Phosvitin kinase activity in Acholeplasma axanthum.

Incubating the soluble fraction derived from A. axanthum with phosvitin and [gamma-32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0-6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca2+ or by cyclic AMP.

Evidence for a phosphoenolpyruvate dependent sugar-phosphotransferase system in the mollicute Acholeplasma florum.

In order to confirm the presence of a phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS) in the mollicute Acholeplasma florum we studied the ability of cell free extracts of this organism to phosphorylate glucose and/or fructose in the presence of PEP. We also cloned and sequenced a DNA fragment coding for a putative polypeptide showing significant similarity with the enzyme II of the beta-glucoside PTS of Escherichia coli. Taken together, these results show that A florum possesses a fructose-PTS, but not a glucose-PTS, and that the amino acid sequence deduced from the DNA fragment is related to beta-glucoside and sucrose enzymes II of PTS from various bacteria.

Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species.

Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake. All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol. The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass. Amongst 20 Mycoplasma species tested, M. agalactiae, M. bovis, M. dispar, M. gallisepticum, M. pneumoniae and M. ovipneumoniae oxidised ethanol. Propanol was also oxidised by M. dispar and isopropanol by M. agalactiae, M. bovis and M. ovipneumoniae. Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone. The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.

Antiserum raised against gyrase A of Acholeplasma laidlawii reacts with phytoplasma proteins.

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.

DNases of Acholeplasma spp.

One strain from each of seven species of Acholeplasma was examined for the presence of DNases. Six of the strains were found to be DNase positive when assayed with DNA-methyl-green-containing agar, indicating that this method can be used to differentiate the seventh strain, Acholeplasma axanthum, from the remaining Acholeplasma spp. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA revealed DNases in the cell extracts of all seven strains and in the supernatant growth medium of five of the strains. The electrophoretic patterns were highly characteristic for each strain and can be used for the rapid identification of different strains of Acholeplasma.

Isoenzymes in two species of Acholeplasma.

The reference strains Type A and Type B and two equine strains of Acholeplasma laidlawii were examined for a wide range of isoenzymes using thin-layer starch-gel electrophoresis; in addition two isoenzymes were examined in two strains of A. equifetale. The type strains A and B of A. laidlawii were differentiated by their lactate dehydrogenase, phosphoglucomutase and aspartate aminotransferase patterns and the two equine strains by their hexokinase, lactate dehydrogenase and phosphoglycerate kinase patterns. The two pairs of strains differed from one another with respect to hexokinase, phosphoglucomutase, adenylate kinase and glucose-6-phosphate dehydrogenase. The two strains of A. equifetale could be distinguished by their isoenzymes of hexokinase. The two species were differentiated by their hexokinase and phosphoglucomutase patterns.

Purification and characterization of a dUTPase from Acholeplasma laidlawii B-PG9.

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.

[The RNAse activity in mycoplasmas of different taxonomic positions]. / RNK-aznaia aktivnost' v mikoplazmakh razlichnogo taksonomicheskogo polozheniia.

Dynamics of the RNAase accumulation in cells and culture fluid of strains of Acholeplasma and Mycoplasma genera has been studied. In certain strains of two genera of mollicutes some differences in dynamics of RNAase formation by them are determined.

Analysis of multiple isoenzyme expression among twenty-two species of Mycoplasma and Acholeplasma.

Crude extracts of triple-cloned, purified cultures of 22 species of Mycoplasma and Acholeplasma were examined for expression of 21 isozyme systems routinely used to type mammalian cells. Nine previously described enzymes (purine nucleoside phosphorylase, adenylate kinase, dipeptidase, esterase, glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and superoxide dismutase) and three enzymes not previously reported in mycoplasma (triose phosphate isomerase, inorganic pyrophosphatase, and acid phosphatase) were detected in some or all of the species examined. These findings provide new information on the enzymatic expressions of these organisms. Three of the isozyme systems (superoxide dismutase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were present in Acholeplasma species but not in any Mycoplasma species. The characteristic pattern of electrophoretic mobility of the 12 isozyme systems also provides a useful biochemical property for identification, characterization, and classification of these mycoplasmas. Mycoplasma isozyme expression for seven of the enzymes were readily detected in various infected-cell culture lines by using either cell extracts or concentrated cell culture fluids. Mycoplasma-specific enzymes found in infected-cell extracts had the same electrophoretic mobility patterns as enzymes obtained from broth-grown mycoplasmas of the same species. Expression of homologous mammalian enzymes was not detectably altered by infection with mycoplasmas.

Acetate kinase activity in mycoplasmas.

Acetate kinase activity was assayed in 13 mycoplasmas. Nine species exhibited the enzymic activity in the direction of either synthesis of acetylphosphate or adenosine triphosphate. On the other hand Mycoplasma orale, Mycoplasma arthritidis, Ureaplasma urealyticum (10 serotypes), and two strains of Anaeroplasma species exhibited only minimal levels of the enzymic activity. In these four species, the enzyme does not seem to play a key role in adenosine triphosphate formation.

On the distribution and characteristics of isozyme expression in Mycoplasma, Acholeplasma, and Ureaplasma species.

A summary of a survey of three genera of mycoplasmatales (Mycoplasma, Acholeplasma, and Ureaplasma) for isozyme expression is presented. Isozyme analysis of mycoplasmas has been employed in at least three distinct areas: (1) as genetic markers for identification, individualization, and taxonomic classification; (2) as markers for cell culture contamination; and (3) as a qualitative measure of the operative metabolic pathways in the diverse species. We have found five ubiquitous enzymes: purine nucleoside phosphorylase, adenylate kinase, inorganic pyrophosphatase, dipeptidase, and esterase. Three enzymes, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and superoxide dismutase, were restricted to Acholeplasma species and were not detected in Mycoplasma or Ureaplasma. Four glycolytic enzymes, glucose phosphate isomerase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase, were restricted to those species of Mycoplasma and Acholeplasma capable of glucose fermentation. Two of these glycolytic enzymes, glucose phosphate isomerase and lactate dehydrogenase, were detected in serovars I and II of U. urealyticum, which is inconsistent with the non-glycolytic activity in this genus.

Properties of the nucleases of mollicutes.

Extracts of the Mollicutes Acholeplasma equifetale, Acholeplasma laidlawii B, Mycoplasma arthritidis. Mycoplasma pulmonis, and Mycoplasma pneumoniae had DNase and endonuclease activity. A. laidlawii B had at least two peaks of DNase activity in sucrose gradients with sedimentation coefficients of 3.1S and 4.3S. These fractions also had endonuclease activity with different substrate specificities. A. laidlawii B may have more than two peaks of endonuclease activity in sucrose gradients.

Activating amphiphiles cause a conformational change of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes according to proteolytic digestion.

1,2-Diacylglycerol 3-glucosyltransferase synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol (MGlcDAG) in the membrane of Acholeplasma laidlawii, which is important for the spontaneous curvature, and is a regulatory site for the lipid surface charge density. A potential connection between activity and a conformational change of this enzyme, governed by essential lipid activators, was studied with purified MGlcDAG synthase in different lipid aggregates. Critical fractions of anionic phospholipids 1, 2-dioleoyl-phosphatidylglycerol (DOPG) and 1,2-dioleoyl-phosphatidylserine (DOPS) were essential for the restoration of enzyme activity, while the zwitterionic 1,2-dioleoyl-phosphatidylcholine (DOPC) and the uncharged diglucosyldiacylglycerol (DGlcDAG) were not. Proteolytic resistance had a very good correlation with the enzyme activity in various lipid-CHAPS mixed micelles. Anionic lipids DOPG and DOPS could protect the exposed MGlcDAG synthase from digestion, whereas DOPC and DGlcDAG could not. Similar features were observed in liposome bilayers. Likewise, the detergent dodecylphosphoglycerol (PGD), with a phosphatidylglycerol-like headgroup, could also stimulate the MGlcDAG synthase activity efficiently with a concomitant protection toward proteolytic digestion. Neither proteolytic resistance nor restored enzyme activity was observed using soluble glycerol 3-phosphate. It is concluded that in addition to critical amounts, both the negatively charged headgroup and hydrophobic chains of the activator amphiphiles, but not a certain aggregate curvature, seem necessary for a proper conformation and the resulting active state of the MGlcDAG synthase.

Evidence for superoxide dismutase and catalase in mollicutes and release of reactive oxygen species.

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas, and ureaplasmas. No superoxide dismutase activities or only traces were detectable in fresh prepared cell lysates, whereas activities were evident after dialysis of the cell lysates. A further increase in superoxide dismutase activities was observed after the cell lysates were heated to 65 degrees C for 30 s. This might be due to the destruction of enzymatic reactions interfering with the activity tests. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxidase activities were detectable. The production of O2- and H2O2 with glucose as substrate was demonstrated for 8 of 10 strains tested. No correlation to the pathogenicities of the strains was indicated. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes.

Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes.

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.

Exclusive occurrence of an extracellular protease capable of cleaving the hinge region of human immunoglobulin A1 in strains of Ureaplasma urealyticum.

The purpose of the study was to further investigate the occurrence of a specific immunoglobulin A1 (IgA1) protease among mycoplasmas of various origin. Fifty-two strains representing the genera Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma were examined. The ability to cause specific cleavage of human IgA1 in the hinge region resulting in intact Fab and Fc fragments was exclusively associated with strains of U. urealyticum. Thus, this trait may be used as a valuable differential characteristic of U. urealyticum. A more pronounced proteolytic activity capable of causing extensive degradation of both human and bovine IgA was demonstrated in the type strain of U. diversum.

Origins of the mycoplasmas: sterol-nonrequiring mycoplasmas evolved from streptococci.

We report the establishment of a phylogenetic relationship between the sterol-nonrequiring mycoplasmas (Acholeplasma species) and streptococci. Three specific antisera prepared against purified Streptococcus faecalis fructose diphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and Pediococcus cerevisiae glyceraldehyde-3-phosphate dehydrogenase were used for comparative enzyme immunological studies; the Ouchterlony double-diffusion technique and the quantitative microcomplement fixation procedure were employed. The reactions obtained provide evidence showing that all seven ACholeplasma species studied (A. laidlawii, A. granularum, A. modicum, A. oculi, A. axanthum. A. hippikon, and A. equifetale) are phylogenetically related to streptococci and that they evolved from streptococci. The data strongly suggest that the acholeplasmas comprise a distinct evolutionary group that has diverged from streptococci belonging to Lancefield group D or N. No reactions were observed between these enzyme antisera and cell extracts from six fermentative Mycoplasma species. These results support the view that mycoplasmas are derived from various bacteria.

Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi.

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-1-P as a donor. The synthesis of deoxyribonucleosides from nucleobases and dR-1-P has been reported from only one other procaryote, Escherichia coli (L. A. Mason and J. O. Lampen, J. Biol. Chem. 193:539-547, 1951). The reverse of this phosphorylase reaction is more widely known, and we found such activity in all mollicutes studied. Some Acholeplasma species but not the Mycoplasma species can phosphorylate deoxyribonucleosides to deoxyribomononucleotides by a PPi-dependent deoxyribonucleoside kinase activity, which was first reported in this group for the ribose analogs (V. V. Tryon and J. D. Pollack, Int. J. Syst. Bacteriol. 35:497-501, 1985). This is the first report of PPi-dependent purine deoxyribonucleoside kinase activity. An ATP-dependent purine deoxyribonucleoside kinase activity is known only in salmon milt extracts (H. L. A. Tarr, Can. J. Biochem. 42:1535-1545, 1964). Deoxyribomononucleotidase activity was also found in cytoplasmic extracts of these mollicutes. This is the first report of deoxyribomononucleotidase activity.

Rapid biochemical tests for characterization of the Mycoplasmatales.

Methods are described for the rapid detection of beta-D-glucosidase and phosphatase in mycoplasma cultures using fluorogenic 4-methylumbelliferone substrates. These methods were applied to a selection of mycoplasma cultures and were compared with the conventionally used tests for these enzymes. Results were similar by both methods, but the fluorogenic tests could be read after 1 h, whereas the conventional tests took several days.

Membrane-bound thioesterase activity in mycoplasmas.

Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 18 degrees C, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibllity that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed.