Reversion to bacillary forms of Serratia marcescens spheroplasts induced by carbenicillin. Scanning electron microscopic study

Bacterial cells of Serratia marcescens were easily induced to form spheroplasts in liquid medium by the addition of carbenicillin. The spheroplasts were unable to divide, but they were able to revert to the bacillary forms in liquid medium not containing carbenicillin. Four phases of the reversion sequence could be differentiated by scanning electron microscopy. (1) After 3 hr of incubation in carbenicillin-free medium, some projections arose out of the spheroplasts, and grew and elongated. (2) Their elongation resulted in a morphological change in the spheroplasts from spherical bodies to long irregular bacillary forms. (3) Further incubation caused several constricted areas in the bacillary form. (4) The long bacillary forms split along the constricted areas to become the parent bacillary forms of S. marcescens. When the long bacillary form that developed during the reversion was retreated with carbenicillin, it was immediately induced to become a spheroplast again.     

Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Natural penicillin resistance of Francisella tularensis

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.     

Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Die Bildung und die Reversion von Sphäroplasten einer Diaminopimelinsäure-auxotrophen Mutante von Escherichia coli

1. The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, dap ⁻, R⁺TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells.
2. The release of the R-factor mediated periplasmic TEM-β-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-β-lactamase activity.

The spheroplasts showed a multiple enhancement of TEM-β-lactamase activity per mg cell protein compared with rod form cells.     

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.     

An improved method for the preparation of mycobacterial spheroplasts and the mechanism involved in the reversion to bacillary form: electron microscopic and physiological study

An efficient method is described for preparing spheroplasts and protoplasts by treating bacillary cells of Mycobacterium smegmatis with precise concentrations of L-glycine (followed by lysozyme). This improved procedure was widely applicable to many rapidly growing mycobacteria by selecting the concentrations of glycine suitable for the individual strains used. The process of reversion of spheroplasts to original bacillary form on solid and in liquid media, as revealed by electron microscopy, appeared to involve the formation of an internal elementary or initial body with subsequent budding from the spheroplast. The internal membrane systems appeared to function in the induction of initial bodies and in the maturation of elementary bodies to become dividing forms. Possible mechanisms involved in the development of bacilli from spheroplasts are discussed.     

SPHEROPLAST FORMATION AND ASSOCIATED ULTRASTRUCTURAL CHANGES IN A SYNCHRONOUS CULTURE OF ANACYSTIS NIDULANS TREATED WITH LYSOZYME1,2

Cells of Anacystis nidnlans were grown in synchronous culture using a light-dark alternation to obtain synchronization. Two synchronous cycles were obtained with, decay of synchrony beginning with the third cycle. Cells of various ages in the growth cycle were treated with lysozyme to form spheroplasts. The percentage of spheroplast formation varied with age of the cells. After extended periods of lysozyme treatment, up to 90% of the cells of all ages showed spheroplast formation. Some cells were resistant to the action of lysozyme regardless of age or length of treatment. An ultrastructure study of the spheroplast was made. The electron-dense inner layer of the cell wall was removed by the action of lysozyme on the glucosamine residues of the cell wall, indicating true spheroplast formation. The photosynthetic apparatus became more pronounced with extended treatment with lysozyme.     

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.     

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.     

Genetic recombination in fused spheroplasts of Providence alcalifaciens.

Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.     

Antigenic structure of Brucella suis spheroplasts

Baughn, Robert E. (University of Tennessee, Memphis), and Bob A. Freeman. Antigenic structure of Brucella suis spheroplasts. J. Bacteriol. 92:1298-1303. 1966.-Immunoelectrophoresis was used to differentiate between the antigenic mosaics of normal cells of Brucella suis and of spheroplasts prepared by treatment with penicillin, glycine, and a combination of these agents. Smooth cells possessed at least 13 antigens, 10 of which were precipitated with homologous antiserum. Three additional antigens were visualized by reaction with spheroplast antisera. Spheroplasts induced with glycine were the least complex, with only six antigens. Penicillin-glycine spheroplasts were similar, but possessed one additional antigen. Penicillin spheroplasts were the most complex, with eight antigens. Although there appeared to be quantitative differences between the antigens of spheroplasts and normal cells, no completely new antigens were detected in spheroplasts. Serum absorption studies indicated that four antigens were associated with the surface of normal B. suis, none of which occurred in spheroplasts.     

Multiplication of Bacteriophage P22 in Penicillin-Induced Spheroplasts of Salmonella typhimurium

The spheroplasts of Salmonella typhimurium (LT2) prepared by treatment with penicillin were capable of adsorbing phage P22 C(1). The normal multiplication of the phage took place, although the burst size was reduced to one-fourth of that in intact cells. Rate of incorporation of (14)C-thymidine into spheroplasts was increased severalfold on phage infection. Multiplication of C(+) also took place, but no lysogeny could be established in spheroplasts. Furthermore, spheroplasts prepared from cells lysogenized with wild-type phage, LT2 (C(+)), and a temperature-inducible C(2) mutant, LT2(tsC(2)), were not inducible. Unlike normal cells, both mitomycin C and actinomycin D interfered with the phage multiplication in spheroplasts. The spheroplast system offers great advantages in the study of the synthesis of nucleic acids and proteins in phage-infected LT2.     

Synthetic Capabilities of Plasmolyzed Cells and Spheroplasts of Escherichia coli

Effects of plasmolysis and spheroplast formation on deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, and phospholipid synthesis by Escherichia coli strain THU were studied. RNA and protein synthesis were severely diminished. DNA and phospholipid synthesis were inhibited, but less so; they could be partly restored. DNA synthesis could be restored by replacing thymine in the medium with thymidine, and phospholipid synthesis, by adding back small quantities of soluble cell extract. Plasmolysis effected marked reductions in rates of growth and macro-molecule synthesis, and temporarily reduced culture viability. Plasmolysis also caused an anomalous stimulation of phospholipid synthesis. Spheroplasts and plasmolyzed cells synthesized small amounts of ribosomal RNA that sedimented normally. However, this ribosomal RNA was very inefficiently packaged to ribosome subunits. Spheroplasts were unable to carry out induced synthesis of beta-galactosidase, and plasmolyzed cells were delayed in this function. Radioautographs examined in an electron microscope showed that DNA synthesis in plasmolyzed cells and spheroplasts was performed by a substantial fraction of the culture populations. That DNA and membrane were associated in the spheroplasts used in this study was suggested by formation of M-bands containing membrane and most of the cell's DNA. The results are discussed in terms of alterations of membrane structure and conformation attending plasmolysis and spheroplasting.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Properties of Infectious T1 Deoxyribonucleic Acid

T1 deoxyribonucleic acid (DNA) infection of spheroplasts was characterized by the following. A small number of the DNA molecules initiated infectious centers, and a small number of the spheroplasts were infected by T1 DNA. Once a favorable encounter of T1 DNA with spheroplast occurred, a minimum of 20 to 30 min was required for T1 DNA to enter the spheroplast. The mature T1 particles produced in the infection of spheroplasts by T1 DNA were released in a burst, but the average burst size was quite small compared with a normal burst of the phage-infected bacteria. T1 DNA preparations, capable of causing viral growth in spheroplasts, did not require detectable amounts of protein for infectivity, were homogeneous in band and boundary sedimentation, and had a guanine plus cytosine content of 48% and a minimal molecular weight of 35 x 10(6). Denatured T1 DNA, like denatured lambdaDNA, did not infect spheroplasts. Renatured T1 DNA was not infectious; this was in marked contrast to renatured lambdaDNA.     

Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.     

Colicin Tolerance Induced by Ampicillin or Mutation to Ampicillin Resistance in a Strain of Escherichia coli K-12

A mutant (G11el) of Escherichia coli selected as being resistant to ampicillin and showing signs of an envelope defect was also found to be tolerant to colicins E2 and E3. The colicin tolerance of G11el could be partially repressed by Mg(2+) ions. Transition from tolerance to sensitivity and vice versa by shifting the concentration of Mg(2+) in the growth medium required several generations. This indicated that synthesis of new envelope material was needed for transition. Previous physiological results have indicated a change in the envelope lipopolysaccharide (LPS) of G11el. However, chemical analyses revealed no differences in carbohydrate composition between LPS from G11el and its parent strain G11al. Genetic experiments showed that the mutation in G11el is located at about 20 min on the E. coli K-12 chromosome. The mutation was dominant over wild type in partial diploids with the mutation located on the episome. Because colicin tolerance was the most striking phenotypic effect as a result of mutation in the actual locus, this gene will be named tolD until the exact gene product is known. Spheroplasts formed from G11al and G11el by ethylenediaminetetraacetate-lysozyme treatment did not adsorb colicin E2; however, penicillin spheroplasts of G11al and G11el were tolerant to colicin E2. Thus, colicin tolerance can be induced biochemically. It is suggested that colicin tolerance often is a secondary consequence of a change in the cell envelope.     

Responses of enzymically isolated aleurone cells of oat to gibberellin a(3)

Oat (Avena sativa L.) aleurone layer cells (spheroplasts) were isolated by maceration of the aleurone layer with a mixture of commercially available cellulase and pectinase. About 20% of the cells present in intact layers were released as spheroplasts and 79 +/- 9% of the spheroplast population was viable as judged by methylene blue staining. The spheroplasts became disorganized in solutions containing less than 0.4 md-mannitol. When the spheroplasts were incubated for 48 hours, total activities of acid p-nitrophenyl phosphatase and acid proteinase increased and protein levels decreased. These changes were not effected by gibberellin A(3). Isolated aleurone layers incubated under the same conditions as the spheroplasts showed reduced responses to gibberellin A(3). It is concluded that the necessary presence of an osmoticum limits the value of spheroplasts as a system for studying the mechanism of action of gibberellin A(3) in the aleurone cell.