Acholeplasma Granularum Research Articles

Distinctions in Mollicutes Purine Metabolism: Pyrophosphate-Dependent Nucleoside Kinase and Dependence on Guanylate Salvage

Cell preparations of Acholeplasma florum L1T, Acholeplasma axanthum S743T, Acholeplasma granularum BTS-39T, Spiroplasma floricola 23-6T, Mycoplasma gallisepticum S6, and Mycoplasma arginini G230T were examined for 13 cytoplasmic enzyme activities involved in the salvage and interconversion of nucleobases, nucleosides, and 5′-mononucleotides. The unique pyrophosphate-dependent nucleoside kinase activity known only in Acholeplasma laidlawii B-PG9 was found in A. axanthum and A. granularum. All the organisms could be divided into three groups based upon their patterns of purine salvage and interconversion activities. All the tested organisms, A. laidlawii, and apparently Mycoplasma mycoides subsp. mycoides lack the ability to synthesize guanylates from other purine mononucleotides, indicating that these members of the class Mollicutes can only salvage guanylates. The plant epiphytes A. florum and S. floricola had an identical purine enzyme pattern which was different from those of all other members of the Mollicutes studied.

Synthesis of Lipids from Acetate Is Not Characteristic of Acholeplasma or Ureaplasma Species

Previous studies of Acholeplasma species indicated that this genus could be separated from Mycoplasma on the basis of the ability to synthesize lipids from acetate. We report that recently described strains of Acholeplasma florum (strain L1 isolated from a lemon flower and strain GF-1 isolated from a grapefruit flower) and two unclassified Acholeplasma strains (strain J233 from coconut palm and Acholeplasma sp. strain PS-1 from an insect) convert little if any acetate carbon to lipid, in contrast to Acholeplasma sp. strain 0502-CL1 isolated from broccoli and six other established Acholeplasma species of animal origin. Thus, the ability to synthesize lipid from acetate in Edward test medium containing horse serum is not characteristic of all members of the genus Acholeplasma (class Mollicutes), but appears to be a property only of a subgroup in the genus containing the species that are most frequently isolated from animals (Acholeplasma laidlawii, Acholeplasma granularum BTS39, Acholeplasma oculi 19L, Acholeplasma equifetale N93, and probably Acholeplasma axanthum S743 and Acholeplasma modicum PG49). Like other Mycoplasma species which we studied, Mycoplasma bovigenitalium PG11, Mycoplasma hominis PG27, Mycoplasma arginini G230, and four sterol-requiring mycoplasmas of insect or plant origin (Mycoplasma sp. strain MQ3 from an insect, Mycoplasma sp. strain pommier from an apple, Mycoplasma sp. strain melaleuca from a Melaleuca flower, and Mycoplasma sp. strain 831-C4 from lettuce) convert little acetate to lipid. Similarly, eight serovars of Ureaplasma urealyticum convert little if any acetate to lipid.

Immunological Heterogeneity of Superoxide Dismutases in the Acholeplasmataceae

The superoxide dismutases from Acholeplasma laidlawii and Acholeplasma hippikon were serologically related, whereas the superoxide dismutase from Acholeplasma equifetale appeared to be unique serologically. Enzymes from Acholeplasma granularum and Acholeplasma axanthum did not show any serological reactivity. Moreover, the acholeplasmic enzymes exhibited electrophoretic heterogeneity, indicating the evolutionary diversity of superoxide dismutases.

Identification of the major antigenic determinants on lipoglycans from Acholeplasma granularum and Acholeplasma axanthum.

The major antigenic determinants of the lipoglycans from Acholeplasma granularum and Acholeplasma axanthum were found to be the oligosaccharide sequences Glcp(beta 1 leads to 2)-Glcp(alpha 1 leads to 4)-Glcp and Glcp(beta 1 leads to 2)-Glcp(beta 1 leads to 2)-Glcp, respectively. The disaccharides sophorose and maltose inhibited hemagglutination by specific antiserum of sheep erythrocytes coated with lipoglycan from A. granularum. Only sophorose possessed this capacity with the lipoglycan from A. axanthum. Periodate oxidation destroyed the ability of both lipoglycans to interact with specific antibody, a result compatible with structural data on the lipoglycans.

Electrophoretic Analysis of Isoenzymes of Acholeplasma Species

Thirteen Acholeplasma strains representing three named species, Acholeplasma laidlawii, Acholeplasma granularum, and Acholeplasma oculi, were examined for the presence of 30 enzymes by horizontal starch gel electrophoresis. A total of 29 enzymes were demonstrated. Twelve of these enzymes have not been reported previously in acholeplasmas (alcohol dehydrogenase, aldolase, alkaline phosphatase, arginase, arginine deiminase, carbarnyl phosphokinase, galactose dehydrogenase, galactose-6-phosphate dehydrogenase, glutamate dehydrogenase [nicotinamide adenine dinucleotide], glutamate dehydrogenase [nicotinamide adenine dinucleotide phosphate], α-glycerophosphate dehydrogenase, and xanthine dehydrogenase). An average group cluster analysis was carried out on the basis of estimates of dissimilarity coefficients. This analysis distinguished among the strains tested, was of some help in identification, and may also be useful in epidemiological investigations.

Survival of Mycoplasma, Acholeplasma and Spiroplasma at different temperatures.

In order to determine the maintenance of viability of Mycoplasma pneumoniae, Mycoplasma salivarium, Acholeplasma laidlawii, Acholeplasma granularum, and Spiroplasma mirum, SMCA strain, outside the human and animal bodies and plant tissues, these mycoplasmal cells were kept in two kinds of solutions and at different temperatures, and their survival periods were measured. The results obtained indicate that, in general, except for M, sali-varium, a solution containing a high concentration of protein, i. e. horse serum and albumin, is more suitable than a solution containing horse serum only for maintaining viability, and that a low temperature is better than a high tem-perature, as expected. In the results obtained here, the survival periods of M. pneumoniae, M. salivarium, A. laidlawii, A. granularum and S. mirum SMCA in phosphate buffered saline containing 1% horse serum and 0.1% bovine serum albumin V (PBS, HS, BSA) were approximately 80, 30, 230, 200 and 100 days, respectively.

Isolation and characterization of the sheep erythrocyte receptor for acholeplasmal lipoglycans.

A receptor specific for lipoglycans from Acholeplasma axanthum and Acholeplasma granularum was isolated from sheep erythrocyte stroma by extraction with n-pentanol and permeation chromatography. The purified receptor appeared as one band on sodium dodecyl sulfate-polyacrylamide gels and stained with Coomassie blue, periodate-Schiff reagent, and Sudan black. It was distinct from the erythrocyte receptor for gram-negative lipopolysaccharides and the glycophorin receptor for certain species of Mycoplasma. Periodate oxidation and trypsin did not affect the receptor activity in intact erythrocytes, but the purified receptor was susceptible to proteolytic digestion. Specific receptors, sensitive to trypsin digestion, could be isolated from rabbit kidney and cultured rabbit epidermal cell membranes. These could be distinguished from the receptor from erythrocytes by their solubility in n-pentanol. The segment of the lipoglycan molecule which binds to these receptors was not lipoidal in nature and was distinct from the specific antigenic determinants of the lipoglycans.

Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and viruses

The effects of tylosin tartrate and tiamutilin were examined in pneumonias induced experimentally in neonatal piglets with a homogenate of pneumonic pig lung, obtained from pigs with naturally acquired enzootic pneumonia. The homogenate contained mycoplasmas, including Mycoplasma hyopneumoniae (M suipneumoniae) and M hyorhinis, and certain bacteria and viruses. The experimental pneumonias generally resembled mycoplasmal pneumonia histologically but were complicated by aspiration pneumonia in some animals. both tylosin tartrate (50 mg/kg) and tiamutilin (10 mg/kg) administered orally twice daily for 10 days, beginning 14 days after intranasal infection, significantly reduced the incidence and severity of macroscopical pneumonic lung lesions. M hyopneumoniae could be isolated from the lungs of the unmedicated piglets, but not from drug-treated piglets. The numbers of M hyorhinis, Acholeplasma granularum, Haemophilus parasuis, Pasteurella multocida and P haemolytica in the lung tissue of the infected piglets were significantly reduced by drug therapy. The role of bacterial in the experimental infection appeared to be that of secondary invaders.

Structure of the oligosaccharide chain of lipoglycan from Acholeplasma granularum

The membrane associated lipoglycan from Acholeplasma granularum is a linear oligosaccharide attached to a diacylglycerol. The polymer has a monomeric weight of 20000 and is composed of glucose, galactose, N-acetylglucosamine, N-acetylfucosamine, glycerol and fatty acid esters. The proposed structure of the oligosaccharide chain is 12 repeating units of 9 sugars: Glc p( β1 → 2)-Gla p( α1 → 4)-Glc p ( α1 → 3,4)- FcNAc( α1 → 3)-Gal p( β1 → 3)-Gal p ( α1 → 3)-Gal p( α1 → 3,4)- GlcNAc( β1 → 3,4)- GlcNAc( β1 → 4)-[Glc p ( β1 → 2)-Glc p( α1 → 4)-Glc p( α1 → 3,4)- FcNAc-( β1 → 3)-Gal p ( α1 → 3)-Gal p( α1 → 3)-Ga p(→1 → 3,4)- GlcNAc( β1 → 3,4)- GlcNAc( β1 β 4)] 10-Glc p( β1 → 2)-Glc p( α1 → 4)-Glc p( α1 → 3,4)- FcNAc( β1 → 3)-Gal p( α1 → 3)-Gal p( α1 → 3)-Gal p ( α1 → 3,4)- GlcNAc( β1 → 3,4)- GlcNAc-diacylglycerol. The position of the linkages (3 or 4) on the amino sugars has not been resolved.

Respiration-associated components of Mollicutes

No cytochrome pigments were detected by difference (reduced minus oxidized) spectroscopy at liquid nitrogen temperature in whole-cell preparations or membrane fractions of Acholeplasma axanthum S273, Acholeplasma equifetale N93, Acholeplasma granularum BTS39, Acholeplasma laidlawii B-PG9, Acholeplasma modicum PG-49, Acholeplasma oculi 19L, Mycoplasma arginini G230, Mycoplasma arthritidis 07, Mycoplasma pneumoniae FH, and Mycoplasma pulmonis JB. All ten Mollicutes species examined contained iron of unknown function (3.0 to 15.3 nmol of iron per mg of protein). Relatively small amounts of acid-labile sulfide were found in all fractions (0.10 to 1.07 nmol of acid-labile sulfide per mg of protein). The data suggest that, as Mollicutes lack cytochrome pigments, they would synthesize most if not all adenosine triphosphate at the substrate level.

Isolation and identification of mycoplasmas from pig lungs in Singapore

A total of 656 lung specimens comprising 196 grossly pneumonic lungs from clinically diseased pigs and 230 grossly pneumonic and normal lungs from abattoirs were cultured for mycoplasmas. Mycoplasmas or acholeplasmas were recovered from 102 lung specimens, of which 28 were serologically identified (by disc growth inhibition and agar gel diffusion tests) as Mycoplasma suipneumonia, 14 as M hyorhinis, 35 as Acholeplasma granularum and 4 as A laidlawii. In addition, 15 isolates were unclassified and six were not typed. Mycoplasmas isolated from the suspect enzootic pneumonic lungs: slaughterhouse pneumonic lungs: normal lungs were in the ratio of 3:2:1. M suipneumonia, A granularum and A laidlawii were isolated from both normal and pneumonic lungs of pigs while M hyorhinis was only isolated from pneumonic ones. A close antigenic relationship between M suipneumonia and A granularum was demonstrated.

Bacterial lipopolysaccharides and mycoplasmal lipoglycans: A comparison between their abilities to induce macrophage-mediated tumor cell killing and [formula omitted] amebocyte lysate clotting

The ability of various bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) to induce macrophage-mediated tumor cell killing and Limulus amebocyte lysate clotting was determined. Lipoglycans from the mycoplasma Acholeplasma axantum or Acholeplasma granularum had no activity or 10 4 to 10 5 less activity than lipopolysaccharides from Escherichia coli 0128:B12, Escherichia coli K235, or Salmonella minnesota R595 in causing Limulus lysate clotting and tumor cell killing by peritoneal macrophages from normal or bacillus Calmette-Guérin-infected mice. Previous studies have shown that the lipid A portion of bacterial lipopolysaccharide is responsible for the effects on macrophage-mediated tumor cell killing and Limulus lysate clotting. The known differences in the lipid structures of bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) may account for the noted differences in the biologic potencies observed here.

An aldehydophosphoglycolipid from Acholeplasma granularum

A novel phosphoglycolipid and a triglucosyl diacylglycerol were found among the lipids of Acholeplasma granularum. The tentative structure of the phosphoglycolipid was determined to be a phosphotriester, [β-d-mglucopyranosyl-(1 → 1)-X-glycerol-3-O-] [d-glyceraldehyde-3-O-][1,2-diacyl-X-glycerol-3-O-kojibiosyl-6-O-]phosphate. The structure assigned to the triglucosyl lipid was β-d-glucopyranosyl-(1 → 3)-α-d-glucopyranosyl-(1 → 2)-→-d-glucopyranosyl-(1 → 1)-diacylglycerol.

Classification and Identification of Ovine and Caprine Mycoplasmas

The purpose of this investigation was to give a survey of the classification of ovine/caprine mycoplasmas as a basis for the identification of strains isolated from sheep and goats. A total of 13 strains representing 13 species and/or serogroups were biochemically examined, and serological cross-titrations were performed using metabolism inhibition, growth inhibition and immunofluorescence. Serogroup 6 (Al-Aubaidi) was found to be identical with Mycoplasma capricolum. The results of identification of 57 isolates, sent to the reference centre from different countries, are given. On the basis of the above investigations and a comparison of some of the classification systems described in the literature, it is concluded that the following species have been isolated from goats and/or sheep: M. agalactiae, M. arginini, M. bovis, M. capricolum, M. conjunctivae, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. ovipneumoniae, M. putrefaciens, Acholeplasma granularum, A. laidlawii and A. oculi. In addition, both Ureaplasmas and strains representing 6 serogroups (groups 5, 7, 10 and 11 of Al-Aubaidi and groups 17 and 18 of Cottew) have been isolated. These serogroups ought to be finally species-classified as soon as possible. kw|Keywords|k]ovine/caprine mycoplasmas; k]classification; k]identification

Localization of Reduced Nicotinamide Adenine Dinucleotide Oxidase Activity in Acholeplasma and Mycoplasma Species

Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (EC 1.6.99.1) was assessed in membrane and supernatant fractions obtained by osmotic lysis and differential centrifugation from Acholeplasma granularum, A. modicum, A. axanthum, and Mycoplasma strain 14. NADH:ferricyanide oxidoreductase activity (EC 1.6.99.1) was also assessed in fractions of A. laidlawii B and the mycoplasma. NADH oxidase and NADH:ferricyanide oxidoreductase activities were localized in the membrane fraction of the acholeplasmas and in the supernatant fraction of the mycoplasma. The localization of NADH oxidase in membrane fractions of cells of Acholeplasma and supernatant fractions of cells of Mycoplasma after osmotic lysis is useful in differentiating these genera.

Localization of Reduced Nicotinamide Adenine Dinucleotide Oxidase Activity in Acholeplasma and Mycoplasma Species

Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (EC 1.6.99.1) was assessed in membrane and supernatant fractions obtained by osmotic lysis and differential centrifugation from Acholeplasma granularum, A. modicum, A. axanthum, and Mycoplasma strain 14. NADH:ferricyanide oxidoreductase activity (EC 1.6.99.1) was also assessed in fractions of A. laidlawii B and the mycoplasma. NADH oxidase and NADH:ferricyanide oxidoreductase activities were localized in the membrane fraction of the acholeplasmas and in the supernatant fraction of the mycoplasma. The localization of NADH oxidase in membrane fractions of cells of Acholeplasma and supernatant fractions of cells of Mycoplasma after osmotic lysis is useful in differentiating these genera.

Hydrolysis of Aesculin and Phosphatase Production by Members of the Order Mycoplasmatales Which Do Not Require Sterol

Two bacteriological methods, one to demonstrate phosphatase production and the other to demonstrate aesculin hydrolysis, were tested on sterol-requiring Mycoplasma and sterol-nonrequiring Acholeplasma strains to determine their utility in differentiating species. The phosphatase test, which gave weak and variable reactions with most of the sterolnonrequiring strains, was judged ineffectual for differentiating these strains. The aesculin test distinguished all Mycoplasma strains and Acholeplasma granularum, both of which gave negative reactions, from most Acholeplasma laidlawii strains and from Acholeplasma axanthum, which gave positive reactions.