Acholeplasma brassicae sp. nov. and Acholeplasma palmae sp. nov., two non-sterol-requiring mollicutes from plant surfaces.

Two mollicutes (strains 0502T [T = type strain] and J233T), which were isolated from the surfaces of broccoli (Brassica oleracea var. italica) plants or the crown tissues of the coconut palm (Cocos nucifera), were capable of sustained growth in serum-free (or cholesterol-free) mycoplasma broth media. Examination by electron and dark-field microscopic techniques revealed that the cells of each strain were small, nonhelical, nonmotile, pleomorphic, and coccoid and that each cell was surrounded by a single cytoplasmic membrane. No evidence of a cell wall was found. The organisms were filterable and grew rapidly in most conventional mycoplasma culture medium formulations containing horse or fetal bovine sera under either aerobic or anaerobic conditions. The optimum temperature for growth of both organisms was 30 degrees C, but multiplication occurred over a temperature range from 18 to 37 degrees C. Both strains catabolized glucose, but did not hydrolyze arbutin, arginine, or urea. The genome size of strain 0502T was 1,215 kbp, and the DNA base composition (guanine-plus-cytosine content) was 35.5 mol%. The genome size of strain J233T was 1,610 kbp, and the DNA base composition was 30.0 mol%. The two isolates were not serologically related to each other or to the type strains of 11 previously described Acholeplasma species. Strain 0502 (= ATCC 49388) is the type strain of Acholeplasma brassicae sp. nov., and strain J233 (= ATCC 49389) is the type strain of Acholeplasma palmae sp. nov.

Mycoplasma lactucae sp. nov., a sterol-requiring mollicute from a plant surface.

Strain 831-C4T (T = type strain), isolated from the surface of lettuce plants (Lactuca sativa) obtained from a retail food market, was shown to be a sterol-requiring mollicute. Morphological examination of this organism by electron and dark-field microscopic techniques showed that it consists of small, nonhelical, nonmotile, pleomorphic coccoid cells, with individual cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew rapidly in all conventional culture medium formulations for mollicutes in either aerobic or anaerobic environments. The optimum temperature for growth was 30 degrees C, but multiplication occurred at 18 to 37 degrees C. Strain 831-C4T catabolized glucose, but hydrolysis of arginine or urea could not be demonstrated. The genome size of strain 831-C4T was determined to be about 569 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was 30.0 mol%. Recent studies in which we compared the 16S rRNA sequences of strain 831-C4T with those of more than 40 other mollicutes indicated that this organism is phylogenetically related to the Spiroplasma-Mycoplasma mycoides clade. Strain 831-C4T was serologically unrelated to the type strains of previously described Mycoplasma species and to 18 other unclassified sterol-requiring isolates cultivated from various animal, plant, or insect sources. Strain 831-C4T (= ATCC 49193) is the type strain of Mycoplasma lactucae sp. nov.

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.

Induction by streptococcal L-forms of two reproductive isolating mechanisms in Drosophila paulistorum.

The six semispecies of the Drosophila paulistorum complex do not normally interbreed, but when hybrids are produced in the laboratory, the males are sterile. In earlier work, cytoplasmic components responsible for this sterility had been transferred by injection of streptococcal L-forms isolated from the Mesitas and Santa Marta flies. To extend these findings, and L-form from the Centro-American flies was injected into three semispecies. Transfer of L-form from one semispecies to another resulted in sterile male progeny. The L-form isolated from Mesitas was injected into all six semispecies. Results simulated "hybrid inviability," i.e., only the Mesitas semispecies produced the expected number of offspring, and the total number of progeny from the other five semispecies was considerably reduced. Despite the small numbers of males, reflecting induced inviability, there was evidence that the transfer of L-forms from one semispecies to another simultaneously caused sterility in male progeny.

Simple staining procedure permits rapid counting of mycoplasma colonies.

A staining procedure employing oil red O and Coomassie R-250 was developed to increase visualization of mycoplasma colonies. This procedure permits CFU determination of mycoplasmas without additional microscopy.

Streptococcal L-forms isolated from Drosophila paulistorum semispecies cause sterility in male progeny.

The Drosophila paulistorum complex contains six semispecies that do not normally interbreed. In the laboratory, crosses between semispecies produce fertile daughters and sterile sons. Microbial endosymbionts have been observed in all D. paulistorum flies that display this male sterility. Streptococcal L-forms have been isolated from the Andean-Brazilian (Mesitas) and Transitional (Santa Marta) semispecies and cultured in artificial medium. Transfer of these L-forms from their native hosts into reciprocal semispecies resulted in sterile male progeny. When L-forms were inoculated into the semispecies from which they had been isolated, most of the male progeny were fertile. Control streptococcal L-forms did not show this sterility pattern.

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.

Freeze-fracture confirmation of the presence of a core in the specialized tip structure of Mycoplasma pneumoniae.

The presence of a specialized terminal region in Mycoplasma pneumoniae was seen in thin sections viewed in an electron microscope. Actively growing cells were examined by the freeze-fracture technique in the absence of fixation to further establish the core as a significant structural entity. Cross fractures revealed a cytoplasmic matrix surrounding a central core structure of about 54 nm. This structure disappeared rapidly in aging cells. The convex protoplastic faces of the membrane around the core had characteristic 5- to 10-nm intramembrane particles evenly distributed across the cell surface, with no apparent difference in the region of the specialized tip. A periodicity previously noted in negatively stained preparations was clearly defined here in thin sections. Attachment of actively growing cells to sheep erythrocytes was seen primarily as a side attachment rather than attachment at the tip alone. This association between the mycoplasma and the sheep erythrocytes seriously deformed the sheep erythrocytes, but no membrane fusion could be detected.

Isolation of acholeplasmas and a mycoplasma from vegetables.

The isolation of Mollicutes from food has not been reported. To isolate Mollicutes in the presence of high levels of unwanted bacteria, we first incubated fresh vegetables in liquid culture media containing lysozyme, ampicillin, and thallous acetate. Culture fluids were than separated from the vegetable samples, subjected to one freeze-thaw cycle, and passed through a filter of 0.4-micron porosity. Filtered samples were cultured in SP4 medium and in a conventional medium containing horse serum. With this procedure 21 acholeplasma isolations representing three species were obtained from endive, broccoli, and kale. Of 35 food samples tested, 11 were positive for acholeplasmas; acholeplasmas isolated from 6 of these samples were recovered only in SP4 medium. In seven single vegetable samples, two or more Acholeplasma spp. were isolated. A. laidlawii was isolated from all three vegetables and A. axanthum was found in broccoli and kale. Four isolates were serologically identified as A. oculi. Mycoplasma verecundum was the only Mycoplasma species recovered. Several isolates could not be typed serologically, as they reacted with antisera to both A. morum and A. hippikon. these isolates may include new Acholeplasma spp.

Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae.

Ultracentrifugation was used to separate three commercial lots of bovine serum fraction (BSF) into components designed to contain lipoproteins. Each BSF lot and component was tested for ability to support the growth of tree strains of Mycoplasma pneumoniae. In general, the level of growth-promoting activity corresponded to the amount of cholesterol present in the BSF or BSF components rather than to the amount or type of lipoprotein Cholesterol was the limiting nutritional factor of BSF with low growth-promoting activity. The addition of cholesterol and bovine serum albumin to BSF with low activity resulted in growth equal to or greater than that observed for BSF with high growth-promoting activity. When cholesterol was added to agar medium containing BSF of low activity, mycoplasma colonies were greater in number, possessed larger mean diameters, and had centers that were more distinct than those observed when this BSF was used alone. Variability in growth-promoting actions of commercial lots of BSF was eliminated by increasing their cholesterol content to an optimum level. An adjustment of the cholesterol and albumin levels of any serum product used in culture media may provide a simple convenient method to improve growth and isolation of mycoplasmas.

Lipoproteins as substitutes for serum in Mycoplasma culture medium.

Crude lipoprotein-containing fractions obtained from sera of three different animal species were tested, in combination with bovine serum in Mycoplasma pneumoniae culture medium. All sera yielded at least one lipoprotein-containing component which was considerably more effective in promoting mycoplasma growth than the unfractionated serum sample from which it was derived. The very low activity of certain whole-serum samples tested in this investigation suggests that toxic substances may be present in whole serum which are not contained in the lipoprotein preparations. The greatest activity appeared in the high-density lipoprotein-containing components of bovine and horse sera and the low-density lipoprotein-containing components of human serum. The high degree of growth-supporting activity of these crude lipoprotein-containing serum components suggests that they may be useful as serum substitutes in mycoplasma culture media.

Lecithin changes in murine Mycoplasma pulmonis respiratory infection.

We examined the lipid content of bronchoalveolar (BA) washes from both mice and rats infected with Mycoplasma pulmonis, an etiological agent of murine pneumonia. During a 30-day period after intranasal inoculation, the total lipid content from infected and control rats (in milligrams per animal) remained relatively equal and unchanged. The saturated, unsaturated, and total lecithin contents in infected rats (in milligrams per animal) all increased. The maximum lecithin values were detected at 7 to 10 days after infection; later, the levels fell to control values. There was essentially no change in any lecithin value from uninfected animals. Although in BA washes from infected animals the mass of disaturated lecithins increased, the percentage of this fraction in the total lecithin pool decreased. The fatty acids of the lecithins from BA washes of infected mice had significantly less palmitic and significantly more oleic and linoleic acids than the lecithins isolated from the BA washes of control animals. Both the relative decrease in the mass of disaturated lecithins in the BA washes and the increase in the percentage of esterified unsaturated fatty acids in the lecithins may be directly related to the reduced lung function reported to occur during the course of murine M. pulmonis pneumonia.

Mycoplasma growth factors in bovine serum fraction.

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major growth factor of bovine serum fraction eluted from the Sephadex column in the void volume. Its growth-supporting activity was greatly enhanced by the presence of bovine serum albumin in the mycoplasma culture media. Other investigators had previously identified the major growth factor in serum as an alpha-lipoprotein. Although density ultracentrifugation revealed the presence of traces of a high-density lipoprotein in bovine serum fraction, another, less dense component, isolated by ultracentrifugation (component 3) and containing cholesterol, cholesteryl esters, free fatty acids, triglycerides, and protein, but no lipoprotein, exhibited considerably more growth-supporting activity than did the high-density lipoprotein, thus indicating that at least two mycoplasma species do not require intact serum lipoprotein for growth. Both the high-density lipoprotein and component 3 exhibited maximum activity only in the presence of bovine serum albumin. A chloroform extract containing component 3 lipids combined with bovine serum albumin to form an effective, partially defined, less complex substitute for serum in mycoplasma culture media.

Oxygen uptake and lung function in mice infected with Streptococcus pneumoniae, influenza virus, or Mycoplasma pulmonis.

Model systems of respiratory infection in mice were established with Streptococcus pneumoniae, influenza virus, and Mycoplasma pulmonis. The LT50 for S. pneumoniae was 2 1/2 days, for lethal influenza 6 days, and for M. pulmonis 5 days. Morbidity in sublethal influenza infections reached a peak during days 5 to 10, with recovery indicated by the third week. The course of each pulmonary infection was followed by use of the animal's maximal ability to consume oxygen (VO2max by determining the weight, compliance, and stability of the excised lung, and in some cases by following O2 consumption of minced tissue. Depression of VO2max began early in each infection; reductions ranged from 9% at the peak of sublethal influenza infection to 50% 12 to 48 hr before the LT50 of fatal infections. The depressions were not relieved by 100% O2. The noninvasive VO2max test, evoked by cold air, was simple, rapid, and reproducible and appeared to serve as a quantitative measure of over-all function during infection. Each type of infection caused an increase in lung weight, with the largest noted during fatal Mycoplasma illness and lethal influenza. The effects on lungs by influenza and M. pulmonis infections were similar but could be differentiated from those with S. pneumoniae. With sublethal influenza, CL was reduced 30% between days 5 to 10, with recovery by the third week. Ctis was not affected. M. pulmonis infections and lethal influenza caused depressions in CL of over 60% by day 4 but only a 30% decrease in Ctis. The data suggest that the decreased compliance in influenza and M. pulmonis infections was due primarily to increased surface tension. In contrast, S. pneumoniae did not affect compliance.

Electron microscopy of Mycoplasma pneumoniae microcolonies grown on solid surfaces.

Mycoplasma pneumoniae sprain CL-8 was studied by using various surfaces for adherence and growth. Cells grown on Epon 812, Formvar, carbon, and glass were of similar morphology. Thin Epon pieces were good material for culturing the organisms and examining thin-sectioned microcolonies by transmission electron microscopy.

Mycoplasma growth inhibition by arginine.

Arginine enhances the growth of nonfermenting mycoplasmas. However, arginine can restrict the growth of glucose-fermenting mycoplasmas and should not be added to media used to cultivate these species.

Tetrazolium reduction as a measure of metabolic activity for glass-adherent Mycoplasma pneumoniae.

The reduction of tetrazolium was used to assay the metabolic capability of developing Mycoplasma pneumoniae cultures on glass. Generally, the amount of tetrazolium reduced correlates with the amount of growth as measured by protein. Until a culture enters the late phase of the growth cycle, the drop in pH of culture medium provides similar information. In this last stage of growth, protein appears to be leveling. The pH continues to fall, but tetrazolium reducing activity decreases. Thus, considering the entire M. pneumoniae growth cycle, formazan production is a more reliable measure of metabolic capability of the organisms than either protein or pH. The reduction of tetrazolium provides a quantitative means of assessing enzymatic activities of glass-adherent M. pneumoniae.