Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Autolytic Formation of Protoplasts (Autoplasts) of Streptococcus faecalis 9790: Release of Cell Wall, Autolysin, and Formation of Stable Autoplasts

A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.     

Regeneration of the cell wall of protoplasts of the fission yeastSchizosaccharomyces versatilis in liquid media and their reversion to cells

Regeneration of the cell wall and reversion of protoplasts with a completely regenerated cell wall to cells were studied by light and electron microscopy in protoplasts of the fission yeastsSchizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even m liquid media The wall regeneration began with the formation of a thin irregular net of flat bundles of long microfibrils and the net was gradually filled with aggregates of short straight microfibrils and small piles of amorphous material. Osmotically resistant organisms with regenerated walls were detected after a 4–6 h cultivation Depending on the nutrient medium used 10–80 % of protoplasts with the regenerated wall were obtained that reverted subsequently to cells. The high percentage of the wall regeneration and reversion to cells was reached by combining cultivation in a poor medium with that in a rich medium Reversion to cells could only occur after the protoplasts had regenerated rigid cell walls These walled protoplasts underwent septation, and, by polar growth, produced cylindrical cells, further dividing by fission.     

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.     

The isolation, culture and regeneration of Petunia leaf protoplasts

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.     

Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.     

Changes in the plasma membrane of regenerating protoplasts of Candida albicans as revealed by freeze-fracture electron microscopy

Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

Effect of restrictive temperature on cell wall synthesis in a temperature-sensitive mutant of Bacillus stearothermophilus.

A temperature-sensitive mutant of Bacillus stearothermophilus, TS-13, was unable to grow above 58 degrees C, compared to 72 degrees C for the wild type. Actively growing TS-13 cells lysed within 2 h when exposed to a restrictive temperature of 65 degrees C. Peptidoglycan synthesis stopped within 10 to 15 min postshift before a shut down of other macromolecular syntheses. Composition of preexisting peptidoglycan was not altered, nor was new peptidoglycan of aberrant composition formed. No significant difference in autolysin activity was observed between the mutant and the wild type at 65 degrees C. Protoplasts of TS-13 cells were able to synthesize cell wall material at 52 degress C, but not at 65 degrees C. This wall material remained closely associated with the cell membrane at the outer surface of the protoplasts, forming small, globular, membrane-bound structures which could be visualized by electron microscopy. These structures reacted with fluorescent antibody prepared against purified cell walls. Production of this membrane-associated wall material could be blocked by bacitracin, which inhibited cell wall synthesis at the level of transport through the membrane. The data were in agreement with previous studies showing that at the restrictive temperature this mutant is unable to alter its membrane fatty acid and phospholipid composition with temperature such that it is not able to maintain a membrane lipid composition which permits normal membrane function at the restrictive temperature.     

Evidence for the synthesis of soluble peptidoglycan fragments by protoplasts of Streptococcus faecalis.

Growing protoplasts of Streptococcus faecalis 9790 were found to synthesize and excrete soluble peptidoglycan fragments. The presence of soluble peptidoglycan derivatives in culture supernatants was determined by (i) incorporation of three different radioactively labeled precursors (L-lysine, D-alanine, and acetate) into products which, after hen egg-white lysozyme hydrolysis, had the same KD values on gel filtration as muramidase hydrolysis products of isolated walls; (ii) inhibition of net synthesis of these products by cycloserine and vancomycin; and (iii) identification of disaccharide-peptide monomer using the beta-elimination reaction, gel filtration, and high-voltage paper electrophoresis. Under the conditions of these experiments the presence of newly synthesized, acid-precipitable (macromolecular) peptidoglycan was not detected. The predominance of monomer (70 to 80%) in lysozyme digests of peptidoglycan synthesized by protoplasts was in sharp contrast to digest of walls from intact streptococci which contain mostly peptide cross-linked products. Biosynthesis and release of relatively uncross-linked, soluble peptidoglycan fragments by protoplasts was related to the absence of suitable, preexisting acceptor wall.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.     

The ultrastructural organization of Brucella L forms and revertant cultures

The submicroscopic organization of Brucella cells in the process of L-transformation and reversion has been studied. As revealed in this study, at its initial stages L-transformation is accompanied by the loss of cell-wall peptidoglycan and by considerable polymorphism of Brucella cells. Further stages are characterized by the presence of a great number of closed annular membrane structures both in the cytoplasm and outside the cells. At late stages of L-transformation the destruction of the cytoplasm and the cells has been found to occur. In revertant cultures the restoration of the cell wall has been noted.     

Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts

Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [¹⁴C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[¹⁴C]glucose and guanosine diphosphate-[¹⁴C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.Abbreviations ECM extracellular material - GLC gas liquid chromatography - GDP-glucose guanosine diphosphate glucose - UDP-glucose uridine diphosphate glucose - U enzyme units as defined by Sigma Chemical Corp., St. Louis, Mo., USA     

A method for isolation of protoplasts from dermatophytes

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.     

Conditional mutants of Staphylococcus aureus defective in cell wall precursor synthesis

Temperature-sensitive (ts) mutants of Staphylococcus aureus with defective cell wall biosynthesis have been differentiated from other ts mutants by their ability to grow at the restrictive temperature (43 C) in the presence of 1 m NaCl. Under all conditions they possess normal colonial and cellular morphology at the level of resolution of the light microscope and are, therefore, not protoplasts. However, differences between mutant and wild-type cells can be seen by scanning electron microscopy. Many of the mutants contained concentrations of nucleotide precursors of peptidoglycan synthesis in excess of those present in wild-type cells, at both 30 and 43 C. The types of peptidoglycan precursors accumulated by six of the mutants have been determined, and specific enzymatic defects in three of these have been identified.     

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.     

Cytological study of early stages of regeneration of protoplasts of fungi and Streptomyces

Early stages of Penicillium chrysogenum 51 and Streptomyces lividans 66 protoplast regeneration on solid media were studied microscopically under conditions of microcompartments. It was shown that at the early regeneration stages there were both rapid reversion into the mycelial form and a retarded one. In P. chrysogenum retarded regeneration resulted in formation of hypha-like structures or protoplast breaking into fragments of various sizes. Some of the fragments restored the cell walls and mycelial organization whereas the others lysed. As a result of the breaking and compartmentalization of the viable areas one protoplasts formed several centers of P. chrysogenum colony reversion. Retarded regeneration of protoplasts in S. lividans 66 resulted in their growth and multiplication in the protoplast-like L-form. On media with penicillin, glycine and horse serum there were isolated colonies of S. lividans L-forms subject to passages or reversion depending on the medium composition.     

Close-packing of plasma membrane particles during wall regeneration by isolated higher plant protoplasts—fact or artefact?

Summary Freeze-fracture preparations of protoplasts isolated from cell suspension cultures and leaf mesophyll tissue have been examined by transmission electron microscopy. During the first 72 hours of cell wall regeneration, the 8–10nm intramembraneous particles were randomly distributed on both the protoplasmic and extracellular fracture faces of the plasma membranes of protoplasts frozen and fractured in the culture medium without glutaraldehyde fixation or cryoprotection. Incubation of living protoplasts in culture medium containing 20% v/v glycerol as cryoprotectant prior to freezing without fixation caused deformation of the plasma membrane in the form of protrusions accompanied by particle aggregation on the protoplasmic fracture face of the membrane. Intramembraneous particle aggregation was not observed in protoplasts fixed in glutaraldehyde prior to incubation in medium containing glycerol. The aggregation of particles into hexagonal close packed arrays and elongate chains is discussed in relation to a previous report in the literature of the possible involvement of intramembraneous particle complexes in microfibril formation by isolated higher plant protoplasts.