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.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.     

Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).     

Conversion of mevalonic acid to gamma, gamma-dimethylallyl pyrophosphate by Mycoplasma

Henrikson, Carl V. (University of South Dakota, Vermillion), and Paul F. Smith. Conversion of mevalonic acid to gamma,gamma-dimethylallyl pyrophosphate by Mycoplasma. J. Bacteriol. 92:701-706. 1966.-Three representative strains of Mycoplasma, M. laidlawii strain B, Mycoplasma sp. avian strain J, and M. hominis type 2 strain O7, were examined for the presence or absence of enzymes associated with the biosynthetic pathway from mevalonic acid to gamma,gamma-dimethylallyl pyrophosphate. M. laidlawii served as a control organism, since it is capable of de novo biosynthesis of carotenoids. All four enzymes, namely, adenosine triphosphate (ATP)-mevalonate 5-phosphotransferase (EC 2.7.1.36), ATP-5-phosphomevalonate phosphotransferase (EC 2.7.4.2), ATP-5-pyrophosphomevalonate carboxy-lyase (EC 4.1.1.33), and isopentenylpyrophosphate Delta(3),Delta(2)-isomerase (EC 5.3.3.2), were demonstrated in this organism. Mycoplasma sp. avian strain J, which contains all enzymes necessary for the biosynthesis of mevalonic acid, lacks the first three of the above enzymes but contains isopentenyl pyrophosphate Delta(3),Delta(2)-isomerase. M. hominis, which lacks the enzymes necessary for the biosynthesis of mevalonic acid, also is deficient in the enzymes involved in its conversion to gamma,gamma-dimethylallyl pyrophosphate.     

Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol

Smith, Paul F. (University of South Dakota, Vermillion), and Carl V. Henrikson. Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol. J. Bacteriol. 91:1854-1858. 1966.-Compounds which inhibit enzymatic reactions in the biosynthetic pathway to carotenoids inhibited growth of a sterol-nonrequiring species, Mycoplasma laidlawii, strain B, and M. hominis, strain 07. Since M. hominis lacks the enzymes for polyterpene biosynthesis, the inhibitory compounds must act also at other sites. Most inhibitors exerted a lytic effect at bactericidal levels. The inhibition of M. laidlawii is reversed by exogenous cholesterol. M. laidlawii exhibited a greatly increased content of cholesterol and a greatly decreased content of carotenoids when grown in the presence of phenethylbiguanide and cholesterol. These results are considered as further evidence for a common function for sterols and carotenols in Mycoplasma.     

Differences in accumulation of radiolabeled amino acids and polyamines by Mycoplasma and Acholeplasma species

Four species in the order Mycoplasmatales, Mycoplasma capricolum, Mycoplasma hominis, Mycoplasma arginini, and Acholeplasma laidlawii, were compared for their ability to accumulate radiolabeled amino acids and polyamines. The use of a novel high-molecular-weight (HMW) medium, from which molecules of less than 12,000 molecular weight had been removed by extensive dialysis, allowed us to discern significant differences among the species in their relative accumulations of [3H]methionine and [3H]leucine and of [3H]spermidine and [3H]putrescine. Accumulation of radiolabeled amino acids in control low-molecular-weight (LMW) medium was small (0.2 to 2% of the label), and the species did not differ in their proportional accumulations of methionine and leucine. Accumulation of methionine was significantly enhanced (5- to 12-fold) in all species in HMW medium. In contrast, leucine accumulation was enhanced sevenfold for A. laidlawii but only twofold for M. hominis and M. capricolum in HMW medium. The nonglycolytic species, M. hominis and M. arginini, accumulated radiolabeled putrescine and spermidine in both media, whereas the glycolytic species, M. capricolum and A. laidlawii, accumulated only radiolabeled spermidine. The ability to accumulate putrescine appeared to be a differential characteristic for nonfermentative, arginine-utilizing mycoplasmas. HMW medium was much more effective than LMW medium for use in radiolabeling M. capricolum proteins with [35S]methionine.     

Properties of Mycoplasma hominis 4330

Mycoplasma strain 4330, one of the earliest strains of pleuropneumonia-like organisms to be isolated from man in the United States, has been found to resemble M. hominis type 1 by serological methods (the growth inhibition and latex agglutination tests). The results of earlier serological studies indicated a similarity between the Campo and 4330 strains which was not detected by use of the cultures currently available. Strain 4330 differs from strains of Mycoplasma recently isolated from man by producing acid from a variety of carbohydrates. This acquisition of biochemical properties may be the result of hundreds of transfers on artificial media during a period of more than a quarter of a century. Identification of the strain was deemed advisable, since two different cultures and a mixed culture existed under the designation "4330." The extraneous organisms were found to be closely related to M. laidlawii by their biological and serological properties.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

Mycoplasma taxonomy studiedy electrophoresis of cell proteins

The electrophoretic patterns of cell proteins in polyacrylamide gels were used for the study of several taxonomic problems in the Mycoplasmatales. The patterns of five Mycoplasma hominis strains showed marked differences that corresponded with their known serological and nucleic acid heterogeneity. The patterns of three M. mycoides var. mycoides strains isolated in different countries were essentially identical. The electrophoretic patterns of several caprine strains resembled those of M. mycoides var. mycoides, supporting their classification as M. mycoides var. capri. Strain B3, a swine isolate, accordingly was tentatively identified as M. mycoides var. capri. The bovine mastitis strain M. agalactiae var. bovis possessed a pattern basically similar to that of the goat mastitis strain M. agalactiae, supporting the inclusion of both strains in one species. Three M. pulmonis strains isolated from rats or tissue cultures showed nearly identical patterns. The pattern of the toxigenic M. neurolyticum (Sabin A) strain resembled but was not identical with that of the nontoxigenic PG28 strain. The avian Mycoplasma species, M. gallisepticum, M. meleagridis, M. synoviae, M. gallinarum, and M. iners showed easily distinguishable and specific patterns, supporting their present classification in different species. Several improvements in the electrophoretic technique are described, and its advantages and limitations as a taxonomic tool are discussed.     

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

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.     

Genetic Differentiation by Nucleic Acid Homology II. Genotypic Variations Within Two Mycoplasma Species

Somerson, Norman L. (National Institutes of Health, Bethesda, Md.), Paul R. Reich, Barbara E. Walls, Robert M. Chanock, and Sherman M. Weissman. Genetic differentiation by nucleic acid homology. II. Genotypic variations within two Mycoplasma species. J. Bacteriol. 92:311-317. 1966.-A deoxyribonucleic-ribonucleic acid (DNA-RNA) homology technique was used to determine genetic relatedness among the nucleic acids of eight mycoplasmas which were serologically classified as Mycoplasma hominis type 1. The DNA preparations from these organisms were each found to be distinct. No subgrouping of the M. hominis type 1 strains could be demonstrated. In contrast, when the nucleic acids from six serologically related mycoplasmas which were isolated from tissue cultures were studied, the DNA from these species could not be distinguished. The DNA buoyant densities of the tissue culture isolates were similar. These isolates were closely related genetically to a porcine mycoplasma, M. hyorhinis.     

Effect of Diethylaminoethyl Dextran on the Growth of Mycoplasma in Agar

The growth of certain strains of Mycoplasma is inhibited by substances present in commercial agar preparations. The addition of diethylaminoethyl (DEAE) dextran (10 mg per 100 ml) to agar media appears to enhance the growth of some strains. Of eight strains initially tested, the presence of DEAE dextran grossly enhanced the growth of three strains. One strain appeared not to be affected, and a clearly enhancing effect was not evident with four strains. Quantitative studies revealed that growth enhancement varied from 10 colony-forming units (CFU) for M. hominis type II (strain Campo) to 10(3.3) CFU for M. pulmonis (strain 880). The growth-enhancing effect is probably due to the ability of DEAE dextran to bind the sulfated polysaccharide moieties in agar and not to the DEAE dextran, per se.     

Characterization of Mycoplasma Strains from Cats

Mycoplasma strains (B1, B2, CS, and S1A) were isolated from the saliva of normal cats. These were compared with a strain (CO) isolated from the eye of a cat with severe conjunctivitis. On the basis of morphology, biochemical reactions, and antigenic composition, two distinct species were recognizable. Strains CO, B1, and B2 were antigenically unrelated to the other species tested; strains CS and S1A possessed antigenic components in common with Mycoplasma arthritidis, M. salivarium, M. hominis, type 1, and M. orale, types 1 and 2. It was tentatively suggested that the two cat species be called M. felis and M. gateae, respectively.     

Binding of lectins to membranes of mycoplasmas from aging cultures

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.     

Physiological and Serological Comparisons Among Strains of Mycoplasma granularum and Mycoplasma laidlawii

Mycoplasma granularum strains grew on a medium devoid of animal serum or of serum fractions containing sterols; all strains possessed properties, including carotenoid biosynthesis, similar to those described for M. laidlawii. Some common antigenic components were noted among M. granularum and M. laidlawii strains by indirect fluorescent-antibody tests. The growth of M. granularum strains was slightly inhibited by antiserum to M. laidlawii PG-8, and the electrophoretic patterns of cell proteins of the M. granularum strains showed a close resemblance to that of M. laidlawii. However, direct fluorescent-antibody procedures performed on colonies grown on a serum-free medium clearly distinguished M. granularum from M. laidlawii. The occurrence of nonsterol-requiring mycoplasmas, in addition to M. laidlawii, raises questions as to the taxonomy of M. granularum and of the saprophytic mycoplasmas in general.     

Studies with Lectins on the Surface Carbohydrate Structures of Mycoplasma Membranes

The surface carbohydrate structures on the cell membranes of various mycoplasma species have been investigated by using lectins, which are sugar-specific proteins. Carbohydrate structures presumably bound to glycolipids, with both galactose and glucose units, were found to be exposed on the surface of Mycoplasma pneumoniae and its temperature-sensitive mutants, M. mycoides var. mycoides and capri, M. pulmonis, M. gallinarum, and M. gallisepticum. Lipid-bound glucose was found on M. neurolyticum. The possible relationship of the lipid-bound surface carbohydrate groups to the known serological cross-reactions and lipid compositions of the various mycoplasma species is discussed. Intact Acholeplasma laidlawii and M. fermentans have no lectin-binding sites exposed on their surfaces; galactose groups were discovered only after Pronase digestion of the organisms, suggesting that their glycolipids are hidden under a protein layer. Neither intact nor Pronase-digested M. hominis reacted with the lectins; this is fully consistent with the lipid composition of this organism, which contains glycolipids. The lectins from Vicia cracca and Phaseolus vulgaris, which react with N-acetyl-galactosamine groups, agglutinated M. gallinarum, M. gallisepticum, M. mycoides var. capri, and M. pulmonis. The agglutinability was lost after Pronase treatment, indicating that the corresponding carbohydrates are presumably protein bound. They may be correlated with the extracellular structures observed by electron microscopy of both sectioned and negatively stained mycoplasma species.     

Experimental pathogenicity of Mollicutes of bovine udder origin in hamster tracheal ring organ culture.

Hamster tracheal-ring organ culture was employed to examine pathogenic effects of 8 isolates of Mollicutes of bovine udder origin. The tested Mollicutes could be categorized into two groups: (i) Mycoplasma F-38, M. mycoides var. capri, M. bovigenitalium mixed with M. bovirhinis, and M. bovigenitalium mixed with M. bovirhinis and Mycoplasma F-38 produced significant ciliostatic effect and infiltration of neutrophils and lymphocytes in lamina propria/subepithelium, hyperplasia and desquamation of epithelial lining cells and loss of cilia; and (ii) A. laidlawii, A. axanthum, an unidentified Acholeplasma and a mixed isolate of M. bovis, M. bovigenitalium, Mycoplasma F-38 and A. laidlawii showed insignificant ciliostatic effects and produced mild histopathological lesions. This correlates with the disease causing potentials of the strains.     

Serological comparison of ten glycolytic Mycoplasma species

Seventeen strains of mycoplasmata representing 11 named species were compared serologically by three parameters: growth inhibition on agar, double immunodiffusion, and complement fixation. In growth-inhibition studies, a strain labeled Mycoplasma histotropicum was found related to and perhaps best classified as M. pulmonis, a relationship confirmed by double immuno-diffusion studies. A comparison of the remaining 10 species demonstrated that two pairs of species could be shown to be closely related by complement fixation and double immunodiffusion but not by growth inhibition; these were: M. granularum-M. laidlawii and M. felis-M. canis. M. pneumoniae and M. gallisepticum were the most serologically unique organisms in this study, showing very few cross-reactions with each other or other species. Overall, taxonomic groupings obtained by comparative serology appeared to correlate with the groupings obtained when the guanine plus cytosine contents of the deoxyribonucleic acid of mycoplasmata were employed as classification criteria. The group of organisms having a guanine plus cytosine content of 23 to 28% (M. canis, M. fermentans, M. hyorhinis, M. neurolyticum, and M. pulmonis) appeared to be generally serologically related. Thus the remarkable heterogeneity observed in the base composition of the deoxyribonucleic acid of order Mycoplasmatales is also reflected and apparently paralleled by a corresponding serological heterogeneity.