Mycoplasma Suipneumoniae and Mycoplasma Flocculare in the Growth Precipitation Test

A number of laboratory and field strains of Mycoplasma suipneumoniae and Mycoplasma flocculare were subjected to a comparative examination by the growth precipitation test. It was found that all the strains could readily be identified by that test. Slight evidence of cross-reaction was noted for a few of the laboratory strains, but not until late in the observation period. Only some of the field strains would form precipitates when primary cultures (from tissue suspensions) were used, but all strains could be identified already in the second and third passages. The test therefore seems well suited for distinguishing the two species from each other.     

Evaluation of Some Serological Techniques for The Identification of Mycoplasma Hyorhinis and Mycoplasma Suipneumoniae

Eighteen strains of Mycoplasma hyorhinis and a strain of Mycoplasma suipneumoniae were tested in 4 serological tests, i. e., disc growth inhibition, metabolic inhibition, indirect haemagglutination and indirect epi-immunofluorescence. Only with immunofluorescence could all tested strains of M. hyorhinis be shown; no cross-reactions between M. hyorhinis and M. suipneumoniae could be detected. The other tests failed in many cases to identify strains of the same species, and they gave cross-reactions between M. hyorhinis and M. suipneumoniae.     

A Serologic Variant of Mycoplasma Hyorhinis Recovered from the Conjunctiva of Swine

In an examination of conjunctival samples from 40 piglets for mycoplasmas, 17 isolates were obtained. Eight could be identified as Mycoplasma hyorhinis, three as Mycoplasma flocculare, and one as Acholenlasma sp. Five strains were not readily identifiable, but together with two previously recovered strains they were found to represent a distinct serogroup. All seven strains were glucose and phosphatase positive. Incubation in a CO2-enriched atmosphere led to enhancement of the growth on solid medium. The serogroup was serologically related to M. hyorhinis, but not to a number of other glucose fermenting species of mycoplasma, and it may therefore be regarded as a new subspecies of M. hyorhinis.     

A Selective Medium for Mycoplasma Suipneumoniae

The isolation of Mycoplasma suipneumoniae (M. snip.) has not hitherto been possible in broth culture if the material also harboured Mycoplasma hyorhinis (M. hyor.), since the latter organism would outgrow M. suip. The present paper reports an effort to solve this problem.     

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.     

Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine-derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.     

Mycoplasma Suipneumoniae Isolated in Denmark

Though several agents may be involved in the etiology of enzootic pneumonia of pigs, recent investigations seem to indicate that the great majority of cases are caused by a Mycoplasma species — called M. suipneumoniae in Britain (Goodwin et al 1965) and M. hyopneumoniae in the U.S.A. (Maré & Switzer 1965).     

Biochemical diversity of Mycoplasma fermentans strains

In this study, the metabolism of a diverse range of Mycoplasma fermentans strains was investigated. It was shown that the ability to utilise glucose, fructose and N-acetylglucosamine differentiated strains, and that the patterns and kinetics of substrate utilisation were correlated with the site of isolation, i.e. joint fluid, respiratory tract, urinary tract or cell culture. Interestingly, isolates from the urogenital tract of AIDS patients used fructose in preference to glucose. There was also some correlation of fructose and N-acetylglucosamine utilisation of isolates with M. fermentans sub-groups, identified in an independent study, and based on the distribution of insertion sequence-like elements in the M. fermentans genome.     

Construction of the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rRNA genes on these maps.

The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.     

Differences in Arginine Requirement for Growth Among Arginine-Utilizing Mycoplasma Species

The essentiality of arginine for initiation of growth of arginine-utilizing, nonglycolytic Mycoplasma species from small populations was studied by growing the organisms in a semisynthetic medium proven to be free from arginine by chemical and biological assays. Initiation of growth of two strains of M. arginini did not require arginine, whereas another strain of M. arginini required 4 mM arginine, as did M. gallinarum. M. hominis grew in 0.4 mM arginine. A species which utilizes both arginine and glucose, N. fermentans, did not require arginine but did require glucose for growth. When mycoplasmata were grown in human heteroploid cell cultures employing medium free from arginine but supplemented with citrulline, similar results were obtained: two M. arginini strains grew in the absence of arginine, whereas growth of M. gallinarum and M. hominis and a third M. arginini strain was dependent on arginine even though mammalian cells were present. The arginine deiminases were heterogeneous serologically: antisera to M. hominis and M. arginini showed reciprocal inhibition of their enzymes but did not inhibit arginine deiminase from M. gallinarum. Antiserum to M. gallinarum inhibited only M. gallinarum enzyme.     

Mycoplasma phocarhinis sp. nov. and Mycoplasma phocacerebrale sp. nov., two new species from harbor seals (Phoca vitulina L.)

A total of 120 mycoplasma strains were recovered from 97 of 265 diseased seals investigated during the seal epidemic in the North Sea and in the Baltic Sea in 1988. Mycoplasmas were isolated from the respiratory tracts (including lungs), hearts, brains, and eyes of the seals. Thirty strains were filter cloned and investigated for their morphological, biochemical, and serological characteristics compared with the characteristics of previously described species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that these strains belong to two new species, for which the names Mycoplasma phocarhinis and Mycoplasma phocacerebrale are proposed. M. phocarhinis (17 strains) did not ferment glucose or hydrolyze arginine but did reduce tetrazolium chloride and potassium tellurite and produced films and spots. M. phocacerebrale (13 strains) metabolized arginine but not glucose and produced phosphatase but did not reduce tetrazolium chloride and potassium tellurite. Both species lysed sheep erythrocytes but did not absorb sheep or guinea pig erythrocytes. The type strain of M. phocarhinis is strain 852 (= ATCC 49639), and the type strain of M. phocacerebrale is strain 1049 (= ATCC 49640).     

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.     

Metabolic Turnover of the Polar Lipids of Mycoplasma laidlawii Strain B

The fatty acyl groups of the membrane polar lipids of Mycoplasma laidlawii B were radioactively labeled by growth in the presence of (14)C-palmitic, oleic, or linoleic acids. No loss of radioactivity from any of the polar lipids occurred during incubation of radiolabeled cells in growth medium containing various nonradioactive fatty acids, although growth and lipid biosynthesis continued throughout the incubation period. The metabolism of the glucose and phosphate moieties was also studied in a similar fashion by utilizing growth in (14)C-glucose or (32)P-inorganic phosphate to radioactively label these groups. As before, no loss of radioactivity from any of the polar lipids occurred during subsequent growth in medium containing unlabeled glucose or phosphate. The results establish that the glyco- and phospholipids of M. laidlawii B are metabolically stable in actively growing cells. The absence of metabolic turnover is discussed in terms of proposed relationships between lipid metabolism and function in this organism.     

Role of arginine deiminase in growth of Mycoplasma hominis.

Arginine has been considered as the major energy source of nonglycolytic arginine-utilizing mycoplasmata. When three strains of Mycoplasma arginini, and one strain each of Mycoplasma arthritidis, Mycoplasma fermentans, Mycoplasma gallinarum, Mycoplasma gallisepticum and Mycoplasma hominis were grown in the medium with high arginine concentration (34 mM) compared with low arginine (4 mM), both the protein content of the organisms and the specific activity of arginine deiminase increased. M. fermentans, the one arginine-utilizing species included in the survey which is also glycolytic, showed an increase in protein content but no increase in specific activity of the enzyme. The glycolytic non-arginine-utilizing M. gallisepticum did not show an increase in either parameter. The Km for arginine deiminase from crude cell extracts was 1.66 X 10(-4)M. The enzyme demonstrated a hyperbolic activation curve subject to substrate inhibition and was not affected by the presence of L-histidine. When mycoplasmic protein and arginine deiminase were determined for M. hominis under aerobic and anaerobic conditions, aerobically grown cells exhibited no detectable enzymatic increases until late in log phase. Higher levels of arginine deiminase were observed earlier in the anaerobic growth cycle. The rate of 14CO2 evolution from [guanido-14C]arginine was not altered in arginine-supplemented cells compared with cells grown in low arginine. In addition, CO2 production did not parallel increased arginine deiminase activity. These observations argue that arginine is used only as an alternate energy source in these organisms.     

Heterogeneity Among Strains of Mycoplasma granularum and Identification of Mycoplasma hyosynoviae, sp. n

Twelve filtrable, pleomorphic organisms isolated from swine joints and respiratory tracts had typical colonial and microscopic characteristics of mycoplasmas. They resisted penicillin and did not revert to cell wall-producing bacterial forms in media devoid of bacterial inhibitors. The morphological and growth characteristics of these mycoplasmas were similar to those described previously for Mycoplasma granularum. However, a new name, M. hyosynoviae, is proposed for them since they differed biologically, serologically, and electrophoretically from the prototype strain of M. granularum. M. hyosynoviae required sterols, was stimulated by gastric mucin, and metabolized arginine; however, it did not metabolize urea, ferment glucose, or reduce tetrazolium. The organism produced "film and spots" on horse serum-supplemented medium and produced alpha hemolysis of guinea pig and sheep erythrocytes; however, it did not digest serum, produce phosphatase, or hemadsorb guinea pig or swine erythrocytes. M. hyosynoviae was distinguished from three other swine mycoplasmas, M. granularum, M. hyorhinis, and M. laidlawii, by means of acrylamide gel electrophoresis, growth inhibition, metabolic inhibition, and immunodiffusion techniques. It was also serologically and electrophoretically distinct from 13 additional non-swine mycoplasmas which require sterols and metabolize arginine.     

Localization of Enzymes in Mycoplasma.

Pollack, J. D. (University of Connecticut, Storrs), Shmuel Razin, and Robert C. Cleverdon. Localization of enzymes in Mycoplasma. J. Bacteriol. 90:617-622. 1965.-Cells of eight parasitic and two saprophytic Mycoplasma strains were lysed by use of osmotic shock, and the membranes were separated from the soluble fraction by use of differential centrifugation. Cell fractions were tested for reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) oxidase, glucose-6-phosphate dehydrogenase, adenosine triphosphatase, ribonuclease, and deoxyribonuclease activities. Adenosine triphosphatase was confined to the membrane fraction of all Mycoplasma strains. The NADH(2) oxidase activity was associated with the membranes of the saprophytic M. laidlawii and with the soluble fraction of the parasitic Mycoplasma strains. NADPH(2) oxidase activity was detected only in the soluble fraction of the parasitic strains. Glusose-6-phosphate dehydrogenase was demonstrated only in the soluble fraction of M. laidlawii. Ribonuclease activity was found usually in both membrane and soluble fractions, but was generally higher in the membrane fraction. In the human and bovine Mycoplasma strains, deoxyribonuclease activity could not be demonstrated in the soluble fraction; in the remaining strains, activity was highest in the soluble fraction. Dissolution of M. laidlawii strain B membranes by sodium deoxycholate significantly increased membrane-NADH(2) oxidase and adenosine triphosphatase activities.     

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.     

Mycoplasma melaleucae sp. nov., a sterol-requiring mollicute from flowers of several tropical plants

Three sterol-requiring mollicutes from floral surfaces of two tropical plant species (Melaleuca quinquenervia and Melaleuca decora) and a single isolate from a flower of the silk oak (Grevillea robusta) were serologically indistinguishable. Strain M1T (T = type strain), isolated from Melaleuca quinquenervia, was chosen for characterization. Light and electron microscopic observations of strain M1T revealed nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew well in SP-4 medium, but no sustained growth occurred in conventional mycoplasma media containing horse serum. The optimum temperature for growth was 23 degrees C, but multiplication occurred over a temperature range of 10 to 30 degrees C. Growth was not observed at temperatures above 30 degrees C. Strain M1T and related strains (strains M5, M10, and SO1) catabolized glucose but hydrolyzed neither arginine nor urea. The size of the strain M1T genome was about 561 megadaltons, while the guanine-plus-cytosine content of the DNA was about 27.0 mol%. The organism was serologically unrelated to the type strains of the 80 previously recognized Mycoplasma species or to 18 other unclassified sterol-requiring strains cultivated from animal, plant, or insect sources. Recent sequencing studies of 16S rRNA demonstrated that strain M1T is a member of a clade that contains the type species of the genus Mycoplasma. Strain M1 (= ATCC 49191) is the type strain of Mycoplasma melaleucae sp. nov.