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.     

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.     

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.     

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.     

Obtaining spheroplasts from the agents of glanders and melioidosis and separation of membrane structures from them]

Spheroplasts were obtained from the causative agents of glanders and melioidosis under the effect of lysozyme and antibiotics. In the capacity of an inducing agent lysozyme was effective in high concentration only (0.4%); preliminary washing and incubation in sucrose were necessary to obtain glanders spheroplasts. Of the antibiotics studied penicillin was more useful for obtaining melioidosis spheroplasts and ampicillin--for glanders spheroplasts. Membrane preparations were derived from the spheroplasts of glanders and melioidosis causative agents.     

Preparation and Properties of Spheroplasts from Aspergillus parasiticus with Special Reference to the de novo Synthesis of Aflatoxins

Spheroplasts were prepared from Aspergillus parasiticus NRRL 3240 using β-glucuronidase from Helix pomatia. They were osmotically fragile spherical structures which lysed when suspended in hypotonic buffers. Purity of the preparation was confirmed by phase-contrast microscopy. Maximal conversion of mycelia to spheroplasts was achieved with 48 and 72 h old cultures. Spheroplasts were metabolically active as indicated by the incorporation of labelled thymidine, uridine and leucine into DNA, RNA and proteins, respectively. A significant incorporation of [methyl-³H] thymidine into trichloroacetic acid-insoluble material suggested the presence of thymidine kinase in this organism. Spheroplasts and lysates demonstrated the ability to incorporate labelled acetate into aflatoxins. Maximum incorporation was observed in those prepared from 96 h old cultures. Lysates were more efficient in de novo aflatoxin synthesis as compared to intact mycelia and spheroplasts.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

The effect of osmotic shock on the accessibility of the murein layer of exponentially growing Escherichia coli to lysozyme.

The restricted access of lysozyme to the murein layer of exponential phase Escherichia coli is enhanced considerably by osmotic shock. When cells suspended in Tris/EDTA/sucrose are diluted 11-fold in water or 10 mM EDTA in the presence of lysozyme, their susceptibility to lysozyme increases by a factor of 50--100, for both Escherichia coli JC411 and W3110, grown to the early exponential phase in unsuppleneted or supplemented minimal media, and in Brain Heart Infusion. Since an 11-fold dilution causes lysis of lysozyme spheroplasts, the effects of a 2-fold dilution have also been investigated. A 2-fold dilution of cell suspended in TrisEDTA/sucrose still increases their susceptibility to lysozyme by a factor of 10--50, but the resulting spheroplasts remain intact. EDTA is necessary to permit lysozyme access to the murein layer during the dilution, which is ineffective in the presence of 5 mM MgCl2. These results are discussed in terms of the formation of lysozyme spheroplasts from young Escherichia coli.     

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual 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.     

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from escherichia coli

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca²⁺ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (>98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cell, and in investigating envelope-related synthesis and assembly processes.     

Spheroplast formation of Mycobacterium smegmatis and morphological aspects of their reversion to the bacillary form.

Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.     

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.     

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.     

Characteristics of chromosomal DNA isolated from Escherichia coli spheroplasts

The chromosomal DNA of Escherichia coli spheroplasts induced by penicillin G was studied biochemically and electron microscopically. Although the spheroplasts were unable to divide, they continued to synthesize chromosomal DNA for several hours even in the presence of penicillin G. Some differences were observed between the chromosomal DNA of the parent cells and that of the spheroplasts in sucrose gradient centrifugation and electron microscopy; two types of chromosomal DNA, a slower sedimenting form and a faster sedimenting form, were released from the gently lysed parent cells. The former was membrane-free folded chromosome and the latter was membrane-associated chromosome. In contrast, the chromosome from the spheroplast showed a single intermediate value of sedimentation coefficient between those of the chromosomal DNA from the parent cell. Cytochrome spreading for electron microscopy showed that the spheroplast chromosomal DNA formed an aggregated mass consisting of several chromosome-molecules of the parent cell.     

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.     

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.     

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.     

The Rcs Stress Response and Accessory Envelope Proteins Are Required for De Novo Generation of Cell Shape in Escherichia coli

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.     

Structure of the cell wall of lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus fermenti, serological group F, were separated by mild conditions of acid hydrolysis; the polysaccharide was composed of glucose and galactose. 2. Soluble cell-wall products were isolated from cell wall lysed by lysozyme and a Streptomyces enzyme preparation. The lysozyme-dissolved fraction contained a greater proportion of mucopeptide. 3. The soluble preparations were heated in dilute acid to hydrolyse the linkage between the polysaccharide and mucopeptide components and then incubated with acid phosphatase. 4. Inorganic phosphate was released from products of Streptomyces enzyme action but not from products of lysozyme action. 5. The phosphate was shown to be present in the mucopeptide as muramic acid phosphate. It is concluded that in the intact wall polysaccharide is joined to muramic acid by a phosphodiester linkage.