Preparation and regeneration of protoplasts from Bacillus megaterium cells dried by sublimation]

Bacterial protoplasts are widely used in genetical research, for instance, in protoplasts fusion experiments and the transfer of heterologous DNA into bacterial cells. The usage of a new fresh grown culture of bacteria in every experiment restricts the reproducibility of the results preventing the technique becoming widespread. The use of antioxidants as components of stabilizing medium for sublimation drying of Bacillus megaterium cells supported cellular viability in bacterial culture. It also made possible preservation of such cellular fundamental properties as the ability to form protoplasts and regenerate the cell wall. Efficiencies of protoplasts formation and generation are similar for lyophilized and fresh grown cells. Cellular properties are conserved for 6 months of storage at least. Experiments with a lot of lyophilized biomass samples are highly reproducible. The potential of the technique was demonstrated in obtaining the hybrid Bacillus megaterium colonies by fusion of protoplasts derived from lyophilized genetically marked strains stored for up to 6 months.     

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.     

Plating of isolated tobacco mesophyll protoplasts on agar medium

Summary A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

A comparative study of submicroscopic structures of protoplasts of various yeast species

A submicroscopic structure was studied of protoplasts of five different yeast species multiplying by budding, formation of cross septum and by a division typical for apiculate yeasts. The protoplasts retain their species specificity. Most considerable changes typical for the conversion of a cell to protoplast are found in membrane cell systems. The reduction of membranes of the endoplasmic reticulum is particularly striking. Both membrane units are frequently separated from each other by lenticular pseudovacuoles. Mitochondria in protoplasts are swollen and their number is reduced approximately two-fold. Defects are often observed in a nuclear membrane. The perinuclear space is usually extended by lenticular pseudovacuoles. A large number of vacuoles is observed in the basic protoplast cytoplasm. The surface of the protoplasts of all species studied is formed only by a cytoplasmic membrane. A partially digested original cell wall often adheres to protoplasts ofSchizosaccharomyces pombe.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

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.     

Functional sieve element protoplasts

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.     

Callus formation from protoplasts of culturedSpinacia oleracea cells

Cell suspensions were initiated from plumule derived calli ofSpinacia oleracea. Some of these cell lines could be maintained in culture for at least three years without a reduced growth rate. A high yield of protoplasts was obtained from the cell suspensions. When protoplasts were cultured in Murashige and Skoog medium with naphthaleneacetic acid and 6-benzyladenine, cell wall formation was observed after three days. The cultured protoplasts produced numerous cell-clusters within two weeks. However only protoplasts isolated from suspensions which were in a rapidly dividing phase were able to divide with a high frequency and give rise to callus colonies.     

Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.     

Formation and Regeneration of Methanococcus voltae Protoplasts

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.     

A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes

The bacterial pathogen Yersinia pseudotuberculosis uses type III secretion machinery to translocate Yop effector proteins through host cell plasma membranes. A current model suggests that a type III translocation channel is inserted into the plasma membrane, and if Yops are not present to fill the channel, the channel will form a pore. We examined the possibility that Yops act within the host cell to prevent pore formation. Yop- mutants of Y.pseudotuberculosis were assayed for pore-forming activity in HeLa cells. A YopE- mutant exhibited high levels of pore-forming activity. The GTPase-downregulating function of YopE was required to prevent pore formation. YopE+ bacteria had increased pore-forming activity when HeLa cells expressed activated Rho GTPases. Pore formation by YopE- bacteria required actin polymerization. F-actin was concentrated at sites of contact between HeLa cells and YopE- bacteria. The data suggest that localized actin polymerization, triggered by the type III machinery, results in pore formation in cells infected with YopE- bacteria. Thus, translocated YopE inhibits actin polymerization to prevent membane damage to cells infected with wild-type bacteria.     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

Isolation and regeneration of Micromonospora olivoasterospora protoplasts

The conditions for preparation and regeneration of the protoplasts of M. olivoasterospora were developed. It was found that effective formation of the protoplasts required preliminary cultivation of M. olivoasterospora in the medium containing glycine in a concentration inhibiting its growth at least by 60-80 per cent. The strains studied markedly differed in their sensitivity to glycine and were highly sensitive to it. The efficacy of the protoplast formation depended on the culture age and increased with the use of the lytic enzyme 3 of Cytophaga dissolvens. The possibility and advisability of the use of prolonged lysis of the Micromonospora cell walls were shown. A rich organic medium was used for regeneration of the protoplasts.     

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Summary Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.     

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.     

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.