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PubMed1966

Formation of a functional electron transport system during growth of penicillin-induced spheroplasts of Haemophilus parainfluenzae.

Wright, Elizabeth A. (University of Kentucky College of Medicine, Lexington), and David C. White. Formation of a functional electron transport system during growth of penicillin-induced spheroplasts of Haemophilus parainfluenzae. J. Bacteriol. 91:1356-1362. 1966.-Penicillin in a lactose medium can be used to cause the formation of spheroplasts in Haemophilus parainfluenzae. The resulting spheroplasts grew under conditions which produced rapid formation of the electron transport system in the normal bacteria. The following elements that are incorporated into a functionally active electron transport system were formed in spheroplasts: formate and l-lactate dehydrogenases, 2-demethyl vitamin K(2), cytochromes b(1) and c(1), and the cytochrome oxidases. The catabolic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and malic dehydrogenase showed slight increases in activity. These experiments indicated that spheroplasts can form a fully functional electron transport system essentially similar to that formed during normal growth. The various components of the electron transport system were formed at different rates in the growing spheroplasts.

Cytochromes↗
PubMed1967

Macromolecule synthesis in yeast spheroplasts.

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.

Adenine↗
PubMed1970

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.

Adsorption↗
PubMed1982

Cell cycle inhibition of yeast spheroplasts.

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, alpha-factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.

Cell Cycle↗
PubMed1984

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 indispensable 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.

Acetylation↗
PubMed2005

Mounting of Escherichia coli spheroplasts for AFM imaging.

The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

Animals↗
PubMed2001

The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium.

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.

Cyanides↗
PubMed1999

Surface charge density and light-scattering of the Plectonema boryanum spheroplasts.

The electrokinetic properties of spheroplasts from the cyanobacterium Plectonema boryanum were examined by particle microelectrophoresis technique. The electrophoretic mobility (EPM) of the particles was determined after incubation with CaCl2 in dependence of iron content in culture media as follows: an iron sufficient medium ('control' variant), an iron-deficient medium ('Fe-starved' variant) and an excess of iron supply medium ('20 x Fe' variant). Strong increase in EPM was observed with micromolar concentrations of divalent cations at '20 x Fe' spheroplasts. This pattern of calcium efficiency was not accompanied with the cation influences on the aggregate ability of particles. The EPM of 'control' spheroplasts strongly decreased with addition of calcium cations. The 'Fe-starved' spheroplasts were characterized with a slight reduction in EPM and a mild change in light-scattering properties of the particles. The data is the direct demonstration of the interaction between calcium cations and spheroplast surface, which could be proposed to play a role in the environmental cycling of iron.

Algorithms↗
PubMed2004

Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts.

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.

ATP-Binding Cassette Transporters↗
PubMed1982

Penicillin-induced unstable intracellular formation of spheroplasts by rickettsiae.

Penicillin G (greater than or equal to 20 micrograms/ml) is rapidly rickettsiacidal for intracellular Rickettsia prowazekii. Light and electron microscopic examinations revealed that penicillin G in culture medium induced a predictable transformation into typical enlarging spheroplasts deficient in the internal, putative peptidoglycan layer of the outer membrane. Under certain conditions, spheroplasts ruptured to discharge contents into host cell cytoplasm and to leave empty shells of defective outer membrane and diffuse amorphous intracytoplasmic antigen. Host cell destruction often accompanied spheroplast rupture. Penicillin G (100 micrograms/ml) caused similar spheroplast formation by Rickettsia rickettsii, but 1,000 micrograms/ml caused neither growth inhibition nor spheroplast formation in Rickettsia tsutsugamushi. The clinical and epidemiological significance of a practical rickettsiacidal drug for the treatment of louse-borne typhus fever is discussed. Practical pharmacologic considerations preclude the use of penicillin for the treatment of typhus or spotted fever.

Animals↗
PubMed1984

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.

Chromosomes, Bacterial↗
PubMed1985

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.

Carbenicillin↗
PubMed1976

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.

Biological Transport, Active↗
PubMed1973

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.

Cell Membrane↗
PubMed1978

Lack of lysogenic induction in "diaminopimelic acid spheroplasts".

As part of an attempt to develop a semi-in vitro system of lysogenic induction, using spheroplasts of Escherichia coli K-12 lysogenic for prophage lambda, we prepared spheroplasts by depriving E. coli dap of diaminopimelic acid (DAP-spheroplasts). DAP-spheroplasts made from E. coli (lambda cI857) were thermally inducible. However, DAP-spheroplasts of E. coli (lambda) were not inducible by UV light. Thus, it appears that a functional cell wall is required for UV induction of prophage lambda.

Coliphages↗
PubMed1979

Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane.

Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30. The rate of incorporation of [3H]arginine into proteins in spheroplasts was about 30% of that of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein. Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein. No prolipoprotein was observed even with a short pulse-labeling with [3H]arginine. On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed. These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope.

Bacterial Proteins↗
PubMed1982

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.

Cell Membrane↗
PubMed1964

DISTRIBUTION OF RADIOACTIVITY IN AUTOLYZED CELL WALL OF BACILLUS CEREUS DURING SPHEROPLAST FORMATION.

Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581-587. 1964.-Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C(14)-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed.

Amino Acids↗