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This communication reports on the release of Mycoplasmavirus L1 after infection of Acholeplasma laidlawii with purified L3 virus. Release also occurred after transfection with certain restriction fragments from MV-L3 and MV-L1 genomes. Since circular molecules are efficiently taken up in polyethylene glycol-mediated transfection, inducing fragments were applied cloned in E. coli plasmids. Release was also observed after electroporation of cells incubated with MV-L1 replicative intermediate DNA and linear MV-L3 DNA isolated from virus particles, respectively. Released MV-L1 viruses were identified after virus plaque formation on indicator lawns according to plaque morphology and hybridization with labeled viral DNA probes as well as by DNA restriction analysis. Uninfected and untransfected cells from six laboratory strains of A. laidlawii (including a MV-L1 resistant one) were examined for the presence of MV-L1 DNA. They all bear MV-L1 DNA integrated in their genomes.

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Lipoglycans , distinguishable from bacterial lipopolysaccharides, are associated with the cytoplasmic membranes of several genera of Mollicutes, namely Acholeplasma, Mycoplasma neurolyticum , Anaeroplasma , and Thermoplasma. Structurally, the lipoglycans are long heteropolysaccharides covalently linked to a lipid. The exact structures of three have been determined. Thermoplasma oligosaccharide is attached to a diglycerol tetraether ; A. granularum to a diacyl glycerol. The lipoglycan from A. axanthum is unique by its possession of glycerol phosphate and galactose phosphate side chains and the occurrence of fatty acids in N-acyl linkages. Only one molecular species of lipoglycan occurs in a given species. These lipoglycans possess a variety of biological activities. The terminal three sugar residues define their antigenic specificity; they attach to specific receptors on mammalian cells; they exhibit pyrogenicity in rabbits and clot Limulus lysate; they stimulate the production of IgM antibody both in vivo and in vitro; they modulate the immune response to T-cell dependent antigens; they exhibit immunosuppressive and immunostimulatory activities.

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The partial characterization of the structure of the lipoglycan (LG) from Acholeplasma axanthum is added to the previous complete structural analysis of the lipoglycan from A. granularum. The terminal sequence of A. axanthum LG is Glcp(beta 1----2)-Glcp(beta 1----2)-Glcp(beta 1----6)-; of A. granularum Glcp(beta 1----2)-Glcp(alpha 1----4)-Glcp(beta 1----4)-. These specific residues define the major antigenic determinants of the LG as determined by blockage of hemagglutination of LG coated erythrocytes by specific oligosaccharides and binding of radiolabeled LG to specific immunoglobulins. The binding of LG to mammalian cells occurs by an interaction between specific eucaryotic cell receptors and the internal sequence of the oligosaccharide chain of LG. Size and sugar chains of LG rather than fatty acid residues appears to define the binding site on the LG.

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Comparison of the lipid composition between members of the Mycoplasmatales reveals a striking diversity of lipid structures, not only between the six genera but among species within the same genus. This is in contrast to nearly all other bacterial groups in which members of the same genus possess essentially the same lipids. There are in fact more similarities between lipids of a given species of mycoplasma and a genus of bacterium than there are between lipids of a given species of mycoplasma and a genus of bacterium than there are between mycoplasma species. Mycoplasmal lipids suggest that these organisms do not represent a phylogenetically related group at all, but are probably degenerative forms of bacteria, particularly gram-positive bacteria, which have lost the ability to synthesize a cell wall.

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Acholeplasma axanthum is one of the few procaryotes, and the only member of the Mollicutes, known to contain phosphosphingolipids. Examination of strain S743 for glycolipids revealed the presence of glucosides of cholesterol and galactosides of glycerol as the predominant glycolipids. The major component is acylated diglucosylcholesterol, followed by monogalactosyldiacylglycerol and monoglucosylcholesterol. The glucose residues of the sterol-based compounds appear to be alpha-linked pyranoses, while the galactose of the glycerol-based lipid is an alpha-linked furanose. The "glycolipid' fraction also contained N-(3-hydroxy)acyl sphinganines with varying degrees of O-acylation. None of these ceramide derivatives was linked to carbohydrate. The major glycolipid, tentatively identified as alpha-D-glucopyranosyl-(1 leads to 3)-(O-acyl)-alpha-D-glucopyranosyl-(1 leads to 3)-cholesterol, along with its deacylated derivative, appears to be the first reported instance of steryl diglycosides among procaryotes, in contrast to the steryl monoglycosides, which are common to other mycoplasmata and some spirochetes.

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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.

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The proteins of 91 strains of Mycoplasma from 19 established species and three groups of uncertain taxonomic position were separated by two-dimensional polyacrylamide gel electrophoresis. Considerable variations in protein patterns were found among strains from the same species or subspecies; the percentage of matching spots (% of congruence) ranged from 42%-100%, but many proteins were strongly conserved in all strains of a given species. Maps of these conserved proteins could be used to identify strains. It was concluded that (1) large colony-type strains of Mycoplasma mycoides subspecies mycoides are more closely related to M. mycoides subspecies capri than to small colony-type strains of M. mycoides subspecies mycoides; (2) Mycoplasma capricolum, Leach group 7, and the F38 group of mycoplasmas are all related to M. mycoides; (3) the 2D group of strains are not related to either M. mycoides or Mycoplasma bovis, (4) M. bovis and Mycoplasma agalactiae are closely related; (5) apart from a small overlap in pattern between Acholeplasma laidlawii and Acholeplasma granularum, the type strains of seven established species of Acholeplasma each had distinct protein patterns that justified their classification as separate species; and (6) analysis of two-dimensional protein patterns is a valuable method for resolving problems in mycoplasma taxonomy and for identifying strains.

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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: Glcp(beta 1 leads to 2)-Glcp(alpha 1 leads to 4)-Glcp(alpha 1 leads to 3,4)-FcNAc(beta 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3,4)-GlcNAc(beta 1 leads to 3,4)-GlcNAc(beta 1 leads to 4)-[Glcp(beta 1 leads to 2)-Glcp(alpha 1 leads to 4)-Glcp(alpha 1 leads to 3,4)-FcNAc(beta 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3,4)-GlcNAc(beta 1 leads to 3,4)-GlcNAc(beta 1 leads to 4)]10-Glcp(beta 1 leads to 2)-Glcp(alpha 1 leads to 4)-Glcp(alpha 1 leads to 3,4)-FcNAc(beta 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3)-Galp(alpha 1 leads to 3,4)-GlcNAc(beta 1 leads to 3,4)-GlcNAc-diacylglycerol. The position of the linkages (3 or 4) on the amino sugars has not been resolved.

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Several Mycoplasma and Acholeplasma species chosen at random and solubilized with sodium dodecyl sulphate showed a common periodic acid-Schiff positive band with an apparent molecular weight of about 64 000, when examined by polyacrylamide gel electrophoresis. Another more cathodic minor band was detected in M. hyopneumoniae and M. flocculare. The common periodic acid-Schiff positive band appeared when a precipitate of serum constituents of the uninoculated growth medium after incubation was examined. The minor band was identified as a serum glycoprotein contaminating mycoplasmas grown in the presence of swine serum. We draw attention to the compounds as a possible source of error in serological tests or in the lymphocyte stimulation response. After lithium diiodosalicylate solubilization and aqueous phenol extraction, polyacrylamide gel electrophoresis showed a periodic acid-Schiff positive band in membranes from M. hyopneumoniae (molecular weight 75 000) and M. hyorhinis (molecular weight 80 000), suggesting the presence of a membrane glycoprotein. Such a glycoprotein was absent from A. granularum. Since the common periodic acid-Schiff positive band was not extracted by aqueous phenol, this growth medium constituent did not contaminate the preparations of membrane glycoproteins. However, the minor band was present in glycoprotein preparations of M. hyopneumoniae grown in the presence of swine serum.

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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, [beta-D-glucopyranosyl-(1 leads to 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 beta-D-glucopyranosyl-(1 leads to 3)-alpha-D-glucopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl-(1 leads to 1)-diacylglycerol.

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The total lipid content of Acholeplasma oculi comprises 13.3% of the dry weight of the organism and is about equally distributed between the neutral lipids plus glycolipids and the phospholipids. The phospholipids were identified as phosphatidyl glycerol and diphosphatidyl glycerol. The glycolipid fraction contained O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol and O-alpha-D-glucopyranosyl-(1 leads to 2)-O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol. The neutral lipid contained pigmented carotenoids. Hot aqueous phenol extraction of lipid-extracted whole cells yielded a polymeric carbohydrate comprising 2.3% of the dry weight of the organism. The A. oculi lipopolysaccharide was found to contain only neutral sugars and no amino sugar, in contrast to other acholeplasmas. The neutral sugars consisted of fucose, galactose, and glucose in a ratio of 2:19:3.

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The neutral lipids of Acholeplasma axanthum contain carotenoid pigments, as evidenced by spectral characteristics, visual color, color reactions, and labeling with [2-14C-A1mevalonic acid. Approximately 80% of the label from [2-14C]mevalonic acid appeared in esterified fatty acids of the glycolipids and polar lipids. These carboxylic acids behaved as hydroxy acids of varying chain length.

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The fatty acid composition of the total lipids obtained from 9 species (22 strains) of Mycoplasma and 3 species (7 strains) of Acholeplasma was determined by gas-liquid chromatography (GLC). The major fatty acids of the Mycoplasma species were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). Lauric acid (C12:0) and myristic acid (C14:0) were present in small amounts in this genus. For the Mycoplasma species, the most prevalent fatty acid was C16:0 or C14:0, and other leading fatty acids were C12:0, C18:0, C18:1 and C18:2. A substantial amount of C12:0 and C14:0 was found in the Acholeplasma species. It was confirmed that C12:0 and C14:0 were synthesized de novo, based on the fact that 14C-acetate was incorporated into these acids. The total percentage of C16 fatty acid was less than that of C18 fatty acids in all the strains of Mycoplasma and Acholeplasma except 4 strains. This may be related to osmo-regulation of the L-forms of Streptococci as mentioned in other reports.

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Phenol-acetic acid-water extracts of 13 recognized mycoplasma and acholeplasma reference strains of bovine habitat were subjected to polyacrylamide gel electrophoresis. A characteristic electrophoretic profile was obtained for each strain and the relative mobility (Rm) values of the major protein fractions were determined. All examined strains had a common protein band of 33.3 Rm value. Comparison of electrophoretic patterns obtained from different reference strains revealed 2 to 7 protein bands with the same Rm values in a number of strains. A possible correlation with antigenic relatedness is discussed.

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Five methods were employed to determine the heterogeneity or homogeneity of lipopolysaccharides from four acholeplasmal species, Acholeplasma axanthum, A. granularum, A. laidlawii, and A. modicum. A axanthum lipopolysaccharide behaved as a single component in all tests. A. granularum exhibited two components of identical composition and antigenic specificity. A. modicum lipopolysaccharide behaved as three components in two tests, but all three were similar in composition and identical serologically. The separable components of lipopolysaccharides from A. granularum and A. modicum probably represent size differences only. A. laidlwii lipopolysaccharide contained two distinct components by all methods. One was identified as the previously reported amino sugar polymer, whereas the other was a lipopolysaccharide containing both neutral and amino sugars.

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Proteins extracted with phenol-acetic acid-water (2:1:0.5, w/v/v) from Acholeplasma laidlawii (PG 8), A. granularum (BTS-39), A. oculi (19L), A. modicum (PG 49), A. axanthum (S743) and the Acholeplasma strains C1 and C112 (which were isolated from aborted horse foetuses) were compared by electrophoresis in horizontal acidic polyacylamide flat gel using the electrophoresis equipment LKB Multiphor 2117. In this system the gels are not prepared in the electrophoresis chamber but between glas plates. For electrophoresis they are applied onto a special cooling plate. This makes it possible to produce a number of identical gels (from the same gel mixture and polymerized under the same conditions) what can be important for comparing investigations. The gels can be stored for more than 4 weeks in the refrigerator at +4 degrees C. Marked differences were observed between the electrophoretic patterns of each of the established species and the horse strains C1 and C112. The results are in agreement with those obtained in serological investigations in which the strains C1 and C112 were different from the established Acholeplasma species.

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Polyacrylamide gel isoelectric focusing (PAGIF) in thin layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp., and eight strains of Ureaplasma urealyticum (T-strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells. PAGIF was performed in the range of pH 3 to 10. Protein patterns were carefully compared, demonstrating resolved and distinguishable species-specific protein bands. The eight serotypes of U. urealyticum (T-strain) gave identical protein patterns in the pH 3 to 10 range. The characteristic "fingerprints" of a species appeared to correlate with the biochemical nature and not the habitat in each case. Arginine-hydrolyzing species seemed to show more diverse focusing than those that ferment glucose, or prefer an acid environment. Characterization and identification of highly resolved species-specific proteins, ease of performance, and reproducibility of this method suggest that PAGIF might be employed as a taxonomic aid.

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