Production Of Spheroplasts Research Articles

CHEMICALLY INDUCED ECDYSIS AND ISOLATION OF NUCLEI FROM THE DINOFLAGELLATES GONYAULAX POLYEDRA AND GONYAULAX TAMERENSIS

Armored dinoflagellates such as Gonyaulax sp. are not good candidates for isolation of intact clean nuclei, due to difficulty in breaking the cells. Previously, isolation of nuclei from Gonyaulax was achieved using vigorous cell disruption by ultrasonication (Rizzo & Hastings, unpublished observations). A detergent method for production of spheroplasts from Gonyaulax was published some time ago (Adamich & Sweeney, 1976), but was not reproducible (Rizzo & Hastings, Morris & Rizzo, unpublished observations). We describe here a modification of the method described by Adamich & Sweeney that is highly reproducible. Ecdysis and production of intact dinoflagellate spheroplasts with a yield of virtually 100% within 5 minutes, has been achieved through the use of anionic and nonionic detergents in hypotonic buffers. This method of ecdysis followed by a gentle spheroplast breakage in a hypotonic buffer allowed the release of nuclei for later biochemical analysis with minimal damage or breakage. The nuclei are subsequently purified by centrifugation in a Ficoll discontinuous gradient.

Ultrastructural characterization of the stages of spheroplast preparation of Borrelia burgdorferi

Spheroplasts were generated from Borrelia burgdorferi by application of Tris-buffered lysozyme and EDTA. Cultures were examined during preparation by scanning electron microscopy (SEM) and by negative staining and/or thin sectioning followed by transmission electron microscopy (TEM). Spirochete and spheroplast proteins were also compared by electrophoresis to determine the effect of outer membrane manipulation during spheroplast preparation on the amounts of two outer surface proteins, OspA and OspB. Periplasmic flagellar release from the confines of the outer membrane began at the earliest steps of the preparation process and increased with continued progress toward the spheroplast condition. With the addition of Tris alone, spirochete uncoiling and outer membrane disruption became apparent. Lysozyme addition resulted in the formation of electron-transparent blebs where the outer membrane separated from the cytoplasmic membrane. Despite the permeability of the outer membrane to the lysozyme and the apparent alterations in its structure, OspA and B showed no decrease in concentration throughout the spheroplast preparation process. It is suggested that the flagellar release that occurs during spheroplast production also occurs in the host during Lyme disease, accounting for early immune responses to flagellin.

The cellular location and specificity of bacterial cytochrome c peroxidases

The locations of cytochrome c peroxidase and catalase activities in the two Gram-negative bacteria Pseudomonas stutzeri (N.C.I.B. 9721) and Paracoccus denitrificans (N.C.I.B. 8944) were investigated by the production of spheroplasts. In both species the cytochrome c peroxidase was predominantly periplasmic: 92% of total activity in Ps. stutzeri and 98% of nonmembrane-bound activity in Pa. denitrificans were found in this cellular compartment. In contrast, the catalase was mostly in the cytoplasmic fraction. Purification of the Pa. denitrificans cytochrome c peroxidase showed it to be the haem c-containing polypeptide of Mr 42,000 that has already been described by Bosma, Braster, Stouthamer & Van Versefeld [(1987) Eur. J. Biochem. 165, 665-670] but was not identified by them as a peroxidase. The visible-absorption spectra of the enzyme closely resemble those of cytochrome c peroxidase from Pseudomonas aeruginosa but the donor specificity is different, with the Pa. denitrificans enzyme preferring the basic mitochondrial cytochromes c to the acidic cytochromes c-551 and reacting well with the Pa. denitrificans cytochrome c-550.

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.

Cell Wall, Membrane, and Intracellular Peptidase Activities of Propionibacterium shermanii

A procedure has been developed for the production of spheroplasts from Propionibacterium shermanii α and P. shermanii ATCC 9614. Thus, it was also possible to produce clean cell wall, membrane, and intracellular fractions. The solubilized cell wall preparation was nearly free from intracellular markers; leakage of aldolase and malate dehydrogenase was below .5% after 90min in lysozyme. The purity and homogeneity of the membrane fraction were visualized with electron micrographs. Polyacrylamide gel electrophoresis and a zymogram technique were used to compare peptidases in the pure fractions. Fifteen dipeptides and 2 tripeptides were used as substrates. Peptidases of P. shermanii α and P. shermanii ATCC 9614 were found in the solubilized cell wall (one band and one band), membrane (three bands and two bands), and intracellular fractions (six bands and seven bands). The solubilized cell wall fractions from P. shermanii α and P. shermanii ATCC 9614 contain peptidases with reflectance values of 0 and 57, respectively. The reflectance 57 peptidase has a broad substrate specificity, that of the reflectance 0 peptidase is slightly narrower. Among the five membrane peptidases two appear to be unique for membranes, those with reflectance values of 28 and 75.An intracellular peptidases of broad specificity with reflectance value of 55 is found in both strains. The enzymes hydrolyzed most of the 15 dipeptides and the P. shermanii ATCC enzyme hydrolyzed the 2 tripeptides. The reflectance 24/25 intracellular peptidase of the two strains has the same, limited substrate specificity. The intracellular peptidases for each strain hydrolyzed all the dipeptides used and L-leucine-L-leucine-L-leucine, plus L-alanine-L-leucine-glycine for that from P. shermanii ATCC 9614.

Inhibition of melanin synthesis in the black yeast Phaeococcomyces sp. by growth on low pH ascorbate medium: production of spheroplasts from "albinized" cells

The black yeast Phaeococcomyces sp. produces pentaketide-based melanin constitutively, resulting in a green–black colony colour on commonly used isolation and culture maintenance agar media. Pentaketide melanins are produced by a multi-enzyme pathway, beginning with the cyclization of conjoined acetate molecules and ending with the polymerization of the final phenolic precursor, dihydroxynapthalene, into melanin. A low pH medium, which contains 0.1% ascorbic acid, causes complete inhibition of melanin formation, resulting in a white colony apearance similar to that of albino mutants of Phaeococcomyces sp. Melanized Phaeococcomyces sp. cells resisted enzymatic cell wall digestion, and spheroplasts could not be formed. In contrast, spheroplasts were produced from cells "albinized" by growth on the low pH medium. Inhibition of melanogenesis in other fungi, by growth on low pH ascorbate medium, was found to be specific to pentaketide melanin producers.

Preparation of mycelial and yeast-like spheroplasts fromMucor rouxii by a lytic enzyme preparation fromPenicillium islandicum

Spheroplasts ofMucor rouxii were prepared from mycelial and yeast-like cells by use of aPenicillium islandicum enzymatic complex. This enzyme preparation, which presents high chitosanase and chitinase activities, was produced by growingP. islandicum either on mycelial or yeast-like walls ofM. rouxii. The presence of magnesium sulfate as an osmotic stabilizer was critical to obtain high yields of spheroplasts from mycelial forms. In the case of yeast-like cells, pretreatment with β-mercaptoethanol followed by magnesium sulfate was essential for extensive spheroplast production.

Inhibition of growth and cell wall synthesis of Mycobacterium smegmatis by d-threonine.

Microbiology and ImmunologyVolume 22, Issue 2 p. 103-107 ArticleFree Access Inhibition of Growth and Cell Wall Synthesis of Mycobacterium smegmatis by D-Threonine Kunihiko Yabu, Corresponding Author Kunihiko Yabu Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Kiyose, TokyoRequests for reprints should be addressed to Dr. Kunihiko Yabu, Department of Biology, Hokuriku University School of Pharmacy, Kanagawa-Machi, Kanazawa-shi, Japan.Search for more papers by this authorShozo Takahashi, Shozo Takahashi Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Kiyose, TokyoSearch for more papers by this author Kunihiko Yabu, Corresponding Author Kunihiko Yabu Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Kiyose, TokyoRequests for reprints should be addressed to Dr. Kunihiko Yabu, Department of Biology, Hokuriku University School of Pharmacy, Kanagawa-Machi, Kanazawa-shi, Japan.Search for more papers by this authorShozo Takahashi, Shozo Takahashi Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Kiyose, TokyoSearch for more papers by this author First published: February 1978 https://doi.org/10.1111/j.1348-0421.1978.tb00353.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Adamek, P., Mison, L., Mohelska, H., and Trnka, L. 1969. Ultrastructural organization of spheroplasts induced in Mycobacterium smegmatis by lysozyme or glycine. Arch. Microbiol. 69: 227– 236. 2Lugtenberg, E.J.J. 1972. Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide. J. Bacteriol. 110: 26– 34. 3Neuhaus, F.C. 1968. Selective inhibition of enzyme utilizing alanine in the biosynthesis of peptidoglycan. Antimicrob. Agents Chemother. (1967): 304– 313. 4Neuhaus, F.C., and Lynch, J.L. 1964. The enzymatic synthesis of D-alanyl-D-alanine. III. On the inhibition of D-alanyl-D-alanine synthetase by the antibiotic D-cycloserine. Biochemistry 3: 471– 480. 5Roos, P., Gavdier, B., Tacquet, A., and Guillaume, J. 1966. Essais d'obtention de spheroplastes de quelques mycobacteries. Ann. Inst. Pasteur Lille 17: 49– 55. 6Sato, H., Diena, B.B., Greenberg, L. 1965. The production of spheroplasts by rapid-growing non-virulent mycobacteria. Can. J. Microbiol. 11: 807– 810. 7Willett, H.P., and Thacore, H. 1967. Formation of spheroplasts of Mycobacterium tuberculosis by lysozyme in combination with certain enzymes of rabbit peritoneal monocytes. Can. J. Microbiol. 13: 481– 488. 8Yabu, K. 1969. Reversal of the growth inhibitory action of glycine and glycylglycine by alanylalanine in Mycobacterium smegmatis. Biochim. Biophys. Acta 184: 460– 463. 9Yabu, K., and Huempfner, H.R. 1974. Inhibition of growth of Mycobacterium smegmatis and cell wall synthesis by D-serine. Antimicrob. Agents Chemother. 6: 1– 10. Citing Literature Volume22, Issue2February 1978Pages 103-107 ReferencesRelatedInformation

Binding of Thienamycin and Clavulanic Acid to the Penicillin-Binding Proteins of Escherichia coli K-12

Thienamycin and clavulanic acid are new beta-lactam derivatives with structures markedly different from those of penicillins or cephalosporins. Both derivatives had the same general mode of action as typical beta-lactam antibiotics since they bound to precisely the same proteins as [(14)C]benzylpenicillin. Thienamycin showed high affinity for penicillin-binding proteins 1, 2, 4, 5, and 6 and a lower affinity for protein 3. Protein 2 had the highest affinity for thienamycin, and concentrations from the minimal morphological change concentration (0.1 mug/ml) up to about 0.6 mug/ml resulted in the conversion of Escherichia coli KN126 into large osmotically stable round cells. Above a concentration of 0.6 mug/ml, rapid cell lysis occurred with the release of the cell contents as spheroplasts. Clavulanic acid showed good affinity for penicillin-binding protein 2, moderate affinity for proteins 1, 4, 5, and 6, and low affinity for protein 3. Protein 2 had the highest affinity for clavulanic acid, and concentrations from the minimal morphological change concentration (30 mug/ml) up to about 50 mug/ml produced a mixture of slightly elongated, swollen, bulging, and lemon-shaped cells. Above a concentration of 50 mug/ml, rapid lysis occurred with production of spheroplasts. The properties of thienamycin and clavulanic acid were compared with those of the penicillins, cephalosporins, and amidinopenicillanic acids.

Comparative Activity of Ampicillin and Cefuroxime Against Three Types of Haemophilus influenzae

On the basis of their susceptibility to ampicillin, strains of Haemophilus influenzae can be divided into three types: type 1 are normally susceptible strains, type 2 produce stable spheroplasts from low inocula, and type 3 are beta-lactamase producers. Because of the production of spheroplasts, standard broth and agar dilution techniques have failed to differentiate between the responses of type 2 and 3 strains to ampicillin, or to identify the superiority of cefuroxime over ampicillin against the beta-lactamase-producing strains. Disk susceptibility tests with heavily seeded plates were also difficult to interpret. To overcome these problems, we developed a medium that supports the growth of H. influenzae, but not survival of spheroplasts, thereby reducing the complicating influence of spheroplast formation. Utilization of the medium made it possible to identify beta-lactamase-producing strains via both minimal inhibitory concentration and disk susceptibility techniques, as well as the superiority of cefuroxime over ampicillin against such strains. In vivo experiments showed that cefuroxime and ampicillin are equally active against infections with type 1 and 2 strains, but that cefuroxime is superior to ampicillin against infections with type 3 beta-lactamase-producing strains.

Spheroplasts of Haemophilus influenzae induced by cell wall-active antibiotics and their effect upon the interpretation of susceptibility tests.

The interpretation of in vitro susceptibility tests of Haemophilus influenzae performed by the agar or broth dilution methods with Levinthal enrichment was found to be markedly influenced by the production of spheroplasts by this species. Using an inoculum of 10(7) organisms/ml, this phenomenon was frequently evident macroscopically as a haziness on agar substrates and in broths containing cell wall-acting agents, such as ampicillin, cephalothin, and penicillin, but was not noted with chloramphenicol. Phase-contrast microscopic examination of the haze from these sources revealed numerous spherical bodies in contrast to the typical cocco-bacillary forms observed in growth controls. With this inoculum size, minimal bactericidal concentrations could not be determined since subculture of 0.1 ml of the hazy broths or the surface haze onto chocolate agar resulted in most instances in the development of a small number of colonies which, upon smear and gram stain, revealed typical Haemophilus morphology. An inoculum of 10(4) organisms/ml abolished the haziness on agar surfaces and in broths and resulted in clear-cut end points. Also, although spherical bodies were still present, they were distinctly less in number as contrasted to tests performed with an inoculum of 10(7) organisms/ml. It is recommended that minimal inhibitory concentration end points in antibiotic susceptibility tests be determined by microscopic, rather than macroscopic, observation of the growth milieu to determine the presence or absence of morphologically typical bacilli which, when observed, is indicative of true in vitro resistance.

Localization of alkaline phosphatase in three gram-negative rumen bacteria.

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.

Controlled production of spheroplasts in the yeast Nadsonia elongata.

Yeast cells of Nadsonia elongata were cultivated in such a way that simultaneously with enzymatic lysis of the cell wall a partial synthesis of cell wall components was taking place. After the initial period of cultivation, which lasted about 10 h and during which the morphology of cells remained unchanged when compared to controls, the cells were transformed into “prospheroplasts”. The prospheroplasts were larger than the control cells and, though they enlarged in volume in distilled water, they still retained the shape of the original cells. However, some changes were found in the ultrastructure of the cell walls of prospheroplasts in comparison with that of the cell walls of intact cells: while in yeast cells the surface was smooth, in prospheroplasts the fibrillar network was revealed as a result of the removal of the amorphous component; the gradual disappearance of the outer cell wall layer and a swelling of the remaining cell wall fragment were seen in ultrathin sections. After about 20-h cultivation the prospheroplasts were transformed into “spheroplasts”. The spheroplasts were osmotically fragile, and did not retain the shape of the yeast cell, even in isoosmotic environment. On the surface of spheroplasts only the fibrillar network composed of separate fibrils was seen. The spheroplasts were the final stage of yeast cell transformation under the conditions employed in the present study. Under the mentioned conditions true protoplasts are never formed. However, if the synthesis of cell wall components could not take place simultaneously with the lysis of the cell wall, the cells were transformed to protoplasts.

Staphylococcal bacteriophage-associated lysin: a lytic agent active against Staphylococcus aureus.

A lytic enzyme active against viable, intact staphylococci is released into culture fluids upon lysis of bacteriophage-infected Staphylococcus aureus PS53 cells. This enzyme, staphylococcal phage-associated lysin (PAL), was partially purified by ammonium sulfate precipitation and gel filtration through Sephadex G-200. PAL is optimally active at pH 6.5 and 30 C, and lytic activity is greatly enhanced by the addition of reducing agents. Lytic activity was observed against all strains of staphylococci tested and against purified staphylococcal cell walls, but no activity was noted against other bacterial species. PAL possesses peptidase activity and results in the production of spheroplasts which can be osmotically stabilized for extended periods by the addition of 7.5% polyethylene glycol 4000.

Use of a chitinase complex and beta-(1,3)-glucanase for spheroplast production from Candida albicans.

Use of a chitinase complex and beta-(1,3)-glucanase for spheroplast production from Candida albicans.

Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli.

Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 m sucrose in 0.01 m tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 m also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment.

Spheroplast production in thymine starvation.

“THYMINELESS DEATH” is the progressive loss of the ability to form colonies in thymine dependent bacteria growing in a medium lacking this pyrimidine1. This treatment is considered to be responsible for the induction of colicine2, lysogenic phage3, and mutagenic activity4. This report links thymine starvation with spheroplast formation in a mutant of Escherichia coli 15 which requires thymine and histidine for growth, and cannot grow in the presence of 100 µg norleucine/ml.

The spheroplasts of bordetella pertussis.

The formation of spheroplasts by Bordetella pertussis in fluid medium containing glycine has been demonstrated. The B. pertussis organisms were grown in a fluid medium containing glycine with aeration on a rotary shaker at 35 °C for 40 hours. Maximum spheroplast production was obtained by increasing the basic inoculum 10-fold to 5 × 107 organisms per milliliter.The B. pertussis spheroplasts are large, round, vacuolated cells resembling those described in other Gram-negative species. They are fragile, autolyze readily at pH values of 7.9–8.3, and are more susceptible to mechanical rupture and enzyme attack than are the bacilli.No adaptation of the bacilli to glycine was observed on successive transfer through increasing concentrations of glycine, and the spheroplasts revert to the bacillary form on transfer to a suitable solid or fluid medium.

Spheroplast induction and lysis of BCG strains by glycine and lysozyme.

Spheroplast induction and lysis of 6 BCG strains of Mycobacterium tuberculosis by glycine and lysozyme was studied in various media. Spheroplast production was noted in only three strains involving 20% of cell populations. Lysis, as distinct from spheroplast induction, occurred in Dubos medium containing 1.5% glycine and 0.01% lysozyme after 24–48 hours of incubation. Estimation from a standard curve indicated 40 to 70% lysis of the total cell population after 7–10 days of incubation.Similarly, lysis of BCG cells occurred when the inducers, glycine, lysozyme, and lithium chloride, were added to nitrogen-starved cultures grown in Aldridge synthetic medium for 7 to 8 days.

The production of spheroplasts by rapid-growing non-virulent mycobacteria.

Spheroplasts were produced in five strains of rapid-growing non-virulent mycobacteria in a variety of media. Optimal spheroplast production occurred after 8–10 days of incubation in the presence of glycine and lysozyme, but in no culture was there complete formation of spheroplasts. While the pattern of spheroplast induction appeared to be specific for each strain and to be highly reproducible, the mechanism of the induction process is unknown.