Formation and reversion of protoplasts in mutant strains ofBrevibacterium sp. M27

In mutant strains ofBrevibacterium sp. M27 requiring amino acids and resistant to antibiotics four methods were verified and a method inducing a high formation of protoplasts and their reversion was developed. Formation of protoplasts is 60–99% and their reversion 17–76%. In induced mutants a correlation between protoplast formation and growth rate could be demonstrated.     

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.     

Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.     

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.     

A novel method for regenerating the protoplasts of thermophilic bacilli

A range of Bacillus thermophiles was tested for the ability to be converted to protoplasts with lysozyme and subsequently, to regenerate to bacillary form. Protoplast formation was straightforward but many of the strains failed to regenerate on commonly-used media. Two medium components were found to be causing the inhibition. Growth of protoplasts as L-forms only occurred if the medium lacked phosphate. However, reversion of L-forms to bacilli was asynchronous and infrequent. Regeneration of cell walls by protoplasts/L-forms to re-establish bacillary form was greatly improved when the medium was gelled with pluronic polyol F127 in place of agar or similar polysaccharides.     

Selection of strains of Streptomyces griseus Kr., the producer of a streptothricin antibiotic grisin, using the method of protoplast fusion

The effect of protoplasting on antibiotic activity of the grisin-producing organism was shown. High frequency of Grn- mutants after strain VG307f protoplasting and no capacity in these mutants for reversion to the initial Grn+ phenotype were shown. The reversion frequency was less than 10(-8). Moreover, it was shown that all the Grn- mutants lost their stability (GrnR) to the effect of their own antibiotic. With respect to strain VG212 there was noted a significant increase in the number of both the minus and the plus variants after the protoplast formation and regeneration. Fusing of protoplasts of strains VG307f and VG212 belonging to the divergent lines in selection of S. griseus Kr. yielded the phage stable strain VG7849 with high levels of the antibiotic production and improved technological properties.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.     

Morphological study of the reversion to bacillary form, of Bacillus megaterium protoplasts.

Protoplasts of Bacillus megaterium readily reverted to bacillary form in liquid media and when plated in a soft-agar layer onto the surface of appropriate agar media. Three phases of the reversion sequence could be differentiated by phase contrast microscopy: (i) increase in size of the individual protoplasts, (ii) non oriented division of the protoplasts and (iii) outgrowth of the bacillary forms. With time-lapse photomicrography, reversion sequences of single protoplasts were demonstrated.     

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.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

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.     

Double-strand break repair machinery is sensitive to UV radiation

The precision of the repair of linearized plasmid DNA was analyzed using a nonsense mutation inactivated beta-glucuronidase (uidA) marker gene delivered to Nicotiana plumbaginifolia protoplasts and Nicotiana tabacum leaves. The reversions at the stop-codon allowed the reactivation of the marker gene. Here we report that irradiation of plant protoplasts or plant tissue prior to the delivery of the DNA repair substrate significantly potentiated the reversion frequency leading to a two to fourfold increase over the non-irradiated samples. The increase in reversion frequency was highest upon the delivery of the linear substrates, suggesting increased sensitivity of the double-strand break (DSB) repair apparatus to UV-C. Moreover, the most significant UV irradiation effect was observed in plasmids linearized in close proximity to the stop codon. The higher reversion frequency in UV-treated samples was apparently due to the involvement of free radicals as pretreatment of irradiated tissue with radical scavenging enzyme N-acetyl-l-cysteine abolished the effect of UV-C. We discuss the UV-sensitivity of various repair enzymes as well as possible mechanisms of involvement of error-prone polymerases in processing of DSBs.     

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.     

Reversion of Streptococcal Protoplasts

The reversion of protoplasts of Streptococcus faecium was examined by light and scanning electron microscopy. Regularly shaped streptococci emerged from the narrow region of large, oval protoplasts.     

F-actin contractile rings in protoplasts of the yeast Schizosaccharomyces

By rhodamine-phalloidin fluorescence, distinct continuous F-actin rings were visualized in 18-20% of the protoplasts of Schizosaccharomyces pombe and S. japonicus var. versatilis, in addition to randomly distributed F-actin dots. Whereas the reversion of ring-lacking protoplasts coincided with the polarization of the dotted F-actin pattern, the ring-containing protoplasts became furrowed as the F-actin rings constricted. The furrowing was more conspicuous in S. japonicus var. versatilis than in S. pombe protoplasts and it was blocked when the reversion was inhibited by Novozyme 234 indicating that the cell wall formation is essential for the F-actin ring constriction.     

凤尾菇和香菇原生质体非对称融合

The protoplasts of auxotrophic mutant monokaryon of Pleurotus. Sajor-caju was used as recipient parenL the protoplasts of wild dikareon of Lentinus edodes were heat-inactivated and used as nuclear donor parent. The fusants of two parents showed some their individual characteristics and physiological difference between themselves. Thrugh protoplast re-isolation and reversion of one randomly selective fusant, two parents had been recovered and the donor parent achieved some new physiological characteristics su…     

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.     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Electron-microscopic and Physical Study

Protoplasts of Bacillus subtilis plated on SD medium form L colonies in quantitative yield and propagate in the L form indefinitely. L bodies or protoplasts placed in 25% gelatin medium form bacillary colonies. Details of the reversion of naked bodies to the walled form are reported. In 25% gelatin medium, reversion begins earlier (about 50% reversion in 4 hr) than the multiplication of bacilli. Thus, virtually all the observed bacillary forms are themselves revertants and not the offspring of a few growing clones. The optimal temperature for reversion is 26 C in 25% gelatin. When cells reverting at 26 C are warmed to 40 C for 3 min, reversion is delayed markedly, whereas viability is unaffected. For electron microscopy, a dense protoplast inoculum was placed on a gelatin surface, incubated, and then fixed in situ. There was no multiplication, but crowding delayed reversion markedly. Successive events of reversion are as follows. The loose nucleoid of the protoplasts condenses in response to the gelatin medium and condenses further and further as reversion proceeds. A thin coat of wall develops around the bodies of various sizes and shapes and then increases uniformly in thickness until a wall of normal aspect is formed. Rod-shaped cells grow out from these bodies-sometimes in several directions at once. A few mesosomes begin to appear only after a thin coat of wall has been formed. These are dense, atypical structures compartmented by membranes. They are located at the cell periphery and do not seem to be in contact with the nucleoids. Quantitative estimates showed that only 20 to 25% of revertant cells or cells grown on gelatin contain even a single mesosome. The others have no mesosome at all. Mesosomes thus do not appear to play a significant role in reversion, and normal mesosome functions must presumably be performed elsewhere in the cell in gelatin-grown bacilli. The role of cell wall, its synthesis, and its chemical nature in successive steps in reversion are discussed.     

Instability of actinomadurae protoplasts generated by lytic enzyme treatment

The effect of lytic enzyme treatment upon protoplast formation and reversion in three species of Actinomadura has been determined. Incubation in the presence of lytic enzyme L2 generates large protoplasts (4 μm diameter) which remain intact for only 2h. In comparison, protoplasts formed by the degradative effects of the enzymes lysozyme and L1 are smaller (2 μm diameter), and remain stable for up to 18h. This results in a greater efficiency of regeneration. Lytic enzyme L2 has been shown to contain impurities, including proteolytic activity, which may affect cell wall regeneration.