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Query: UMLS:C0026936 (Mycoplasma)
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Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.
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PMID:Protein phosphorylation in Mycoplasma gallisepticum. 284 67

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.
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PMID:Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum. 316 71

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.
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PMID:Adenosine triphosphatase activity of mycoplasma membranes. 422 19

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)
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PMID:Synthesis of adenylate nucleotides by Mollicutes (mycoplasmas). 665 57

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).
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PMID:Suspected utility of enzymes with multiple activities in the small genome Mycoplasma species: the replacement of the missing "household" nucleoside diphosphate kinase gene and activity by glycolytic kinases. 1242 76

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants (rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP-heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.
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PMID:Identification, cloning and characterization of rfaE of Actinobacillus pleuropneumoniae serotype 1, a gene involved in lipopolysaccharide inner-core biosynthesis. 1279 93

Most ATPases, involved in energy-driven processes, act in the cytoplasm. However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells. In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region. With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg(2+)-dependent ATP binding site is demonstrated. In addition to ATP, ADP also could bind to OppA. The presence of an ATPase activity on the surface of M. hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4',4'-diisothiocyanostilbene-2',2'-disulfonic acid and influenced by the ATP analog 5'-fluorosulfonyl-benzoyladenosine. Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells. Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis.
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PMID:OppA, the substrate-binding subunit of the oligopeptide permease, is the major Ecto-ATPase of Mycoplasma hominis. 1476 96

A genomically and chemically detailed mathematical model of a "minimal cell" would be useful to understand better the "design logic" of cellular regulation. A "minimal cell" will be a prokaryote with the minimum number of genes necessary for growth and replication in an ideal environment (i.e., preformed precursors, constant temperature, etc.). The Cornell single-cell model of Escherichia coli serves as the basic framework upon which a minimal cell model can be constructed. A critical issue for any cell model is to describe a mechanism for control of initiation of chromosome replication. There is strong evidence that the essence of chromosome replication control is highly conserved in eubacteria and even extends to the archae. A generalized mechanism is possible based on binding of the protein DnaA-ATP to the origin of replication (oriC) as a primary control. Other features, such as regulatory inactivation of DnaA (RIDA) by conversion of DnaA-ATP to DnaA-ADP and titration of DnaA by binding to other DnaA boxes on the chromosome, have emerged as critical elements in obtaining a functional system to control initiation of chromosome synthesis. We describe a biologically realistic model of chromosome replication initiation control embedded in a complete whole-cell model that explicitly links the external environment to the mechanism of replication control. The base model is deterministic and then modified to include stochastic variation in the components for replication control. The stochastic model allows evaluation of the model's robustness, employing a low standard deviation of interinitiation time as a measure of robustness. Four factors were examined: DnaA synthesis rate; DnaA-ATP binding sites at oriC; the binding rate of DnaA-ATP to the nonfunctional DnaA boxes; and the effect of changing the number of nonfunctional binding sites. The observed DnaA synthesis rate (2000 molecules/cell) and the number of DnaA binding sites per origin (30) are close to the values predicted by the model to provide good control (low variance of interinitiation time), with a reasonable expenditure of cell resources. At relatively high binding rates for DnaA-ATP to the DnaA boxes (10(16) M(-1) s(-1)), increasing the number of DnaA binding sites to about 300, improved control (but little further improvement was seen by extension to 1000 boxes); however, at a low binding rate (10(10) M(-1) s(-1)), an increase in DnaA boxes had an adverse effect on control. The combination of all four factors is probably necessary to obtain a robust control system. Although this mechanism of replication initiation control is highly conserved, it is not clear if simpler control in a minimal cell might exist based on experimental observations with Mycoplasma. This issue is discussed in this investigation.
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PMID:Robust control of initiation of prokaryotic chromosome replication: essential considerations for a minimal cell. 1547 Jul 9

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.
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PMID:Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087). 1610 22

Several species of mycoplasmas glide on solid surfaces, in the direction of their membrane protrusion at a cell pole, by an unknown mechanism. Our recent studies on the fastest species, Mycoplasma mobile, suggested that the gliding machinery, localized at the base of the membrane protrusion (the "neck"), is composed of two huge proteins. This machinery forms spikes sticking out from the neck and propels the cell by alternately binding and unbinding the spikes to a solid surface. Here, to study the intracellular mechanisms for gliding, we established a permeabilized gliding ghost model, analogous to the "Triton model" of the eukaryotic axoneme. Treatment with Triton X-100 stopped the gliding and converted the cells to permeabilized "ghosts." When ATP was added exogenously, approximately 85% of the ghosts were reactivated, gliding at speeds similar to those of living cells. The reactivation activity and inhibition by various nucleotides and ATP analogs, as well as their kinetic parameters, showed that the machinery is driven by the hydrolysis of ATP to ADP plus phosphate, caused by an unknown ATPase.
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PMID:Gliding ghosts of Mycoplasma mobile. 1617 85


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