Spheroplast Cells Research Articles

Analyses of the Effect of Peptidoglycan on Photocatalytic Bactericidal Activity Using Different Growth Phases Cells of Gram-Positive Bacterium and Spheroplast Cells of Gram-Negative Bacterium

We conducted photocatalytic experiments focusing on the peptidoglycan layer to elucidate the details of the mechanism of photocatalytic sterilization. The previous study of our laboratory suggested that the presence of the peptidoglycan layer increases the bactericidal effect. To further verify it, the following experiments were performed: experiments on cells with different peptidoglycan layer thickness used Lactobacillus plantarum cells with different growth phases, experiments on cells with the thin peptidoglycan layer used Escherichia coli cells and spheroplast cells from which the peptidoglycan layer was removed from E. coli cells. The bactericidal effects increased as the growth progresses of L. plantarum. It was confirmed by TEM that the thickness of the peptidoglycan layer increased with cell growth. The survival rates of E. coli intact cells were significantly lower than those of spheroplast cells. These results strongly suggest that the peptidoglycan layer enhances the photocatalytic bactericidal effect. As a result of allowing the photocatalytic reaction to act on peptidoglycan, the amount of hydroxyl radical was smaller, and the amount of hydrogen peroxide was higher than in the absence of peptidoglycan. It is suggested that peptidoglycan may convert produced hydroxyl radical to hydrogen peroxide.

Double Tubular Contractile Structure of the Type VI Secretion System Displays Striking Flexibility and Elasticity.

Antimicrobial treatment can induce many bacterial pathogens to enter a cell wall-deficient state that contributes to persistent infections. The effect of this physiological state on the assembly of transenvelope-anchored organelles is not well understood. The type VI secretion system (T6SS) is a widespread molecular weapon for interspecies interactions and virulence, comprising a long double tubular structure and a transenvelope/baseplate complex. Here, we report that cell wall-deficient spheroplasts assembled highly flexible and elastic T6SS structures forming U, O, or S shapes. Upon contacting the inner membrane, the T6SS tubes did not contract but rather continued to grow along the membrane. Such deformation likely results from continual addition of sheath/tube subunits at the distal end. Induction of TagA repressed curved sheath formation. Curved sheaths could also contract and deliver T6SS substrates and were readily disassembled by the ClpV ATPase after contraction. Our data highlight the dramatic effect of cell wall deficiency on the shape of the T6SS structures and reveal the elastic nature of this double tubular contractile injection nanomachine.IMPORTANCE The cell wall is a physical scaffold that all transenvelope complexes have to cross for assembly. However, the cell wall-deficient state has been described as a common condition found in both Gram-negative and Gram-positive pathogens during persistent infections. Loss of cell wall is known to have pleiotropic physiological effects, but how membrane-anchored large cellular organelles adapt to this unique state is less completely understood. Our study examined the assembly of the T6SS in cell wall-deficient spheroplast cells. We report the elastic nature of contractile T6SS tubules under such conditions, providing key insights for understanding how large intracellular structures such as the T6SS accommodate the multifaceted changes in cell wall-deficient cells.

Application of Environmental Scanning Electron Microscope-Nanomanipulation System on Spheroplast Yeast Cells Surface Observation.

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.

NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae

Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent.

Extracellular and cell-associated forms of Gluconobacter oxydans dextran dextrinase change their localization depending on the cell growth

Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium.

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C6 to C13 were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C11) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C10) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C12) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H+-ATPase. Octanol (C8) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C9–C12) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.

Photocatalytic disinfection of water polluted by Pseudomonas Aeruginosa

Photocatalysis by titanium dioxide (TiO2) is a water treatment method. Pseudomonas aeruginosa is a microorganism resistant to chlorine and UV-C irradiation. TiO2 photocatalytic technology can destroy bacteria, which are resistant to oxidative destruction of cell membrane caused by sole UV irradiation. This study aims to investigate the total mineralization of the bacterium (P. aeruginosa) to the extent of death and cell-mass destruction using TiO2 photocatalytic oxidation process. In this work the effects of parameters such as amount of TiO2, irradiation time, initial concentration of bacterium, presence of ascorbic acid and effect of cell wall on removal of P. aeruginosa were studied. The data, which were obtained in this study, showed that the optimum concentration of TiO2 was 325 ppm. Also at the initial concentration of TiO2 equal to 325 ppm and initial microorganism MPN / 100 ml equal to 300 and after 75 min UV irradiation time, P. aeruginosa removal efficiency was 94.3 %. Removal efficiency of P. aeruginosa in the absence of TiO2 or UV irradiation was very low. Decreasing the concentration of microorganisms increased its removal efficiency. Removal efficiency of spheroplast cells was more than intact cells of P. aeruginosa, which shows the important role of cell wall on cell resistance against chemical agents. Ascorbic acid had inhibitory effect on this process.

Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy

Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

Antifungal activity of alkanols against Zygosaccharomyces bailii and their effects on fungal plasma membrane

A series of aliphatic primary alkanols from C(6) to C(13) were tested for antifungal activity against a food spoilage fungus Zygosaccharomyces bailii using a broth dilution method and were compared for their effects against Saccharomyces cerevisiae and Z. rouxii. Decanol (C(10)) was found to be the most potent fungicide against Z. bailii at a minimum fungicidal concentration of 50 microg/ml (0.31 mM), whereas undecanol (C(11)) was found to be the most potent fungistatic at a minimum inhibitory concentration of 25 microg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. Octanol (C(8)) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The primary antifungal action of alkanols comes from their ability to disrupt the native membrane-associated function of integral proteins nonspecifically as nonionic surface-active agents (surfactants). The antifungal activity of decanol against Z. bailii was slightly enhanced in combination with anethole.

Progress in the mitotic cycle of yeast spheroplasts in liquid culture

When inoculated into a rich medium with standard concentrations of nutrients at a sufficiently low cell density, spheroplast cells prepared from synchronized cells of Saccharomyces cerevisiae IFO 0971 carried synchronously out at least two successive rounds of DNA synthesis and nuclear division. Spheroplast cells performed their first round of DNA synthesis during roughly the same time as did normal cells, but entered the first round of nuclear division significantly later than normal cells. Following this first round, spheroplast cells initiated the second round of DNA synthesis without significant delay, but entered the second round of nuclear division again with significant delay compared with normal cells. The seemingly specific delay in initiating nuclear division in the mitotic cycle of spheroplast cells cultivated in this medium increased linearly as the medium was diluted with respect to nutrient concentration. Addition of d-glucose (but not of l-glucose) to a diluted medium partially suppressed the increasing delay in initiating nuclear division in spheroplast cells cultivated in such a medium. In contrast to spheroplast cells, the timing of nuclear division initiation in the mitotic cycle of normal cells did not vary with the degree of dilution of the medium. Addition of hydroxyurea to a spheroplast culture which had completed the first round of DNA synthesis did not prevent (most) cells from commencing nuclear division, and spheroplast cells of such a culture displayed sedimentation patterns of newly synthesized DNA strands characteristic of a full-sized material, indicating that spheroplast cells in liquid culture complete DNA replication early in the mitotic cycle. On the basis of these results we propose that in spheroplast cells the potential to carry out a reaction(s) specifically involved in the initiation of nuclear division is at a lower level than that of normal cells. The expression of this potential is believed to be governed by the concentration of nutrients in the surrounding medium due to the reduced capacity of the cells to take up nutrients, when compared with the capacity of normal cells.

Mitotic Behaviour of Yeast Spheroplasts in Liquid Culture

The mitotic behaviour of spheroplasts prepared from synchronized cells of Saccharomyces cerevisiae IFO 0971 in a liquid medium varied with cell density. When incubated at low cell density, spheroplasts entered the first round of mitosis in partial synchrony and most proceeded to the second round. When incubated at high cell density, they showed significant retardation of mitosis, although they apparently carried out one normal round of DNA synthesis. These and other observations indicate that in spheroplast cells incubated at high cell density, some biochemical reactions necessary for the initiation of mitosis are retarded. An additional observation suggested that spheroplast cells may undergo normal mitosis only in the presence of relatively high concentrations of nutrients in the surrounding medium.

Mechanism of potent mutagenic action of N-Methyl- N′-nitro- N-nitrosoguanidine on intracellular phage Lambda

N-Methyl- N′-nitro- N-nitrosoguanidine efficiently induces mutations from “clear” to “virulent” in phage λ only during the intracellular growth phase. Lambda DNA extracted from infected bacteria after treatment with MNNG ‡ produced a mutant yield about 100-fold higher than the spontaneous level upon transfection of MNNG-treated spheroplast cells, whereas the yield diminished an order of magnitude when assayed on untreated spheroplasts. As measured by 14C incorporation after treatment with [ methyl- 14C]MNNG, λ DNA packed in head protein was methylated to about 3% by an MNNG dose of 0.6 mg/ml but was barely mutagenised; whereas intracellular λ DNA was methylated to no more than 0.6% by an MNNG dose of 0.09 mg/ml and was highly mutagenised. Lambda phages treated in vitro with ethyl methanesulfonate produced a rather low mutant yield on untreated cells but the yield increased about tenfold on MNNG-treated cells. Mutability of untreated λ on cells having received an F′ factor was enhanced efficiently by ultraviolet light, but not so by MNNG, previously applied to the F′. Surprisingly similar MNNG dose-effect curves exist for enhancing spontaneous, mispairing (MNNG or EMS induced) and misrepair (ultraviolet light induced) mutagenesis of λ. From these and other data we conclude that MNNG hypermutagenesis results from a synergistic increase in mispairing probability of appropriately methylated bases (by action of MNNG in vivo) in the target gene within an MNNG-induced intracellular environment that has an enhanced mutagenic capacity.

GROWTH AND SURVIVAL OF ENTEROBACTERIA IN SELENITE‐CYSTINE BROTH CONTAINING THIOSULFATE

SUMMARY Concentrations of 0.1 or 0.2% selenite‐cystine and 0.16 or 0.32% thiosulfate in trypticase soy broth were bactericidal for seven strains of Escherichia coli, a Shigella sp. culture, and Salmonella cholerae‐suis. Higher concentrations of both reagents resulted in the inhibition of six strains of Salmonella and a culture of Arizona sp. The growth and length of the lag period of cultures of Enterobacter, Klebsiella, Proteus and Pseudomonas were not significantly affected. In mixed cultures of E. coli and Salmonella enteritidis, E. coli was selectively inhibited by the selenite‐cystine‐thiosulfate mixture even when present in large numbers. Uptake of selenium‐75 by intact and spheroplast cells of E. coli and S. enteritidis in selenite‐cystine medium in the presence and absence of thiosulfate was not significantly different. It was concluded that the cell wall does not play a major role in the uptake of selenite by these organisms. The formation of “seleno‐polythionates” has been demonstrated in the selenite‐cystine‐thiosulfate mixture. Use of this medium to isolate Salmonella from contaminated food samples indicated that it was not as effective as commercial selenite‐cystine broth or tetrathionate broth.