Acholeplasma vituli sp. nov., from bovine serum and cell cultures.

Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli.

Mollicutes-wall-less bacteria with internal cytoskeletons.

The structure and motility of the Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma) are briefly reviewed. The data are presented from the perspective of prokaryotic and eukaryotic motors, cytoskeletons, and cell motility. The Mollicutes are eubacteria derived from Clostridia by regressive evolution and genome reduction to produce the smallest and simplest free-living and self-replicating cells. Structurally, the Mollicutes are characterized by a complete lack of a cell wall and the presence of an internal cytoskeleton. Spiroplasma, which are helical cells with a flat, ribbon-like cytoskeleton, are amenable to structural and geometrical analysis. Motility and shape changes can be explained and modeled by the cytoskeleton acting as a linear motor.

Ultrastructural changes in mollicutes induced by the peptide antibiotic herbicolin A.

Electron microscopy of negatively stained mycoplasma, ureaplasma, and acholeplasma cells showed ultrastructural changes after 10 min of treatment of the organisms with the peptide antibiotic herbicolin A in concentrations ranging from 10 micrograms/ml for Mycoplasma capricolum to 600 micrograms/ml for Ureaplasma urealyticum. The morphological changes were shown to be reversible at low concentrations of the antibiotic but irreversible at high concentrations.

Characterization of a mycoplasma virus (MV-O1) derived from and infecting Acholeplasma oculi.

A new Mycoplasmatales virus, referred to as MV-O1, was isolated during cloning of Acholeplasma oculi 19L. The virus formed plaques only on strains of A. oculi, i.e. the original clone, A. oculi 19L, a subclone of A. oculi 19L (A.oculi-i), A. oculi Goat 5 and wild isolate (K-2) of A. oculi, but not on other acholeplasmas, including strains of A. laidlawii, nor on five human mycoplasma species tested. The virus required horse serum for multiplication as well as for plaque formation and passed through a 100 nm filter. Electron microscopy revealed enveloped, spherical particles 80-130 nm in diameter. The buoyant density of purified virus was 1.23 g ml-1 in CsCl, and agarose gel electrophoresis indicated that the viral nucleic acid was DNA.

The effect of mycoplasmas and acholeplasmas of equine origin on organ cultures of chicken-embryo trachea.

Chicken-embryo tracheal organ cultures were inoculated with equine strains of Mycoplasma arginini, M. equigenitalium, 2 strains of M. subdolum, Acholeplasma laidlawii and 3 strains of A. oculi. All strains established and multiplied in the explant cultures, but only M. subdolum and A. oculi produced a cytopathic effect on ciliated epithelial cells, causing sloughing of cells and cilia after 6 days. There was a correlation between ciliostasis and increase in titre of both M. subdolum and A. oculi and this relationship was not observed with M. equigenitalium and A. laidlawii. All the strains of acholeplasma multiplied to some extent in organ culture media, but reached higher titres in the presence of explants. Cells infected with the M. subdolum strain showed sloughing of cilia, vacuolization, and increase in size of mitochondria, followed by disorganization of epithelium and marked destruction of subcellular organelles. Mycoplasmas were closely attached to the epithelial surface of the tracheal explant 8 days after infection.

Insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas.

The insect cell lines Dm-1 (Drosophila melanogaster), AS-2 (Aceratagallia sanguinolenta) and AC-20 (Agallia constricta) were infected with spiroplasmas, mycoplasmas and Acholeplasma laidlawii. In Dm-1 cultures maintained at 25 degrees C in M1A medium, all strains multiplied except M. hyorhinis and the uncultivable sex-ratio organism. Spiroplasma citri R8A2, S. floricola BNR-1 and OBMG, S. apis PPS-1 and the strains BC-3, corn stunt spiroplasma (CSS) and 277F produced cytopathogenic effects (CPE), whereas S. mirum SMCA, M. orale, M. arginini and A. laidlawii did not. Cytadsorption was found with the cultivable spiroplasmas and A. laidlawii. At 30 degrees C SMCA, M. orale, M. arginini and A. laidlawii killed the Dm-1 cultures. M. hyorhinis grew without any CPE. In AS-2 and AC-20 cultures grown at 28 degrees C in LB medium, R8A2, B88, 277F, BNR-1 and PPS-1 multiplied and reached titres of 2 X 10(8) to 4 X 10(9) CFU/ml. They produced CPE leading to culture death. CSS did not grow. R8A2 reached higher titres in AS-2 cultures than in fresh LB medium. This stimulating factor was studied by means of conditioned medium. All 6 spiroplasmas cytadsorbed to AS-2 and AC-20 cells. B88 and 277F adsorbed heavily, while the other 4 strains adsorbed only slightly. Fluorescent DNA staining with "Hoechst 33258" revealed the presence of non-helical forms inside the cells.

Cytoplasmic helical structure associated with Acholeplasma laidlawii.

A distinct spiral protein structure was found in three species of Acholeplasma, but was not found in the Mycoplasma species studied. The spirals, which are 14 nm in width and of variable length from 50 to 300 nm, are formed by a helical arrangement of 7-nm subunits. A rosette-like structure 45 nm in diameter also composed of 7-nm subunits was found in close association with the spirals and may be a taut in vivo form of the spiral. The electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gels indicated that the spirals are composed of a predominant polypeptide with an apparent molecular weight of 100,000. No evidence can be found for inferring actin-like properties for this structure.

Ultrastructural localization of tellurite reduction in Acholeplasma species.

Aerobic reduction of tellurite by five Acholeplasma species and two Mycoplasma species was investigated by light and electron microscopy. Among the Acholeplasma species, colonies of A. laidlawii and A. oculi exhibited a heavy, macroscopically visible reduction of tellurite, whereas the reaction of A. axanthum was weaker. A. granularum and A. modicum did not reduce the substrate under the experimental conditions employed. The two subspecies of M. mycoides also reacted with tellurite, as did also the investigated strain of M. bovigenitalium although to a lesser extent. Ultrastructurally, reduction sites were localized to the cytoplasmic membrane in the three tellurite positive Acholeplasma species and apparently to the cytoplasm of M. mycoides subsp. mycoides. Reduction sites could not be demonstrated in M. mycoides subsp. capri and in M. bovigenitalium. The results support previous evidence obtained by biochemical methods which indicates membrane localization of redox enzymes in Acholeplasmas.

Lysis of Acholeplasma laidlawii by antibodies and complement.

Membranes of Acholeplasma laidlawii were used to determine the membrane components involved in immune lysis and to eventually detect enzymatic changes in the membrane components during this reaction. In a previous publication we reported that A. laidlawii can be killed by antibody and complement and that antibodies to membrane proteins are by far more effective than antibodies to membrane lipids in the complement-dependent killing of this organism. In this report we demonstrate that membrane damage occurs after the combined action of antibody and complement. In addition, the cytoplasmic enzyme hexokinase is released during immune killing. As visualized by electron microscopy, the organisms lost their cytoplasm and were transformed into ghosts. In the majority of organisms, tears in the membrane could be seen. Ultrastructural lesions of about 8.0 to 10 nm in diameter were visible when the organisms were incubated with antiserum and complement. 14C-labeled fatty acids were incorporated during growth into A. laidlawii membrane lipids. After incubation of labeled organisms with antiserum and complement, release of radioactive material into the supernatant and changes in the lipid composition were not observeed. Enzymatic degradation of membrane lipids of A. laidlawii during immune lysis was not detected.