INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,Jan. 1991, p. 45-49 Vol. 41, No. 1 0020-7713/91/010045-05$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Acholeplasma seiflertii sp. nov. a Mollicute from Plant Surfaces FRANCOISE BONNET,l COLETTE SAILLARD,l JEAN CLAUDE VIGNAULT,' MONIQUE GARNIER,l PATRICIA CARLE,l JOSEPH M. BOVE,l* DAVID L. ROSE,2 JOSEPH G. TULLY,2 AND ROBERT F. WHITCOMB3 Laboratoire de Biulogie Cellulaire et Mofe'culaire, Institut Nationale de la Recherche Agronomique, F-33140 Pont-de-la-Maye, France'; Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland 21 7012; and Insect Pathology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 207053

Two mollicutes (strains F7T [T = type strain] and F28) isolated from floral surfaces of plants growing in Morocco and France were capable of sustained growth in serum-free (or cholesterol-free) mycoplasma broth media. The two isolates were found to be genomically and serologically related. Morphologic examination of the organisms by electron and dark-field microscopic techniques showed that each strain consists of small, nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of cell walls was observed. Growth in serum-free or cholesterol-free medium was sustained only when the medium contained a Tween 80 fatty acid mixture (0.01 or 0.04%). The organisms grew rapidly in most conventional mycoplasma culture medium formulations containing horse or fetal bovine serum or a bovine serum fraction and under either aerobic or anaerobic environments. The optimum temperature for growth was 28"C, but multiplication occurred over a temperature range from 20 to 35°C. Both strains catabolized glucose and mannose, but did not hydrolyze arbutin, arginine, or urea. The molecular mass of the genome of strain F7T was determined to be about 886 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was found to be 30.0 mol%. The two isolates were serologically unrelated to type strains of the 11 previously described Acholeplasma species and to 10 other unclassified sterol-nonrequiring mollicutes culti- vated from various animal, plant, or insect sources. Strain F7 (= ATCC 49495) is the type strain of Acholeplasma seiflertii sp. nov.

The occurrence of nonhelical, wall-less prokaryotes (class between insects and plant hosts during feeding excursions. Mollicutes) on plant surfaces was first well documented in The first insect-derived acholeplasma to be characterized, 1979 (9,15) when organisms with the general features of both Acholeplasma entomophilum (27), has now been identified Acholeplasma and Mycoplasma species were isolated from as a frequent gut inhabitant in a variety of insect hosts (30) the surfaces of several tropical floral plants. Subsequent and a resident on a number of plant surfaces (31). characterization of these strains and other new isolates from In this report, we describe the features of two related plant surfaces (22) showed that some of the strains were acholeplasma strains that were isolated from floral surfaces related to established sterol-nonrequiring Acholeplasma spe- of plants growing in France and Morocco and propose that cies that occur in vertebrate hosts (e.g., Acholeplasrna these organisms should be considered members of a new axanthum , Acholeplasma oculi, and Acholeplasma laid- Acholeplasma species. lawii) (9, 22). Other isolates represented either new Achole- plasma species (14, 33) or possibly sterol-requiring Myco- MATERIALS AND METHODS plasma species (9, 22, 26). While the presence of sterol-requiring Mycoplasma spe- Mycoplasma strains. Strain F7T (T = type strain) was cies on plant surfaces was suspected from the studies isolated in June 1980 from flowers of a sweet orange tree reported in 1979 to 1982 (15, 22), the possibility that these (Citrus sinensis) located in Morocco. Strain F28 was isolated organisms might represent sterol-requiring, nonhelical forms from the surfaces of large umbelliferous flowers of Angelica of the genus Spiroplasrna could not be excluded at the time. sylvestris growing wild near Bordeaux, France. Both isolates The occurrence of Mycoplasma species on plant surfaces were purified by conventional filtration cloning techniques was confirmed recently when two of the strains from the (23). The type strains of previously described Acholeplasma early studies (17, 29) were fully characterized. In addition, species and a collection of currently unclassified, nonhelical, sterol-requiring mollicutes were isolated from insect guts sterol-nonrequiring mollicutes of diverse animal, plant, and and from hemolymph samples (30), and four of these strains insect origins were used in various parts of this study (28). were characterized recently as new insect-derived Myco- Culture medium and cultivation techniques. The proce- plasma species (28, 34). dures used for primary isolation of mollicutes from plant or The natural occurrence of acholeplasmas in the guts and floral surfaces have been described previously (2). SP-4 hemolymph of insects was established by Clark and col- culture medium (32),which contains fetal bovine serum, was leagues in the early 1980s (6, 30). The observations of these used in the initial isolation of both strains. Other culture workers indicated that a wide variety of acholeplasmas could media used included the Edward formulation of conven- be found in insect gut fluids and on plant or flower surfaces, tional 20% horse serum mycoplasma broth (lo), serum leading to speculation that an exchange of mollicutes occurs fraction broth containing 1%bovine serum fraction (25), and serum-free medium with or without special supplements of a fatty acid mixture (polyoxyethylene sorbitan monooleate- * Corresponding author. Tween 80; Sigma Chemical Co., St. Louis, Mo.) (14, 27).

45 46 BONNET ET AL. INT. J. SYST.BACTERIOL.

Most of the culture media used for growth and characteri- incubation at 30°C. Direct agar plate immunofluorescence zation of strains F7T and F28 contained 500 U of penicillin tests (12) were performed on strain F7T colonies on horse per ml. A solid medium of each of these formulations was serum agar medium by using fluorescein-conjugated antisera made by adding 0.8% Noble agar (Difco Laboratories, to the acholeplasmas mentioned previously (27). Finally, Detroit, Mich.). Agar cultures were incubated at 30°C either disk growth inhibition tests with strain F7T also were per- aerobically (with 5% carbon dioxide in a GasPak system formed with antisera to approximately 98 Mycoplasma spe- [BBL Microbiology Systems, Cockeysville, Md.]) or anaer- cies or putative mycoplasmas (28). obically (hydrogen GasPak system). Electrophoretic analysis of cell proteins. The techniques To measure the temperature requirements for growth of used to compare patterns of mollicute cell proteins by the two strains, a series of 10-fold dilutions of each organism one-dimensional polyacrylamide gel electrophoresis have in serum fraction broth was prepared. One series of the been described previously (16). Strains F7T and F28 were diluted mycoplasma was incubated at each of eight temper- grown in SP-4 medium and concentrated by centrifugation, atures (10, 18, 25, 28, 30, 32, 35, and 37°C). The relative and 100-p1 portions of resuspended cell pellets (about 25 pg number of organisms, measured by noting color changes (red of protein) were subjected to sodium dodecyl sulfate-poly- to yellow) and turbidity in the highest dilution of SP-4 broth acrylamide gel electrophoresis analysis. Following electro- (number of color-changing units per milliliter), was recorded phoresis, the gels were stained with Coomassie blue and after incubation for 3 weeks. photographed. Early passages of strains F7T and F28 were also main- Genomic analysis. The techniques used to extract and tained for at least five passages on SP-4 or Edward type purify chromosomal DNAs from mollicutes and to determine medium which contained no penicillin or other antibiotics. genome sizes by analyzing the renaturation kinetics of At each subculture, the organisms were plated onto conven- denatured DNAs have been described previously (3,4). The tional blood agar and incubated aerobically at 37°C. After 2 guanine-plus-cytosine contents of purified DNAs of strains to 10 days, the plates were examined for bacterial colonies F7T and F28 were determined by using buoyant density and evidence of reversion. techniques (5). Purified DNA from Spiroplasma citri (ge- Morphological studies. Strain F7T was grown overnight nome molecular mass, approximately 1,000 megadaltons (30°C) in SP-4 broth, and the resulting logarithmic-phase [mDa]; base composition, 26 mol% guanine plus cytosine) cultures were examined by dark-field microscopy, using a was used as a reference standard. DNA-DNA hybridization magnification of x 1,250. For electron microscopic examina- was used to compare strains F7T and F28, as well as to tion, the organism was grown in approximately 10 ml of measure the levels of relatedness of these two organisms to horse serum broth and cells were pelleted by centrifugation. several other plant-insect acholeplasmas. The techniques The cell pellet was then fixed for 2 h in 3% glutaraldehyde, used for hybridization experiments with mycoplasmal DNA postfixed in 1% osmium tetroxide for 1 h, dehydrated in have been described previously (8). acetone, embedded in Epon-araldite, sectioned, and stained with 1% aqueous uranyl acetate and Reynold lead citrate. RESULTS AND DISCUSSION Sterol requirement. Growth requirements for sterol were assayed by using a standard broth culture method (24) and Cultural and morphological properties. Both strain F7T and serum-free broth medium supplemented with either 0.04% strain F28 grew rapidly on mycoplasma medium formula- Tween 80 fatty acid mixture or various concentrations of tions containing serum (fetal bovine or horse serum) or cholesterol. All cultures in this test were incubated at 30°C. bovine serum fraction. No sustained growth was observed in The protein contents of mycoplasma cell pellets were as- serum-free mycoplasma broth, but when the serum-free sayed with a Bio-Rad protein kit (Bio-Rad Laboratories, broth was supplemented with 0.01 to 0.04% Tween 80 fatty Richmond, Calif.). The ability of the two floral isolates to acid mixture, both strains could be maintained indefinitely. show sustained growth in serum-free (and cholesterol-free) Broth cultures in serum-free base medium supplemented broth with and without fatty acid supplements was also with Tween 80 supplements usually reached peak logarith- measured. In this test, each organism was passaged through mic-phase growth levels within 48 h at 30°C. Growth oc- a series of 23 10-fold dilutions in serum-free broth (volumes, curred over a temperature range of 20 to 35"C, with optimum 0.2 ml of 1.8 ml) containing either 0.01 or 0.04% Tween 80 or growth observed at 28°C. No growth was apparent in broth in serum-free broth without supplements. The inoculum cultures maintained at 37°C. consisted of 0.2 ml of a culture grown in bovine serum Colonies of strain F7T on Edward type horse serum agar fraction broth. or SP-4 agar plates exhibited typical fried egg morphology Tests for biological and biochemical properties. The proce- (Fig. 1).Growth occurred on agar plates containing serum- dures used to demonstrate carbohydrate fermentation (1) free base medium and Tween 80 supplements, but even after and hydrolysis of arbutin (18), arginine, and urea (1) have incubation for 5 to 6 days the colonies were usually very been described previously. The techniques used to assess small. Anaerobic incubation (GasPak system) was required filtration characteristics (23) and hemadsorption (11) also for optimum growth on solid medium. have been reported previously. When logarithmic-phase cultures of strains F7T and F28 in Serological tests. Antiserum to strain F7T was raised in SP-4 medium were examined by dark-field microscopy, we rabbits by using standard techniques (21). Hyperimmune found that they contained predominately coccoid forms. antisera to all 11 previously described Acholeplasma species Preparations of cells sedimented from broth cultures and and to 10 other unclassified acholeplasmas were available examined by electron microscopic techniques also contained from the reference collection maintained at the National predominately coccoid elements. No evidence of cell walls Institute of Allergy and Infectious Diseases laboratory in was observed in these preparations; representative round Frederick, Md. (28). Antisera from this collection were cells varied in diameter from 300 to 500 nm and were utilized in standard disk growth inhibition tests (7) against surrounded by a single cytoplasmic membrane (Fig. 2). both strain F7T and strain F28 by using a mycoplasma agar Sterol requirement. The growth responses of strains F7T medium containing 1% bovine serum fraction and aerobic and F28 in serum-free base media containing fatty acid or VOL.41, 1991 ACHOLEPLASMA SEIFFERTII SP. NOV. 47

TABLE 1. Growth responses of strains F7T and F28 to cholesterol"

Amt of cell protein Supplement(s) added to (mg/100 ml of medium) serum-free base medium Strain F7T Strain F28 Bovine serum fraction (1%) (control)' 1.02 5.25 Noneb IG' IG Albumin (1%) and palmitic acid IG IG (10 pglml)b Albumin and palmitic acid plus: Tween 80 (0.04%)' 0.46 1.15 Cholesterol (1 pg/ml) 0.82 1.34 Cholesterol (5 pglml) 0.83 1.54 Cholesterol (10 pg/ml) 1.10 2.10 Cholesterol (20 pglml) 0.91 2.325 All cultures were incubated at 30°C. These preparations contained no cholesterol. IG, Insufficient growth.

strain F28 rapidly ferm&ted-glucose and mannose with the cholesterol supplements are shown in Table 1. Neither strain grew in the serum-free base broth alone or when palmitic production of acid and a consequent decline in the pH of the culture medium. No evidence of arbutin, arginine, or urea acid and albumin were added to the medium. Significant growth yields occurred when the broth base was supple- hydrolysis was observed. Colonies of strain F7T on agar mented with 0.04% Tween 80 fatty acid mixture, and the medium hemadsorbed guinea pig erythrocytes. Passage of a strain F7= broth culture containing lo9 color-changing units addition of cholesterol (1 to 20 kg/ml) frequently provided per ml through 450- and 300-nm-pore-size membrane filters some slight additional stimulation of growth. In tests in reduced the viable cell titer to about one-fifth and about which 0.01% Tween 80 was added to the base broth (data not 100-fold, respectively (to 2 x lo8 and 2 x color-changing shown), similar stimulation of the growth of both strain F7T lo7 units per ml, respectively). Filtrates recovered after passage and strain F28 was observed. The lack of a growth require- ment for cholesterol was confirmed by serially diluting of the strain through a 220-nm-pore-size membrane filter contained 2 x lo4 color-changing units per ml, and a filtrate cultures of strains F7T and F28 in serum-free medium recovered after passage through a 100-nm-pore-size filter supplemented with Tween 80. Each organism continued to was negative for growth. Similar results were observed in grow through 23 10-fold dilutions in medium containing filtration tests with strain F28 (data not shown). Serological tests. Growth inhibition and plate immunoflu- orescent tests carried out with antisera or conjugates to the acholeplasmas and mycoplasmas listed recently (27) indi- cated that neither strain F7T nor strain F28 is related serologically to 11 previously described species belonging to the genus Acholeplasma or to a group of 10 unclassified, sterol-nonrequiring acholeplasmas that represent putative species in the genus. Growth inhibition tests also were used to establish a serologic relationship between strain F7T and strain F28. Antiserum prepared to strain F7T produced homologous growth inhibition zones of 5 to 6 mm, while growth of strain F28 antigen produced zones which ranged in size from 4 to 5 mm with this antiserum. Analysis of cell proteins. Figure 3 shows the patterns of the cellular proteins of strains F7T and F28. The patterns are quite similar, with differences primarily involving the inten- sity of some bands. Genomic analysis. Genome size measurements of the DNA of strain F7T, in which we used renaturation kinetics, indicated that this organism had an average genome molec- ular mass of about 886 MDa (individual values ranged from 713 to 1087 MDa). Similar values were obtained for strain FIG. 2. Electron micrograph of a sectioned and stained cell F28 (range, 756 to 1,054 MDa; average, 897 MDa)- The base Dellet of strain F7T. The section was stained with 2% aaueous uranvl composition of the DNA (guanine-plus-cytosine content) of ketate and Reynold lead citrate. The arrow indicates' a unit me& both strains was determined to be 30 2 1mol%, as measured brane. Bar = 100 nm. by the buoyant density method. Reciprocal hybridization 48 BONNET ET AL. INT. J. SYST.BACTERIOL.

31) and to share with these two acholeplasmas the unique growth requirement for increased amounts of some fatty acids (0.01 to 0.04% Tween 80) (14, 27). Strains F7T and F28 were isolated from floral surfaces, and no isolates that are serologically related to these two organisms have been reported in insects so far. Until further isolations repre- sentative of this organism are made from insects, arthro- pods, or other environments or locations, the true habitat and whether this organism is involved in a plant-insect cycle will remain unknown. The properties described above for strains F7T and F28 fulfill the essential criteria (13) for species belonging to the class Mollicutes, including absence of a cell wall, filter- ability, lack of reversion to walled bacteria when organisms are grown in antibiotic-free media, penicillin resistance, and production of typical colonial forms on agar. The lack of any growth requirement for sterol or serum and the lack of helicity place these organisms in the family Acholeplasma- taceae (25). The inability of strains F7T or F28 to hydrolyze urea, in addition to the properties outlined above, mandates assignment of the organisms to the genus Acholeplasma. Finally, the results of serologic comparisons of strains F7T and F28 with a collection of antisera to all previously described Acholeplasma species and with antisera to other unclassified strains representing putative species in the genus demonstrate that these strains are not related to other organisms in the genus at the species level. Therefore, we propose the name Acholeplasma seiffertii for these organ- isms, in recognition of the contributions which Gustav Seiffert made from 1937 to 1940 to our understanding of the occurrence of filterable, non-sterol-requiring procaryotes in FIG. 3. One-dimensional polyacrylamide gel electrophoresis of soil and compost (19, 20). The taxonomic description below total cell proteins of strains F7T and F28. Gels were stained with summarizes the properties of this species. Coomassie blue. The masses of molecular markers (in daltons) are Acholeplasma seiflertii sp. nov. Acholeplasma seiffertii indicated on the right and on the left. (seif.fer’ti.i. N. L. gen. n. seiffertii, of Seiffert, in honor of Gustav Seiffert, a German microbiologist who performed pioneer studies of sterol-nonrequiring mollicutes that occur comparisons of the DNAs of the two strains revealed high in soil and compost.) Cells are primarily of coccoid, varying levels of genomic relatedness (Table 2). The DNAs from in diameter from 300 to 500 nm. Cells lack true cell walls. these two organisms also appeared to be unrelated to the Nonmotile. Colonies on solid medium containing 0.8% No- DNAs of a cluster of other acholeplasmas, including strains ble agar usually have the appearance of fried eggs. Chemoor- of Acholeplasma florum. ganotroph. Acid produced from glucose and mannose. Does Habitat. The two mollicutes characterized in this study not hydrolyze arbutin, arginine, or urea. Agar colonies appear to have a plant habitat similar to that of Achole- hemadsorb guinea pig erythrocytes. plasma florum and Acholeplasma entomophilurn strains (30, Cholesterol or serum not required for growth. Temperature range for growth, 20 to 35°C; optimum growth occurs at 28°C. Serologically distinct from previously described Achole- TABLE 2. Levels of homology between DNAs of strains F7= plasma species. and F28 and DNAs of other plant-insect acholeplasmas Isolated from floral surfaces of a sweet orange (Citrus % Hybridization with tritium- sinensis) in Morocco and wild angelica (Angelica sylvestris) Unlabeled DNA from: labeled DNA froma: in France, but the true habitat is not known. Pathogenicity Strain F7T Strain F28 for plants or insects has not been determined. The average genome molecular mass is 886 MDa. The Strain F7T 100 (80.4)b 80.5 guanine-plus-cytosine content of the DNA is 30 2 1 mol%, Strain F28 85 100 (77) as determined by the buoyant density method. Acholeplasma fIorum L1 9 11.5 The type strain is strain F7 (= ATCC 49495). Acholeplasma fIorum MQ-3 10.8 12.0 Acholeplasma laidlawii PG8 7.8 8.3 Strain 0502 6.9 6.6 ACKNOWLEDGMENT Strain 5233 8.0 10.2 We thank Colis Blood for his contributions to the preparation of Strain PS-1 8.7 9.9 antisera to the new acholeplasmas. Salmon sperm (control) 6.0 5.5 REFERENCES Averages of three experiments. Hybridization was carried out under high-stringency conditions (37°C). 1. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and J. E. Faber. The numbers in parentheses are homologous experimental hybridization 1970. Standardized bacteriologic techniques for characteriza- percentages, which were normalized to 100%. tion of Mycoplasma species. Int. J. Syst. Bacteriol. 20:35-58. VOL. 41, 1991 ACHOLEPLASMA SEIFFERTII SP. NOV. 49

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