Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).     

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.     

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.     

Nisin resistance distinguishes Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth media.

The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined. When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition. The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min. Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species. At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A. laidlawii and Acholeplasma oculi. However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP. Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration. Inhibition of oxygen uptake was greater for A. laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol. Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.     

Nisin resistance distinguishes Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth media

The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined. When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition. The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min. Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species. At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A. laidlawii and Acholeplasma oculi. However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP. Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration. Inhibition of oxygen uptake was greater for A. laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol. Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Interaction of mycoplasma virus type 2 with cellular components of Acholeplasma laidlawii strain JA1.

Mycoplasma virus type 2 was shown to adsorb specifically to intact cells, membranes, and lipoglycan of Acholeplasma laidlawii strain JA1 but not to these components of Acholeplasma oculi. The oligosaccharide chain of the lipoglycan defined the specificity of the receptor site since deacylation not only did not reduce adsorption but increased it threefold. Actual adsorption of virus to lipoglycan was demonstrated by sucrose density gradient separation of the virus-lipoglycan complex. A strain of A. laidlawii, JA1r, resistant to infection with mycoplasma virus type 2, was incapable of adsorbing the virus and was devoid of lipoglycan.     

Cultivation of Mycoplasmas on Cellulose Ester Substrates

The ability of mycoplasmas to grow on cellulose ester substrates was evaluated. Mycoplasma pneumoniae, M. hominis, M. arthritidis, M. gallisepticum, and Acholeplasma laidlawii grew on Millipore (mixed cellulose ester) filters and Sepraphore III (cellulose polyacetate) membranes.     

Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2.

Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii. Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase. A. laidlawii was grown on linoleic acid. Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol. The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature. When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately. The hydrolysis of the residual 30% is again strongly temperature dependent. Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2. Under conditions in which all the lipid is in the solid state, no hydrolysis occurs. When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed. The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A. laidlawii. Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase. The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.     

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.     

Detection of Cholesterol in Cell Membranes by Use of Bacterial Toxins

A method is described for the detection of cholesterol in membranes from erythrocytes, mycoplasmas, and bacterial cells by a ferritin-labeling technique. Membranes treated with cereolysin, a bacterial hemolysin which specifically binds to cholesterol, and then treated with ferritin-antitetanolysin, were specifically ferritin-labeled for cholesterol. A similar antigen-antibody system, streptolysin O-ferritin-antistreptolysin, was also used successfully with erythrocyte membranes. There was an uneven distribution of ferritin in erythrocyte membranes suggesting that the distribution of cholesterol may not be entirely random. Mycoplasma gallisepticum was intensely labeled, but Acholeplasma laidlawii with or without cholesterol in the membranes was not labeled, suggesting an unusual location for cholesterol in A. laidlawii membranes. As controls, two of three species of bacterial membranes lacking cholesterol were not ferritin-labeled.     

Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.     

Mycoplasmal infection of insect cell cultures

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection. Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%. A. laidlawii, Mycoplasma orale, M. arginini, but not M. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A. laidlawii and M. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages. A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium. After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C. At p5 no difference could be found. Uridine phosphorylase assays of Dm-1 cultures infected with A. laidlawii, M. hyorhinis, M. orale, and M. arginini gave clearly positive results only with A. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Sensitivites in vitro to antimicrobial drugs of bovine mycoplasmas isolated from respiratory and genital tracts

A total of 155 Mycoplasma strains were examined for sensitivity to nine antibiotics and four nitrofurans by the agar dilution method. They consisted of 69 strains of Mycoplasma bovirhinis, 33 strains of M. bovigenitalium, 49 strains of Acholeplasma laidlawii and four strains of A. modicum isolated from the nasal secretions, tracheas and lungs of calves manifesting respiratory symptoms and from bovine genital tracts collected at a slaughterhouse. As a result, furamizole and mitomycin C showed the strongest growth-inhibiting effect on all the strains. They were followed in this effect by kitasamycin tartrate, spiramycin adipate, tylosin tartrate, tetracycline-HCl and chloramphenicol. Furthermore, these five drugs were followed in the effect by furazolidone, nitrofurantoin and sodium nifurstyrenate. Fradiomycin sulfate and kanamycin sulfate showed only little effect on all the strains. Erythromycin lactobionate showed a strong growth-inhibiting effect on the Acholeplasma strains, but not on the Mycoplasma strains. There were some cross resistant strains of the Acholeplasma species to the effects of the macrolides.     

Inactivation of the vascular permeability-increasing activity of bradykinin by mycoplasmas

Mycoplasma pneumoniae, M. genitalium, M. fermentans, M. hominis, M. salivarium, M. orale, Ureaplasma urealyticum and Acholeplasma laidlawii inactivated the vascular permeability-increasing activity of bradykinin when the mixture of bradykinin and mycoplasma cells was injected after incubation at 37 degrees C for 1 h. Cell components responsible for inactivation of the activity of bradykinin were found to be arginine-specific aminopeptidase and carboxypeptidase.     

Divalent cations in native and reaggregated mycoplasma membranes

The Mg(2+) content of membranes of several Mycoplasma and Acholeplasma species varied between 0.88 and 1.98 mug of Mg(2+) per mg of protein, depending on the species and on growth conditions. Ca(2+) could be detected only when it was added to the growth medium. The Mg(2+) content of isolated A. laidlawii membranes could be increased almost threefold by dialysis against 20 mm Mg(2+), whereas aggregated A. laidlawii membranes contained about six to eight times more Mg(2+) per mg of protein than the native membranes. This was taken to indicate that the molecular organization of the lipid and protein in the reaggregated membranes differs from that of the native membranes. Between 60 and 83% of the Mg(2+) in native and reaggregated A. laidlawii membranes was associated with the lipid fraction extracted with chloroform-methanol. The removal of over 80% of membrane protein by Pronase digestion did not release any significant amount of Mg(2+). Hence, most of the divalent cation appears to be bound to membrane lipids, most probably to phospholipids. Ethylenediaminetetraacetic acid released the bulk of Mg(2+) bound to the native and reaggregated A. laidlawii membranes, except for about 0.5 mug of Mg(2+) per mg of protein which was too tightly bound. Hence, a small but fairly constant amount of Mg(2+) is unavailable for chelation.     

Ribosomal RNA genes in Mycoplasma

Using Southern blotting analysis with labelled mycoplasmal ribosomal RNA as probe, two fragments (1 Kb and 5 Kb) were detected in an EcoR I digest of Mycoplasma capricolum DNA. This analysis revealed that the 5 Kb fragment carries both 16S rRNA sequences and the entire 23S rRNA gene of this mycoplasma. The 1 Kb fragment contains 16S rRNA sequences only. The 5 Kb EcoR I fragment has been cloned and used to characterize the structure of rRNA cistrons in various Mycoplasma strains. These experiments clearly demonstrate a substantial homology of Mycoplasma capricolum rRNA sequences with the E. coli rRNA cistron on one hand, and with Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii on the other hand. This analysis also reveals two rRNA cistrons in Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii whereas one rRNA cistron is present in Mycoplasma capricolum.     

Disposition of exposed antigens on the faces of isolated Mycoplasma gallisepticum membranes.

The transverse disposition of exposed protein antigens on the two faces of isolated Mycoplasma gallisepticum membranes have been investigated by using indirect immunoferritin labeling to accomplish visualization of the antigens at the ultrastructural level. Comparison between the labeling patterns obtained with unabsorbed specific mycoplasma antiserum and antiserum from which antibodies directed against outer side determinants had been removed revealed that the majority of protein antigens were the same on the opposed membrance faces or at least displayed extensive interside cross-reactivity. The relatively scarce tagging of isolated Acholeplasma laidlawii membranes, contrary to membranes on intact organisms observed in this investigation, precluded conclusions regarding the disposition of membrane antigens of this species. The advantages and limitations of the employed method in disposition studies and the factors influencing the transverse distribution of membrane proteins in mycoplasmas are discussed.     

Reduced nicotinamide adenine dinucleotide oxidase activity in membranes and cytoplasm of Acholeplasma laidlawii and Mycoplasma mycoides subsp. capri.

The properties of the membrane-bound reduced nicotinamide adenine dinucleotide (NADH) oxidase of Acholeplasma laidlawii were compared with those of the corresponding cytoplasmic activity of Mycoplasma mycoides subsp. capri. The striking differences in pH optima, susceptibility to inhibitors and detergents, and heat inactivation between the NADH oxidase activity, with oxygen as an electron acceptor, and the NADH oxidoreductase activity, with dichlorophenol indophenol (DCPIP) as an alternate electron acceptor, support the presence of more than one catalytic protein in both the membrane-bound and soluble enzyme systems. The detection of more than one band positive for the NADH-nitroblue tetrazolium oxidoreductase reaction on electrophoresis of either the membranes of A. laidlawii or the cytoplasm of M mycoides subsp. capri also points in the same direction. The membrane-bound enzyme system differed, however, form the soluble one because it had a lower ratio of oxidase activity to oxidoreductase activity, and because it was less susceptible to heat inactivation and more readily incorporated incorporated into reaggregated membranes. In addition, the specific activity of the membrane-bound enzyme system increased as the culture aged, whereas that of the soluble system decreased as the culture aged. It is suggested that the different location in the cell could be responsible for some of the differences between the membrane-bound NADH oxidase activity of A. laidlawii and that found in the cytoplasm of M. mycoides subsp. capri.