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.     

Improved Microscopy of Mycoplasma In Vitro

Techniques were developed for continuous microscopic observation of mycoplasmata growing in vitro in Rose chambers by using an inverted phase microscope. The methods permitted direct microscopic observation of undisturbed growth of mycoplasmata in liquid medium. Inocula of mycoplasmata were passed through 0.22-mum filters before culture to provide a suspension of discrete particles. The sequential growth of Mycoplasma pneumoniae was followed from points or single straight lines, with development of branching, a net-like confluence of filaments, large bodies occurring in the center of developing colonies, and finally coccoid forms. Other species of Mycoplasma which did not attach as readily to glass could be observed also by inverted phase microscopy. Umbonation of colonies (a "friedegg" appearance) occurred in liquid medium, indicating that this appearance was not due simply to interaction with the agar medium, but may reflect a qualitative difference in growth patterns between center and periphery. For growth on solid medium, direct observation of colonies in uncovered plates of agar medium was made by using inverted phase microscopy. This was found helpful in detecting small colonies and in observing relationships between colonies.     

Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium.

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.     

Gliding motility of Mycoplasma sp. nov. strain 163K.

The gliding movements of Mycoplasma sp. nov. strain 163K cells were characterized by photomicrographic and microcinematographic studies. The capability of gliding proved to be a very stable property of strain 163K. Cells were continuously moving, without interruption by resting periods, on glass as well as on plastic surfaces covered with liquid medium. Gliding cells always moved in the direction of their headlike structure; their course did not indicate any preference for a certain direction. Under appropriate growth conditions, cells showed linear and circular movements. Under inadequate conditions, cells glided in narrow circles or entered into zigzag trembling and tumbling movements. Organisms glided as single cells, in pairs, and in multicellular configurations. Movement patterns and gliding velocity were significantly affected by the cultivation and preparation time, the medium viscosity, and the storage and observation temperature. The number of passages on artificial media and the composition of the media used did not have a striking influence on gliding motility, but movements were effectively inhibited by homologous antiserum. The data obtained suggest that at least some of the structures associated with gliding are heat sensitive and located on the cell surface, that the gliding mechanism requires an intact energy metabolism, and, finally, that gliding motility is an extremely stable genetic property of Mycoplasma sp. nov. strain 163K.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Formation of Filaments by Pseudomonas putida

When Pseudomonas putida 40 was grown on a variety of liquid media in which oxygen became a limiting factor during growth, the latter stages of growth involved the elongation of cells without septation, which can result in the complete filamentation of the culture (up to several hundred micrometers long). The filaments appeared to consist of a chain of protoplasts within a common sacculus. Later these filaments were capable of a rapid fragmentation by septation to give a population of ordinary rods with a corresponding increase in the number of viable particles but no appreciable change in total bacterial mass. Filamentation did not occur if slow growth rates were maintained by restriction of oxygen availability from the beginning of growth. In complex media filaments were not formed during growth on 1% peptone alone, but the addition of 0.1 M phosphate or 6.6 × 10⁻⁴ M EDTA induced extensive filamentation that was reversed by the addition of 6.6 × 10⁻⁴ M Mg²⁺. In minimal media a much higher Mg²⁺ concentration than that required for active growth or present in the complex media was usually required for filamentation. A very narrow range of Mg²⁺ concentration promoted filamentation, and this optimum differed markedly depending on the carbon source used. Other medium variations which influenced the level of filamentation are reported. We found that most strains of P. putida (including the neotype strain) and P. fluorescens gave filaments under the conditions developed with strain 40, whereas several strains of P. aeruginosa failed to give filaments on the same media.     

Adaptation of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> to unfavorable growth conditions: Changes in morphological and physiological characteristics

Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions results in altered morphological and physiological characteristics of the cells. M. gallisepticum populations in a complete nutrient medium contain pear-shaped vegetative cells ( d ～ 0.3 μm; l ～ 0.8 μm) with pronounced polar and cytoskeletonlike structures. Such mycoplasma cells are able to induce damage in a bacterial genome, causing an SOS response of the test strain (Escherichia coli PQ37). In a starvation medium, M. gallisepticum produces nanoforms, small coccoid cells (d ～ 0.15–0.2 μm) without either polar or cytoskeleton-like structures. Unlike vegetative cells, nanoforms do not induce genome damage. Alleviation of unfavorable growth conditions results in a reversion of nanoforms to vegetative cells.     

Mycoplasma penetrans under nutritional stress: influence on lipid and lipoprotein profiles and on the binding to and invasion of HeLa cells

By serial passages through media containing decreasing concentrations of horse serum, the sterol-requiring Mycoplasma penetrans strain HF-2 was adapted to grow in a serum-free medium supplemented with bovine serum albumin, cholesterol and free fatty acids. Chemical analysis of membrane preparations obtained from the native and adapted strains revealed two major differences. (1) The polar lipid fraction of the native strain contains, in addition to the de novo-synthesized phospholipids, exogenous lipids incorporated unchanged from the growth medium, whereas exogenous lipids were not detected in the adapted strain. (2) Protein analyses of the native and adapted strains showed that upon adaptation, the 42-kDa membrane lipoprotein, one of the two major lipoproteins of this organism, was missing. Studies on the adhesion to, and invasion of HeLa cells by the native and adapted strains revealed that whereas the adherence to HeLa cells of the adapted strain was almost the same as that of the native strain, the invasiveness of the adapted strain into HeLa cells was very low or nonexistent.     

Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.     

Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).     

Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain

The Gram-positive bacterium Bacillus cereus is a facultative anaerobe that is still poorly characterized metabolically. In this study, the aerobic vegetative growth and anaerobic vegetative growth of the food-borne pathogen B. cereus F4430/73 strain were compared with those of the genome-sequenced ATCC14579 strain using glucose and glycerol as fermentative and nonfermentative carbon sources, respectively. Uncontrolled batch cultures on several defined media showed that B. cereus strains had high amino acid or pyruvate requirements for anaerobic fermentative growth. In addition, growth performance was considerably improved by maintaining the pH of the culture medium near neutrality. Spectra of fermentation by-products were typically (per mole of glucose) 0.2-0.4 acetate, 1.1-1.4 L-lactate, 0.3-0.4 formate, and 0.05-0.2 ethanol with only traces of succinate, pyruvate, and 2,3-butanediol. These spectra were drastically changed in the presence of 20 mmol nitrate x L(-1), which stimulated anaerobic growth. During anaerobic and aerobic respiration, the persistent production of acetate and other by-products indicated overflow metabolisms. This was especially true in glucose-grown cells for which respiratory complex III made only a minor contribution to growth. Surprisingly, oxygen uptake rates linked to the cytochrome c and quinol branches of the respiratory chain were maintained at high levels in anaerobic, respiring, or fermenting cells. Growth and metabolic features of B. cereus F4430/73 are discussed using biochemical and genomic data.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.     

Morphological changes of synchronized Campylobacter jejuni populations during growth in single phase liquid culture

Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.     

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.     

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.     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Characteristics of a stable, filamentous mutant of a coccoid blue-green alga

Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatum strain BG1, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.