Isolation,structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase

The action of the lysoamidase bacteriolytic complex on Staphylococcus aureus VKM B-209P cells has been studied to obtain protoplasts. The cells in the midlogarithmic phase were the most sensitive to lysoamidase action. It led to local destruction of cell wall due to hydrolysis of the peptidoglycan. Protoplast formation occurred in two steps in the presence of 1 M sucrose. First, osmotically fragile spheroplasts were formed. Then, the protoplasts were released from the destructed cell wall. The protoplast yield was about 80%. The protoplasts preserved the intact ultrastructure and were able to synthesize peptidoglycan fibrillae. Mainly the spheroplasts that maintained the cell-wall residues reversed into bacterial forms. The protoplasts had respiratory activity similar to cells. Respiration of cells and protoplasts was stimulated by various substrates. High rates of oxygen consumption were observed with -glycerophosphate and ethanol as substrates.     

Hydrolysis of a Staphylococcus aureus cell wall peptidoglycan by 209 P lysoamidase

Hydrolysis of Staphylococcus aureus 209 P cell wall peptidoglycan was accompanied by the liberation of 1.3 mol of C-terminal and 1.2 mol of N-terminal glycine per mole of Glu as well as of 0.5 mol of N-terminal and 0.3 mol of C-terminal alanine. Gel chromatography on Sephadex G-25, ion-exchange chromatography on QAE-Sephadex A-50 and paper electrophoresis of S. aureus peptidoglycan hydrolysates gave seven homogeneous fractions; these fractions were structurally defined. Lysoamidase hydrolyzed bonds Mur-Ala, Gly-Gly and Mur-GlcN in the peptidoglycan molecule. Hydrolysis of glycan chains was accompanied by the formation of large fragments, (GlcN-Mur)9 and (GlcN-Mur)28. The lytic effect of lysoamidase on S. aureus peptidoglycan is coupled with bacteriolytic enzymes of lysoamidase: acetmuramyl amidase, glycyl--glycine endopeptidase and acetyl--muramidase.     

用中性红标记酵母原生质体初探

用２％蜗牛酶处理酵母细胞６０分钟,啤酒酵母Ｙ２９的原生质体形成率为９０％,再生率为９．５％;糖化酵母ＩＢ的原生质体形成率为８６％,再生率为１２％。用５００ｐｐｍ中性红染液对Ｙ２９菌株的整细胞和原生质体染色１５分钟,细胞的着色率为８４％,存活率为１２％,而原生质体的着色率为７５％,再生率为６．４％,经染色后的原生质体体积缩小,在交变电场中排队所需的场强电降低。     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Factors affecting protoplast formation and regeneration by four species of Streptomyces

Four species of Streptomyces, Streptomyces canescens, S. limosus, S. griseus and S. griseolus , were used to study the effects of glycerine and gelatin on the formation and regeneration of protoplasts. For each species efficient protoplast formation with high protoplast concentrations and low levels of non-protoplast units was obtained with mycelia grown in medium without glycerine. The low regeneration frequencies of protoplasts of S. canescens and S. limosus on R2 medium were increased substantially by the addition of 1% gelatin. The use of single colonies, rather than spores, to establish mycelial cultures was found routinely to produce good protoplast preparations.     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的:采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法:经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶(0.8%溶菌酶+1.2%蜗牛酶+1.6%纤维素酶)处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果:在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论:采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的：采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法：经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶（0.8%溶菌酶＋1.2%蜗牛酶＋1.6%纤维素酶）处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果：在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论：采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

谷子胚性愈伤组织粘液提取物对原生质体培养的影响

将谷子胚性愈伤组织粘液提取物添加到谷子原生质体培养基中,其对原生质体培养的影响表明该提取物有助于原生质体形成细胞壁;并且该类有粘液分泌的念伤组织的原生质体游离所需的酶液浓度低、处理时间短。由原生质体形成完整细胞的数量在一定范围内与谷子原生质体培养的植板率相对应;通过增加形成完整细胞的数量可较大幅度地提高原生质体培养的植板率。     

Effects of nutrients,cell density and culture techniques on protoplast regeneration and early protonema development in a moss,Physcomitrella patens

To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.     

Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts

The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied. The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells. The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock. The heat-shock induces modification of 88 and 86 Kd heat-shock proteins. It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.     

Characteristic features of protoplast formation and regeneration in Fusidium coccineum

The methods for preparation and regeneration of protoplasts were tested with respect to the strains of F. coccineum markedly differing in their capacity for antibiotic production, sporulation and the growth rate. It was found that the substrate used for the culture growth had a significant effect on the cell wall and sensitivity of the mycelium to lytic enzymes. An enzyme from Hellix pomatia and its combination with lysozyme were used for lysing the culture. The cytological investigation of the time course of the culture lysis revealed a stage-by-stage pattern of protoplast formation by means of fragmentation of the hyphal contents till a ball was formed. Two to 4 protoplasts differing in their size and structure were formed within a cell. The pH value and osmotically stabilizing component had some effect on the rate of protoplast formation. Highly productive strains were characterized by formation of protoplasts heterogenous in their size and by decreased frequency of regenerations. The enzyme-free protoplasts preserved their viability and capacity for germination in osmotically stabilizing media for 72-96 hours of storage at 4 degrees C. On solid media the regeneration frequency reached 38 per cent. The regenerated cells formed colonies morphologically similar to those of the intact culture.     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Cytologic control of protoplast formation and regeneration in Streptomyces erythraeus, strain BTCC-2

Experiments on protoplast formation and regeneration in S. erythraeus, strain BTCC-2 (Saccharopolyspora erythrae) were performed under microscopic control at all the stages. It was shown that the highest protoplast titer was provided by the mycelium grown in one step in the absence of glycine. For characterizing the protoplasts formed by the mycelium grown under different conditions, their regeneration capacity was estimated by microscopic examination of the protoplasts after 15-20-hour growth in microchambers and evaluation of the regeneration efficiency 7-10 hours later. Of interest was the fact of spontaneous development of colonies consisting of the protoplast-like cells (L-cells) in 15-20 hours. Such colonies were formed only by the protoplasts grown from the mycelium incubated in one step in the absence of glycine or in the presence of 0.1 per cent of glycine. Such conditions provided also the maximum efficiency of the protoplast regeneration. The long-term storage of protoplasts led to a decrease in their viability.     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.     

Protoplast isolation and regeneration in Streptomyces clavuligerus

The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. The latter promoted improved protoplast viability. A systematic survey was made of the components of regeneration medium R5, previously used for S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus protoplasts resulted from these experiments. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.     

The isolation of protoplasts of the fission yeastSchizosaccharomyces byTrichoderma viride and snail enzymes

The formation of protoplasts of the fission yeastsSchizosaccharomyces pombe andSchizosaccharomyces versatilis after the combined application of snail enzymes andTrichoderma viride enzymes in an osmotic stabilizer (0.4m KC1, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.     

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species,Sonneratia alba,S. caseolaris and S. ovata: development of a bioassay method for allelopathy,the protoplast co-culture method

A bioassay method for allelopathy, the ‘protoplast co-culture method’ was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl₂, MgSO₄ and CaCl₂) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3 % sucrose and 0.6–0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the ‘sandwich method’ with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.     

Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts

A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3′ chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.