The motility of mollicutes

Recent experiments show that the conformation of filament proteins play a role in the motility and morphology of many different types of bacteria. Conformational changes in the protein subunits may produce forces to drive propulsion and cell division. Here we present a molecular mechanism by which these forces can drive cell motion. Coupling of a biochemical cycle, such as ATP hydrolysis, to the dynamics of elastic filaments enable elastic filaments to propagate deformations that generate propulsive forces. We demonstrate this possibility for two classes of wall-less bacteria called mollicutes: the swimming of helical-shaped Spiroplasma, and the gliding motility of Mycoplasma.     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Cytoskeleton of mollicutes

Mollicutes are a class of bacteria that lack a peptidoglycan layer but have various cell shapes. They perform chromosome segregation and binary fission in a well-organized manner. Especially, species with polarized cell morphology duplicate their membrane protrusion at a position adjacent to the original one and move the new protrusion laterally to the opposite end pole before cell division. The featured various cell shapes of Mollicutes are supported by cytoskeletal structures composed of proteins. Recent progress in the study of cytoskeletons of walled bacteria and genome sequencing has revealed that the cytoskeletons of Mollicutes are not common with those of other bacteria. Mollicutes have special cytoskeletal proteins and structures that are sometimes not shared even by other mollicute species.     

Superoxide dismutase activity and photosensitizing properties of 2,5-dihydroxybenzolsulfonate

2,5-dihydroxybenzolsulphonate was shown to actively interact with the superoxide radical O2-. formed under UV-radiation of glycyltryptophan aqueous solutions or under visible light illumination of glycyltryptophan and riboflavine. The constant of the rate of reaction of 2,5-dihydroxybenzolsulphonate with O2-. was determined to be equal to 1.3 X 10(9) M-1s-1. A photosensitized effect of 2,5-dihydroxybenzolsulphonate under illumination with the light with 280 nm was found. The sensitization efficiency of glycyl-tryptophan oxidation increases when the experiments are carried out in the oxygen atmosphere.     

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.     

Photosensitization with derivatives of haematoporphyrin

This review describes recent progress in delineation of the structure of the active component(s) in the tumour-localizing photosensitizer HPD (haematoporphyrin derivative), along with suggestions concerning the likely determinants of accumulation of this product by different tissues.     

Biodistribution of photosensitizing agents

• 1.1. The features of neoplasia which predict for drug responsiveness are rapid growth and/or inefficient repair of damage, especially to DNA.
• 2.2. PDT has the advantage of yielding responses regardless of the growth fraction of a tumor, and repair appears to play only a minor role.
• 3.3. While an entirely different spectrum of tumors can be targeted with PDT, the perhaps unavoidable accompaniment is that a new set of rules for efficacy will need to be established.
• 4.4. The selectivity of PDT is based on the need for irradiation which can be directed, along with the short tissue half-life of the cytotoxic product, singlet oxygen. Sensitizers which target specific cellular organelles could promote PDT efficacy, if in vitro data (Woodbum et at., 1992b Photochem. Photobiol. 55, 697–704) can be translated into clinical practice.
• 5.5. It remains to be established whether total drug distribution to neoplastic tissues or concentration in specific sub-cellular sites is the more important factor.
• 6.6. Questions relating to the role of biodistribution as a factor in efficacy of PDT sensitizers of photosensitizers remain to be explored. Just as the political cartographers are grappling with changes in territorial boundaries of known lands, we continue to clarify the rules relating to PDT boundaries. In this regard, it is clearly important for determinants of pharmacokinetics and biodistribution to be evaluated and understood.
• 7.7. Once clinical reports on the “second generation” agents are published, we may get a better picture, although it is not unusual for clinical reports to raise more questions than they answer.
• 8.8. It seems safe to conclude that there is nothing “magic” about HPD, and that additional efficacious photosensitizers for PDT can be produced.
• 9.9. If we find that a wide variety of different structures are useful in the clinic, a likely conclusion is that there are multiple mechanisms of tumor localization. Since the nature of neoplasia is so diverse, this possibility should not be surprising.
• 10.10. In view of the finding, cited above, that liposomes show about the same degree of tumor selectivity as does Photofrin, it may be feasible to target any efficient photosensitizer for neoplastic tissues by development of an appropriate delivery system.

     

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.     

Mechanisms of genetic instability of mollicutes (mycoplasma)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.     

Effect of maize stunting mollicutes on survival and fecundity of Dalbulus leafhopper vectors

The effect of corn stunt spiroplasma (CSS) on survival and fecundity of three Dalbulus leafhopper species was determined. CSS significantly reduced the survival, as measured by the time to 50% (t₅₀) and 25% (t₂₅) survival, and by the scale parameter (b) of the Weibull model, for D. elimatus and D. gelbus. Fecundity of these two species, as measured by the net and gross reproductive rates, was also reduced by CSS. CSS did not significantly affect the corn leafhopper, D. maidis. In a separate experiment, maize bushy stunt mycoplasma (MBSM) reduced the survival and fecundity of D. maidis at temperatures from 20 to 29 °C. The effect of MBSM on D. maidis survival was less severe than CSS on D. elimatus and D. gelbus; t₂₅, but not t₅₀, was reduced by MBSM. Survival times and the cohort generation time generally declined with increasing temperature. Fecundity, however, generally increased with increasing temperature.     

The effect of photosensitizing dyes on the 3H-thymidine incorporation of cells grown in vitro

The photodynamic inactivation of ³H-thymidine incorporation in mouse embryo (ME) and mouse L cells by acridine orange (AO), methylene blue (MB) or neutral red (NR) has been studied by estimating the number of nuclei capable of incorporating ³H-thymidine during a 24 h period following light exposure. In the dark NR and AO reduced the number of ME-nuclei incorporating ³H-thymidine but MB caused an increase in non-scheduled DNA synthesis. The dark effect on L cells was less but the photoinactivation of thymidine uptake was proportionally greater in these cells. Polyoma virus was shown to be capable of growing in cells whose thymidine uptake was reduced or completely stopped by photoinactivation with NR. However, if the NR damage was very great, or when AO was used to photosensitize cells, the synthesis of viral DNA was interfered with.     

Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes

Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials. Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method. A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures. In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range. The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains. Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.     

Regulation of carbon metabolism in the mollicutes and its relation to virulence

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts.

Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.     

Separation and analysis of haematoporphyrin derivative components by high-performance liquid chromatography

High-performance liquid chromatography has been used to separate and analyse the components of haematoporphyrin derivative, a material used in cancer phototherapy. Both haematoporphyrin derivative in the solid form and the solution derived from it have been quantitatively analysed on reversed-phase columns. The factors (low pH, presence of ion-pairing reagent and solvent) that are of importance in optimising these separations are discussed.