Conditions Of Osmotic Shock Research Articles

Antibacterial Effects of ZnO Nanodisks: Shape Effect of the Nanostructure on the Lethality in Escherichia coli.

The role of the shape of the nanostructure on the antibacterial effects of ZnO nanodisks has been investigated by detailed mass spectrometry-based proteomics along with other spectroscopic and microscopic studies on E. coli. The primary interaction study of the E. coli cells in the presence of ZnO nanodisks showed rigorous cell surface damage disrupting the cell wall/membrane components detected by microscopic and ATR-FTIR studies. Protein profiling of whole-cell extracts in the presence and absence of ZnO nanodisks identified several proteins that are upregulated and downregulated under the stress of the nanodisks. This suggests that the bacterial response to the primary stress leads to a secondary impact of ZnO nanodisk toxicity via regulation of the expression of specific proteins. Results showed that the ZnO nanodisks lead to the over-expression of peptidyl-dipeptidase Dcp, Transketolase-1, etc., which are important to maintaining the osmotic balance in the cell. The abrupt change in osmotic pressure leads to mechanical injury to the membrane, and nutritional starvation conditions, which is revealed from the expression of the key proteins involved in membrane-protein assembly, maintaining membrane integrity, cell division processes, etc. Thus, indicating a deleterious effect of ZnO nanodisk on the protective layer of E. coli. ZnO nanodisks seem to primarily affect the protective membrane layer, inducing cell death via the development of osmotic shock conditions, as one of the possible reasons for cell death. These results unravel a unique behavior of the disk-shaped ZnO nanostructure in executing lethality in E. coli, which has not been reported for other known shapes or morphologies of ZnO nanoforms.

From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes.

Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications.

Rapid dynamics of cell-shape recovery in response to local deformations.

It is vital that cells respond rapidly to mechanical cues within their microenvironment through changes in cell shape and volume, which rely upon the mechanical properties of cells' highly interconnected cytoskeletal networks and intracellular fluid redistributions. While previous research has largely investigated deformation mechanics, we now focus on the immediate cell-shape recovery response following mechanical perturbation by inducing large, local, and reproducible cellular deformations using AFM. By continuous imaging within the plane of deformation, we characterize the membrane and cortical response of HeLa cells to unloading, and model the recovery via overdamped viscoelastic dynamics. Importantly, the majority (90%) of HeLa cells recover their cell shape in <1 s. Despite actin remodelling on this time scale, we show that cell-shape recovery time is not affected by load duration, nor magnitude for untreated cells. To further explore this rapid recovery response, we expose cells to cytoskeletal destabilizers and osmotic shock conditions, which uncovers the interplay between actin and osmotic pressure. We show that the rapid dynamics of recovery depend crucially on intracellular pressure, and provide strong evidence that cortical actin is the key regulator in the cell-shape recovery processes, in both cancerous and non-cancerous epithelial cells.

Identification and Analysis of the Transcriptional Regulatory Networks Governing Mechanosensitive Channels in E. coli

Mechanosensitive (MS) channels are membrane proteins which are gated by mechanical stress in the cell membrane. Channel opening in response to mechanical stresses makes it possible for molecules to pass through the membrane, allowing an organism to alleviate solute imbalances which place osmotic stress on the membrane and can cause cell lysis and death. Two of the seven known MS channels in E. coli, MscL and MscS, are individually capable of rescuing these bacteria from sudden changes in osmolarity (osmotic shock). If MscL and MscS are both sufficient for ensuring survival under osmotic shock conditions, what is the purpose of the other five MS channels? In the interest of exploring the physiological roles of the various MS channels, we hypothesize that understanding how they are regulated will shed light on their function. Specifically, we investigate the regulatory context of the MS genes by identifying the regulatory architecture and transcription factors responsible for controlling MS gene output. We use a method known as Sort-Seq to locate transcription factor binding sites with base pair resolution, and DNA affinity chromatography to determine transcription factor identity. Understanding how the MS genes are regulated will give us clues as to the specific stressors which they defend the cell against. This work provides a specific case study of a method which we think will have much broader reach in mapping out regulatory networks genome-wide.

Osmotic Stress Effect over Carbohydrate Production in a Native Starin of &amp;lt;i&amp;gt;Scenedesmus&amp;lt;/i&amp;gt; sp.

The production of biofuels is currently presented as a possible answer in the search for sustainable alternatives for the total or partial substitution of fossil fuels. One of the most successful biofuels that have been developed is bioethanol. However, bioethanol production has been limited since it relies on the use of sugar cane or cereals. These materials are important sources of food and their demand as both a biofuel and a foodstuff has led to the price increase and may lead to possible shortages. Our group has focused on searching for native microalgae as sources of carbohydrates and bioethanol, with the goal of finding a sustainable source of bioethanol. Currently, twelve different strains which reach growth rates between 0.7 - 1.8 g/L and present carbohydrate production under osmotic shock conditions have been isolated. In this work, we demonstrate the results obtained with the Chlorella sp. [1] strain and the results obtained with the Scenedesmus sp. strain. The Scenedesmus sp. strain showed an increase in the production from 22 to 650 mg/sugar/g of biomass (dry weight), after 24 hours of osmotic shock with 0.1 M NaCl. The osmolytes which were produced after osmotic shock were identified as sucrose and trehalose, both of which are fermentable. These results demonstrate that this strain, through the photosynthetic pathway and osmotic shock, is a potential source of fermentable sugars.

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.

Staphylococcus aureus AgrA Binding to the RNAIII- agr Regulatory Region

The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2–3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.

Separation of detubulation and vacuolation phenomena in amphibian skeletal muscle.

Sartorius muscle fibres from cold-adapted Rana temporaria were exposed to variants of an established detubulation procedure (Koutsis et al. (1995) J Muscle Res Cell Motil 16, 519-528) to test the extent to which detubulation and tubular vacuolation phenomena could be separated using different conditions of osmotic shock. A control procedure was optimised to a 28-min exposure to 400 mM glycerol-Ringer. This was followed by a recovery step involving its replacement by a Ca2+/Mg(2+)-Ringer solution and steady cooling over 30 min from room temperature (approximately 18 degress C) to approximately 10 degress C, followed by the restoration of the normal Ringer solution. This procedure successfully abolished the action potential after-depolarisation component, reflecting a loss of tubular conduction ('detubulation') in 74.3 +/- 5.9% of the fibres studied. Omitting the cooling during the recovery step sharply reduced the incidence of detubulation. So did omitting either the high-[Ca2+] and/or [Mg2+] in the recovery solutions in test procedures, but to significantly different extents (P < 5%). Yet trapping of fluorescent Sulfhorhodamine B dye in 'closed' vacuoles persisted albeit with reduced proportions of fibre volume occupied by vacuoles. Furthermore, the variations in recovery conditions produced similar levels of vacuolation despite smaller vacuole sizes in the cooled fibres (P < 0.05). These findings demonstrate that fibre vacuolation and detubulation are phenomena that are potentially separable through varying the conditions of osmotic shock, with detubulation requiring significantly more stringent conditions than vacuolation.

The thylakoid membrane in a wide pH range

The structural organisation of thylakoid systems of pea chloroplasts subjected to osmotic shock after incubation in a wide range of pH values (from 1.0 to 12.0) was studied. It was found that the percentage of thylakoid profiles stacked into granas is 86 % at pH 4.0, 72 % at pH 7.4 and 25 % at pH 10.0. In the pH range from 6.0 to 10.0 under the conditions of osmotic shock, a complete disordering of thylakoid systems, unstacking, and fragmentation of thylakoids are observed. Decreasing medium pH to 5.5-3.5 leads to a shortening of agranal thylakoids, the appearance of numerous lipid drops, and a shrinkage of thylakoid systems, which cease to respond to osmotic shock. Thylakoid systems remain structured, and the stacking in granas is retained. At pH levels from 3.0 to 1.0, thylakoid systems appear as spherical clusters of tightly stacked vesicles, whose structural organisation also does not change under osmotic shock. Increasing the pH of the medium to 11-12 leads to a complete unstacking of thylakoids and a swelling of single thylakoid sheets, which also do not respond to osmotic shock. A correlation between the release of acyl lipids from the thylakoid membrane and the loss of the native response of the membrane to osmotic shock is discussed.

Changes in the structure and the functional state of thylakoids under the conditions of osmotic shock

The effect of osmotic shock on the ultrastructure and functions of C-class pea chloroplasts has been examined. When incubated in a non-sucrose medium for 30 s or more, thylakoids were found to pass to a stable deformed state. This state was characterized by an altered orientation of thylakoids to each other with the lumen thickness remaining the same as in the normal state. Experiments with shorter incubation periods (10–20 s) revealed a swelling of thylakoids, which probably represented an intermediate stage. The deformation of the thylakoid system was accompanied by a decrease in the non-cyclic ATP synthesis but by an increase in the rate of cyclic photophosphorylation. Besides, the deformed thylakoids demonstrated an acceleration of the basal electron transport, as well a rise in the light-induced H+ and imidazol uptake. The data obtained are discussed in the light of membrane interactions fixing the configuration of a thylakoid.

Effect of osmotic shock on the redox system in plasma membrane of Dunaliella salina

The unicellular halotolerant alga Dunaliella salina had the ability to oxidize NADH and reduce Fe(CN)63−. The redox reactions were to some extent stimulated by slight hyperosmotic shock (2.0 mol/L → 2.6 mol/L NaC1), but markably inhibited by abrupt hyperosmotic shock (2.0 mol/L → 3.5 mol/L NaC1) and hypoosmotic shock (2.0 mol/L → 1.0 mol/L NaC1; 2.0 mol/L→0.67 mol/L NaC1). With the adaptation of algal cells to osmotic shock by accumulating or degrading intracellular glycerol, the plasmalemma redox activities were also restored. The O2 uptake stimulated by NADH could be promoted by FA and SHAM. Hypoosmotic shock increases the basal respiration rate of alga cells, but weakened the stimulating effects of NADH, FA and SHAM on O2 uptake. On the other hand, hyperosmotic shock reduced the basal respiration rate, but relatively enhanced the above effects of NADH, FA and SHAM. H+ extrusion of alga cells was inhibited by NADH and stimulated by Fe(CN)63−. Vanadate and DES could inhibit H+ efflux, but had little effect in the presence of NADH and Fe(CN)63−. Both hyper- and hypoosmotic shock stimulated H+ extrusion. This effect could be totally inhibited by vanadate and DES, but almost unaffected by 8-hydroxyquinoline. It was suggested that H+-ATPase probably played a more important role in H+ extrusion and osmoregulation under the conditions of osmotic shock.

Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication

Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Bypass of receptor-mediated resistance to colicin E3 in Escherichia coli K-12

Colicin E3 was found to kill, under conditions of osmotic shock, cells lacking a functional outer membrane receptor (bfe). Under such conditions, component A of the colicin, carrying endonucleolytic activity, also killed bfe cells, whereas fragment T2, obtained by tryptic digestion of the colicin and also active endonucleolytically, was inactive. Tolerance to the colicin caused by defects in the outer membrane could be overcome by osmotic shock, whereas tolerance probably caused by an altered plasma membrane could not.

Characterization of two psychrophilic Pseudomonas bacteriophages isolated from ground beef.

Characterization studies were performed on two psychrophilic phages which were isolated from ground beef samples. Phage inactivation by exposure to heat, low pH, osmotic shock conditions, and freezing showed that these two isolates were different. One-step growth experiments indicated that one isolate had a burst size five times as large (500) and a latent period two times as long (4 hr) as the other when tested at 7 C. Nucleic acid type was 2-deoxyribonucleic acid for both. Electron micrographs showed one to belong to Bradley's phage group A and the other to phage group C.

Characterization of Two Psychrophilic Pseudomonas Bacteriophages Isolated from Ground Beef 1

Characterization studies were performed on two psychrophilic phages which were isolated from ground beef samples. Phage inactivation by exposure to heat, low pH, osmotic shock conditions, and freezing showed that these two isolates were different. One-step growth experiments indicated that one isolate had a burst size five times as large (500) and a latent period two times as long (4 hr) as the other when tested at 7 C. Nucleic acid type was 2-deoxyribonucleic acid for both. Electron micrographs showed one to belong to Bradley's phage group A and the other to phage group C.