanding the enzymatic profiles of Mollicutes is crucial for their classification and understanding their biological roles.
- Previous studies have not comprehensively assessed enzyme activities across key Mollicutes genera.
Purpose of the Study:
- To assay cytoplasmic and membrane fractions of selected Mollicutes genera (Entomoplasma, Mesoplasma, Mycoplasma, Acholeplasma) for various enzyme activities.
- To identify signature enzyme activities for distinguishing between Mollicutes genera.
- To construct a preliminary classification scheme for Mollicutes based on metabolic enzyme profiles.
Main Methods:
- Enzyme assays were performed on cytoplasmic and membrane fractions for NADH oxidase (NADH ox), phosphofructokinase (PFK), deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (G6Pde), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase, hypoxanthine-guanine phosphoribosyl transferase, dUTPase, and uracil-DNA glycosylase (UNG).
- Comparative analysis of enzyme activities was conducted across different Mollicutes genera and species.
- Phylogenetic relationships derived from rRNA analyses were integrated with enzyme data.
Main Results:
- NADH ox activity was primarily cytoplasmic in most strains, except for Acholeplasma species where it was membrane-localized.
- Distinct patterns of ATP- and PPi-dependent enzyme activities (PFK, dGUOK) and the presence/absence of enzymes like dUTPase and G6Pde differentiated genera.
- Acholeplasma strains showed variations, with some exhibiting characteristics suggesting a distinct metabolic subgroup or misclassification.
Conclusions:
- A preliminary scheme for distinguishing Mollicutes genera based on signature functional enzyme assays was developed.
- The enzyme-based classification provides insights into the metabolic potential of Mollicutes, complementing rRNA-based phylogeny.
- The study highlights metabolic diversity within the Acholeplasma genus and identifies Mesoplasma entomophilum as having restricted dTTP synthesis.
