The chromatographic properties of the DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]

The DNA-dependent DNA-polymerase (DNA polymerase I which is not sorbed on the column with DEAE-cellulose, and DNA-polymerase II, which is absorbed by this column and is eluted from it by 0.3 M of NaCl), have been isolated from Acholeplasma laidlawii PG-8. DNA-polymerase I in homogeneous state was obtained as a result of the stepwise treatment by heparin-sepharose (elution at 0.35 M of NaCl) and poly-U-sepharose (elution at 0.3 M of NaCl). It was presented on the electrophoregram by one polypeptide with molecular weight of 72 kDalton. The second form of DNA polymerase was also obtained in homogeneous state as a result of sequential treatment on heparin-sepharose (elution at 0.3 M of NaCl) and on poly-A-sepharose (elution at 0.25 M of NaCl): the protein which had manifested polymerase activity was a polypeptide with molecular weight of 45 kDalton.     

The isolation and partial purification of 2 DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]

Two forms of DNA-dependent DNA-polymerase have been isolated and partially purified from the limited amount of biomass of cells Acholeplasma laidlawii PG-8, a typical representative of genus Acholeplasmataceae, as a result of successive chromatography on the columns with DEAE-cellulose DE-52 and Green A-sepharose. The first form of DNA-polymerase is eluted from the ion-exchange column with NaCl concentration of 0.1 M from the column with Green A-sepharose of 0.27 M, while the second form-with NaCl concentrations of 0.6 and 0.4 M, respectively. The both enzymatic activities are able to implement DNA synthesis. The conditions of DNA-polymerase production proved to be rather convenient for isolation of the concentrated and highly active enzymes.     

RNAse substrate specificity in Acholeplasma laidlawii PG-8]

A substrate specificity of RNAses of A. laidlawii PG-8 to polynucleotides - poly (C), poly (U), poly (A) has been studied. Due to the data obtained both intracellular and extracellular RNAses of A. laidlawii possess similar specificity to different polynucleotides. Both RNAses preferentially break cytidine-bonds. Specificity of the studied enzymes in respect to polyuridylic and polyadenylic acids was less expressed.     

Analysis of the DNA gyrase genes ofAcholeplasma laidlawii PG-8B

A 5-kb region of theAcholeplasma laidlawii PG-8B genome was sequenced. The region contained the genes for RecF, DNA gyrase subunits A and B (GyrA and GyrB), and a fragment of the ATP-binding subunit of the hypothetical ABC transporter. In phylogenetic analysis,A. laidlawii GyrA and GyrB formed statistically significant, stable clusters with the corresponding proteins ofClostridium acetobutylicum, Staphylococcus aureus, Bacillus subtilis, andStreptococcus pneumoniae. A laidlawii PG-8B clones resistant to fluoroquinolone (FQ) antibiotic ciprofloxacin (Cff) were obtained on a selective medium. The clones carried mutations in the quinolone resistance-determining region (QRDR) ofgyrA, which resulted in substitutions Ser83→Ala, Ser83→Phe, or Asp91→Asn. No mutations were found ingyrB QRDR of the resistant clones.     

Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.     

The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Respiratory activity of Acholeplasma laidlawii cells and its role in the active transport of carbohydrates]

The respiratory activity of the Acholeplasma laidlawii cells was studied in order to elucidate a possible mechanism of coupling of transport with energy. The respiration of the cells is stimulated by ethanol, glucose, NADH, lactate, and pyruvate. The substrates of the Krebs cycle have no effect on the respiration. The respiratory activity, stimulated by ethanol and glucose, is inhibited by the inhibitors of the respiratory chain, SH reagents, and the inhibitors of glycolysis. The results of experiments with inhibitors suggest that the respiratory chain in the A. laidlawii cells is reduced and terminated by flavoprotein. This is confirmed by the results of spectroscopic analysis of cytochromes. Respiration coupled with phosphorylation did not play any important role in the active transport of carbohydrates. Probably, the energy, necessary for the transport of carbohydrates, is supplied by the substrate phosphorylation. This explains the activation of respiration by glucose, which is so sensitive to arsenate. The respiration of the A. laidlawii cells is not stimulated by some carbohydrates (fructose, 3-O-methyl-D-glucose).     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

On the possible role of respiratory activity of Acholeplasma laidlawii cells in sugar transport

1. Out of 20 exogeneous substrates only ethanol and, to a much lesser extent, lactate and pyruvate were shown to be capable of stimulating the respiration of Acholeplasma laidlawii cells. However, none of these substrates changed the initial rate of active transport of 3-O-methyl-d-glucose (3-O-MG).
2. From inhibitory analyses and spectroscopic data, it is apparent that the respiratory chain of A. laidlawii has no cytochromes and is probably not responsible for oxidative phosphorylation.
3. Valinomycin and nigericin stimulated cell respiration only in the presence of K⁺-ions, while monensin stimulated it in the presence of Na⁺-ions.
4. 3-O-MG transport was shown to be sensitive to uncouplers, ATPase inhibitors and arsenate are resistant to a majority of respiratory inhibitors tested. This suggested that there was no relationship between respiration and carbohydrate transport in the A. laidlawii cells. Further evidence was provided by the absence of respiratory stimulation during the transport of non-metabolizing carbohydrates.

     

The enzymatic synthesis of membrane glucolipids in Acholeplasma laidlawii.

In membranes of the prokaryote Acholeplasma laidlawii, the physiological regulation of the two major membrane lipids, monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), is governed by factors affecting the equilibria between lamellar and non-lamellar phases of the membrane lipids. The synthesis of the glucolipids is considered to be a two-step glucosylation: (i) DAG+UDP-Glc----MGlcDAG+UDP; and (ii) MGlcDAG+UDP-Glc----DGlcDAG+UPD. This was corroborated by in vivo pulse labelling experiments showing turnover of MGlcDAG but not DGlcDAG. The enzymatic synthesis of MGlcDAG was localized to fresh or freeze-dried membranes in vitro. Synthesis of DGlcDAG was minor in such membranes but of substantial magnitude in intact cells. Synthesis of MGlcDAG was stimulated by small amounts of SDS but completely inhibited upon solubilization of the membranes by a variety of detergents. The inhibitory effect of several UDP-Glc analogs on glucolipid synthesis demonstrated the importance of UDP-Glc as the sugar donor. Synthesis of both glucolipids was lost in freeze-dried plus lipid-extracted cells but restored when lipids were transferred back to the extracted cell membrane. By selectively adding specific lipids, a strong dependence on the acceptor lipid DAG, as well as the need for general matrix lipids for enzyme activity, was established. In addition, the anionic phosphatidylglycerol (PG), but not the other phospholipids, had a strong stimulatory effect. The presence of different phosphorylating agents stimulated the synthesis of DGlcDAG and partially inhibited that of MGlcDAG. This, together with the lipid dependency, may constitute mechanisms for the regulation of the enzyme activities in vivo.     

The distribution of molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii

A double-label technique has been applied to study the distribution of different molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii. After growth on oleic acid, 16% of the total phosphatidylglycerol contains two oleic acid residues and 84% contains one oleic acid and one saturated fatty acid. The dioleoyl phosphatidylglycerol is present in equal amounts in the outer and the inner layer of the membrane. Phosphatidylglycerol which is associated with membrane proteins consists exclusively of the class containing only one oleic acid.     

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.