Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

Identification ofEscherichia coli spheroplasts by the fluorescent-antibody staining technique

Spheroplasts ofEscherichia coli were produced by penicillin or lysozyme-ethylenediaminetetraacetic acid and examined by the direct fluorescent-antibody staining technique. Most spheroplasts stained with somatic-O fluorescent antibody exhibited brilliant peripheral fluorescence with localized areas of irregular staining. Electron micrographs showed that these spherical structures had considerable amounts of cell wall fragments associated with them. Two strains ofE. coli employed in the present study required different concentrations of penicillin for the conversion of all cells in an exponential culture to spheroplasts. Slight differences in lysozyme sensitivity were also encountered with these strains. The direct fluorescent-antibody staining technique was effective for the rapid identification ofE. coli spheroplasts in mixed cultures.     

E. coli spheroplast-mediated transfer of cloned cauliflower mosaic virus DNA into plant protoplasts

Summary Saishin (Brassica chinensis L.) mesophyll protoplasts and E. coli spheroplasts harbouring hybrid plasmid with tandemly dimerized cauliflower mosaic virus DNA were mixed in ratios of 1:1,000 and incubated for 20 min at 30° C in the presence of 20% polyvinyl alcohol. Subsequently, protoplasts/spheroplasts mixture was washed with high pH-high Ca buffer. After 3 days of culture, 8% of Saishin protoplasts were transfected as monitored by immunofluorescence technique. When plant protoplasts and bacterial spheroplasts were mixed in ratios of 1:100 or 1:2,000, 1% or 5% of protoplasts were transfected, respectively.     

Cell cycle inhibition of yeast spheroplasts

Summary Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthesized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mitochondrial DNA synthesis is inhibited in MATa spheroplasts by the application of the yeast mating pheromone, -factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.     

Stabilizing Effect of Colicin E2 on the “Spheroplast Membrane”

Cells of Escherichia coli W3110 and its thymineless mutant, both of which are colicin E2 sensitive, were treated with colicin E2, and then converted to spheroplasts. These spheroplasts seemed to be more stable than those from untreated cells; suspensions of spheroplasts of untreated cells were lysed spontaneously and the turbidity was reduced by approximately 45% on incubation with ethylenediaminetetraacetic acid-lysozyme, whereas suspensions of spheroplasts of colicin E2-treated cells showed 25% reduction in turbidity. This change was irreversible and 5 min of treatment with colicin E2 at 37 C was necessary for stabilization. This process was inhibited by 2,4-dinitrophenol or streptomycin. Cells harboring the colicin E2 factor were not affected by treatment in this way with colicin E2. Alteration of composition of phospholipids was not observed.     

Isolation problems and structural organization of membrane units in Mycobacterium sp. smegmatis

Summary Membrane units from lysed spheroplasts induced by lysozyme or glycine from Mycobacterium spec. smegmatis were isolated in a biological active state by differential centrifugation and by density gradient technique. They were compared morphologically with membraneous fractions obtained from mycobacterial cells disintegrated under a high hydrostatic pressure.Higher homogeneity of membraneous structures isolated from spheroplasts was confirmed. Three types of membraneous structures could be distinguished. They include empty ghosts of spheroplasts, tubular structures containing cytoplasmic material and fragments of typical membraneous structures relatively free of contaminants. By studying protoplasts in the process of lysis it was determined that these structures correspond with cytoplasmic membranes and mesosomes.Differences between lysozome and glycine induced spheroplasts as a starting material for isolation of membraneous structures include the proportion of contamination by other cellular components, reasons of which are discussed.     

Protoplast-like structures formation from two species of enterobacteriaceae by fosfomycin treatment

A procedure for protoplasts formation from Escherichia coli and Serratia marcescens by treatment with fosfomycin alone is described. This method gives high and low yields of stable protoplasts from E. coli and S. marcescens respectively. In the last case numerous spheroplasts were obtained. Electron micrographs of intact cells, protoplasts and spheroplasts are shown.     

Translocation of colicin from the receptor to the inner cell membrane: function of the peptidoglycan layer

Sensitivity of spheroplasts (prepared in two ways) of a colicin-sensitive strain, of colicinresistant and of colicin-tolerant mutants and of strains immune to colicins E1 and E2 was estimated and compared. Generally, the removal of the peptidoglycan layer brought about a slight nonspecific support for colicin translocation across the cell wall in sensitive,tolB tolerant and immune bacteria.tolB spheroplasts were colicin E1-sensitive, but E2-insensitive. Spheroplasts were always fragile and lysed spontaneously, especially those produced by lysozyme. Bacteria carryingtolA, tolQ andtolR mutations kept their colicin insensitivity as spheroplasts, just as the resistant ones. Bacteria rendered colicinogenic and hence colicin-immune turned to high colicin sensitivity in spheroplast form. The results indicate a change in plasma membrane associated with the spheroplast formation.     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

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.     

Ultrastructural organization of spheroplasts induced in Mycobacterium sp. smegmatis by lysozyme or glycine

Summary The conversion of Mycobacterium sp. smegmatis cells into spheroplasts was achieved at a high rate by their treatment with either lysozyme (100 g/ml) or glycine (2%) in a liquid Dubos medium without albumine, stabilized by sucrose (up to 0.34 M). The dynamic of conversion was followed in hanging-drop preparations.Electron microscopic studies of induced spheroplasts were performed. Two types of membranes (80 Å and 130 Å in the width) together with tubular mesosomes localized either intracytoplasmatically or released were revealed.Differences between lysozyme and glycine induction in conversion rate and in time function were noted. Glycine spheroplasts were moreover characterized by numerous cell wall residues adhering to the cell surface and by the incidence of cytoplasmic exfoldings present in a considerably higher amount than in lysozyme induced spheroplasts.Based on these studies suggestions to the mechanism of inductive processes were pointed out.     

Spheroplast of Acetic Acid Bacteria

A procedure, preparing spheroplast of acetic acid bacteria, was established to elucidate the membrane structure of the organisms. Of the acetic acid bacteria, only Acetobacter aceti cells were converted into spheroplasts by the sucrose-EDTA-lysozyme system. To Gluconobacter suboxydans, a method exchanging sucrose in the system for NaCl was indispensable. This NaCl-EDTA-lysozyme system was adequate for almost all acetic acid bacteria, which were converted efficiently into spheroplasts. The existence of EDTA was not essential to the genus Gluconobacter.     

Action of penicillin onSerratia marcescens

The action of penicillin onSerratia marcescens was studied. In culture media containing sucrose (0.33m) and in the presence of magnesium ions, cell wall lesions occurred giving rise to osmotically fragile spheroplasts. However, in the absence of sucrose and magnesium ions it was still possible to induce some spheroplast formation. Quantitative aspects of the conversion of rods into spheroplasts were studied as well as physical properties of the spheroplasts.     

In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin

Spheroplasts of the unstable l-form of Proteus mirabilis with fragile, shape defective cell walls grown in medium containing 120 mg/l penicillin G and then killed and permeabilized by ether treatment, were capable of in vitro synthesis of peptidoglycan from the precursors UDP-GlcNAc and UDP-MurNAc-l-Ala-d-Glu(ms-A₂pm-d-Ala-d-Ala). The in vitro peptidoglycan was extensively peptide-crosslinked, indicating a continuing function of peptidoglycan transpeptidase in the spheroplasts. The seven penicillin-binding proteins (PBPs) of P. mirabilis with their functions as multiple peptidoglycan transpeptidases were shown to be saturated in the spheroplasts and thereby functionally inactivated by the penicillin of the growth medium to a very different degree. Complete or almost complete saturation occurred with the PBPs 1A, 1B, and 3, for which functions as indispensible transpeptidases in Escherichia coli have been postulated. In contrast, PBPs 5 and 6 were not saturated in the l-form spheroplasts. Transpeptidase function has been described previously in PBP 5 of P. mirabilis. The working hypothesis is proposed that synthesis of the functionally defective peptidoglycan of l-form spheroplasts in the presence of penicillin takes place with transpeptidase function of PBP 5.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Efflux of potassium ions from cells and spheroplasts of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast treated with silver and copper ions

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H⁺-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K⁺ transport systems during preparation of spheroplasts.     

A simple protocol to characterize bacterial cell‐envelope lipoproteins in a native‐like environment

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival. The biochemical characterization of membrane proteins, either integral, lipidated or peripheral is challenging due to their mixed protein‐lipid nature, making it difficult to purify and obtain considerable amounts of samples. In contrast to integral membrane proteins, lipidated proteins are usually purified as truncated soluble versions, neglecting the impact of the membrane environment. Here we report a simple and robust protocol to characterize bacterial lipidated proteins in spheroplasts from Escherichia coli using a β‐lactamase as a model. The Metallo‐β‐lactamase NDM‐1 is an enzyme anchored to the inner leaflet of the outer membrane of Gram‐negative bacteria. Kinetic parameters and stability of the lipidated NDM‐1 and the soluble unbound version (NDM‐1 C26A) were measured in spheroplasts and periplasm, respectively. These studies revealed that membrane anchoring increases the K_M of the enzyme, consequently decreasing the catalytic efficiency, while not affecting its kinetic stability. This approach can be used to characterize lipidated proteins avoiding the purification step while mimicking its native environment. This approach also helps in filling the gap between in vitro and in vivo studies.     

Oxidation of exogenous c-type cytochromes by intact spheroplasts of Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize reduced c-type cytochromes derived from horse heart, tuna, Saccharomyces oviformis, Candida krusei, Rhodocyclus purpureus, Rhodopseudomonas plustris and Paracoccus denitrificans with characteristics similar to those observed with isolated membranes. Rates of cytochrome c oxidation by the spheroplasts were only 10% of those measured with isolated membranes in which thylakoid-bound cytochrome oxidase contributes to the overall rates. Small amounts of an endogenous c-type cytochrome were released upon lysozyme treatment of the cells. The results appear to indicate the presence of cytochrome oxidase in the cytoplasmic membrane of A. nidulans.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - cyt cytochrome(s)