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PubMed1990

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein.

Bacterial Outer Membrane Proteins↗
PubMed1978

Transfection of Escherichia coli spheroplasts: infectious lambda prophage DNA.

High mol. wt. DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious. Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E. coli (lambda c857 tsxisam6) lysogen yielded 200 times as many plaques on sup+ than on sup- spheroplasts. Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not. About 10(4) genome equivalents of E. coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments.

Coliphages↗
PubMed1984

Isolation and structure of glucan from regenerating spheroplasts of Candida albicans.

Regenerating spheroplasts of Candida albicans formed organized glucan nets in liquid culture. The nets consisted of interwoven microfibrils about 50 nm wide, but of an undetermined length. Partial acid hydrolysis of the polysaccharide showed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues, but no intrachain beta(1----3) and beta(1----6) linkages. Periodate oxidation and GLC of the methylated glucan indicated a highly branched polymer (9.5% branch points). Sequential enzymic degradation of the isolated nets confirmed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues. Degradation by (1----3)-beta- and (1----6)-beta-glucanase released 23% (w/w) and 30% (w/w) respectively of the carbohydrate as glucose equivalents. The residual material was degraded by chitinase. Equal amounts of N-acetylglucosamine and glucose equivalents were detected in the chitinase hydrolysate, suggesting a possible linkage between glucan and chitin. Our data indicate that the cell wall of C. albicans contains at least two highly branched glucans with predominantly beta(1----3) or beta(1----6) linkages.

Candida albicans↗
PubMed1979

Additivity of action between polysorbate 80 and polymyxin B towards spheroplasts of Pseudomonas aeruginosa NCTC 6750.

When polymyxin B and polysorbate 80 were used together against spheroplasts of Pseudomonas aeruginosa, the activities were found to be additive. These substances have previously been reported to act synergistically against P. aeruginosa, but little or no intrinsic activity towards intact cells has been attributed to polysorbate 80. We suggest that in addition to enhancing polymyxin B penetration to the cytoplasmic membrane, polysorbate 80 may also act as an antimicrobial agent when polymyxin-induced damage to the outer membrane facilitates the surfactant's passage through the cell envelope.

Polyethylene Glycols↗
PubMed1979

Influence of suspending media upon the susceptibility of Pseudomonas aeruginosa NCTC 6750 and its spheroplasts to polymyxin B.

The lytic and bactericidal actions of polymyxin B on whole cells and spheroplasts of Pseudomonas aeruginosa varied markedly with the suspending media, and there was little correlation between them. Relative rates of lysis of these preparations and also of Bacillus megaterium protoplasts suggested that polymyxin causes progressive damage to the cytoplasmic membrane, such that membrane permeability towards various ions increased as follows: K(+) > Na(+) > NO(3) (-) > Cl(-), Ca(2+), H(2)PO(4) (-)/HPO(4) (2-). Impermeant compounds, such as NaCl and sucrose, protected whole cells against lysis but not against death. It is suggested that lysis of whole cells by polymyxin is a secondary effect, resulting from entry of solutes normally excluded by the cytoplasmic membrane and the fragility of the damaged outer membrane. Because the degree of lysis varies with the external solutes, it should be treated with caution as a descriptor of polymyxin activity.

Bacillus megaterium↗
PubMed1976

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.

Anti-Bacterial Agents↗
PubMed1978

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.

Cell Membrane↗
PubMed1998

Response of Salmonella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48.

The bacteriocin AS-48 was not active against intact cells of Salmonella choleraesuis LT2 at neutral pH, but it was very effective on spheroplasts, suggesting that the outer membrane (OM) acts as a protective barrier. Cells sublethally injured by heat or treated with OM-permeabilizing agents (i.e., EDTA and Tris) became sensitive to AS-48. The combination of two or more treatments decreased the amount of AS-48 required for cell killing. The activity of AS-48 against heat-injured cells did not change significantly in the pH range of 4.0 to 8.0. AS-48 showed bactericidal activity against intact cells of Salmonella at pH 4.0. The potency of AS-48 increased greatly when the bacteriocin was dissolved at pH 9.0.

Anti-Bacterial Agents↗
PubMed2002

Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

BH3 Interacting Domain Death Agonist Protein↗
PubMed1974

Binding of streptococcal competence factor by the spheroplast membrane of a group H streptococcus.

The binding of streptococcal competence factor (CF) was found to be specific for a strain of streptococcus which was capable of undergoing competence induction. Three other streptococcal strains which could not be induced to competence, did not bind CF. CF binding was independent of time, temperature, age of the culture, and type of growth media employed. Several observations indicated that the receptor sites for CF are located in the bacterial membrane: (i) the retention of CF by spheroplasts, (ii) the binding of CF by isolated membrane fractions, and (iii), the degradation of CF binding capacity of membranes by different chemicals and enzymes.

Bacterial Proteins↗
PubMed1974

Biotin uptake by cold-shocked cells, spheroplasts, and repressed cells of Saccharomyces cerevisiae: lack of feedback control.

Cold-osmotic-shocked cells and spheroplasts of Saccharomyces cerevisiae (ATCC 9896) display a biotin uptake system similar to that observed in intact cells. 2-Mercaptoethanol was found to inhibit biotin transport. Cells repressed for biotin uptake by growth in excess biotin (25 ng/ml) possess an energy-dependent transport system that has a K(m) for biotin of 6.6 x 10(-7) M and a V(max) equal to 39 pmol per mg (dry weight) per min. A similar K(m) (6.4 x 10(-7) M) but a considerably higher V(max) (530 pmol per mg (dry weight) per min) was determined for biotin uptake by cells grown in sufficient biotin (0.25 ng/ml). The V(max) rates of biotin uptake by both repressed and derepressed cells were increased approximately 35-fold in the presence of glucose. These yeast cells appear to regulate their biotin uptake by two mechanisms. An exit system provides for immediate adjustments, whereas turnover of the transport system and repression of new synthesis establishes a slower adaptation to changes in the environment. Feedback inhibition was ruled out as a mechanism of regulation of transport.

Biological Transport, Active↗
PubMed1977

Fusion of yeast spheroplasts.

Spheroplasts of two different auxotrophic strains of Saccharomyces cerevisiae, both of mating type a, were fused with the aid of polyethylene glycol and calcium ions. After reversion to vegetative cells in solid media, the resulting zygotes were shown to be diploid cells of mating type aa.

Calcium↗
PubMed1977

Deoxyribonucleic acid synthesis in permeabilized spheroplasts of Saccharomyces cerevisiae.

Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.

Adenosine Triphosphate↗
PubMed1981

Parasexual genetic analysis of Candida albicans by spheroplast fusion.

Doubly auxotrophic strains of Candida albicans were selected from mutagenized cultures. Spheroplasts prepared from the auxotrophic strains were fused with polyethylene glycol. Prototrophic derivatives formed by this fusion protocol from auxotrophic strains were selected by complementation on minimal medium. These prototrophs had a cell volume twice that of the original strain and were shown to be heterozygous at four loci. Prototrophs obtained by this procedure infrequently gave rise to auxotrophic recombinants whose cell volume remained twice that of the original strain. It is suggested that these auxotrophic recombinants arise from mitotic crossing-over. This paper is the first report of a parasexual cycle in C. albicans.

Candida albicans↗
PubMed1987

Helix hand fidelity in Bacillus subtilis macrofibers after spheroplast regeneration.

Left- and right-handed Bacillus subtilis macrofibers produced by strains FJ7 and C6D were converted to spheroplasts. Intact cells were regenerated and macrofibers were produced under conditions conducive for production of left- and right-handed structures. The resulting helix hand phenotypes always corresponded to those expected on the basis of the parental genotype.

Bacillus subtilis↗