Accumulation of plant galactolipid affects cell morphology of Escherichia coli

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.     

ELECTRON MICROSCOPY OF CELLULAR DIVISION IN ESCHERICHIA COLI.

Conti, S. F. (Brookhaven National Laboratory, Upton, N. Y.) and M. E. Gettner. Electron microscopy of cellular division in Escherichia coli. J. Bacteriol. 83:544-550. 1962.-Exponentially growing cells of Escherichia coli were fixed in formalin, exposed to uranyl nitrate, dehydrated at low temperatures with ethanol, and embedded in methacrylate. Polymerization was carried out at -70 C, by exposure of specimens to radiation from a cobalt(60) source. Electron micrographs revealed that cellular division occurs by the centripetal growth of the cell wall. The fine structure of the cytoplasm, nuclear apparatus, cell wall, and cytoplasmic membrane was also studied.     

Formation and Ultrastructure of Extra Membranes in Escherichia coli

A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).     

Evaluation of in vitro antibacterial activity of some disinfectants on Escherichia coli serotypes

Three disinfectants commonly used in poultry farms (formalin, TH4+, and Virkon-S) were chosen for the present study. The effect of disinfectant concentration and the duration of exposure to these disinfectants on the survival of Escherichia coli serotypes (O114:K-, O86, O55:K39, and O86:K60) were investigated. Formalin (0.6%), TH4+ (0.06%), and Virkon (0.5%) all killed the four serotypes within 5 min of exposure. As the disinfectant concentration decreases, the length of exposure time to kill serotype increases. At 0.03%, 0.007%, and 0.03% of formalin, TH4+ and Virkon-S concentrations failed to kill the four E. coli serotypes within 360 min, respectively. An improvement of the inhibitory effect of these disinfectants occurred when added together with the inoculum instead of an established population. The influence of formalin, TH4+, and Virkon-S on the cell morphology of E. coli O55:K39 was investigated by using transmission electron microscopy. Formalin-treated cells exhibited normal cell morphology, with the exception that the treated cell was less fimbriated, and more destruction of pili increased when formalin concentrations were doubled. Cells treated with TH4+ (0.03%) showed destruction of the cell wall and cell surface membrane after 5 min. Cell filamentation occurred at 0.015% and increased with the increase of exposure time to this drug. Spheroplasts were observed only when cells were treated with 0.125% Virkon-S for 60 min, and cell lysis started to occur when 0.25% Virkon-S was applied for 15 min. Scanning electron microscope study revealed that Virkon-S at 0.03% and TH4+ at 0.007% completely prevented the adherence of E. coli O55:K39 serotype to chicken tracheal organ, whereas formalin (0.03%) disinfection minimized the adherence of E. coli cells to tracheal explants after 360 min of incubation.     

Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.     

Thermally enhanced radioresponse of cultured chinese hamster cells: inhibition of repair of sublethal damage and enhancement of lethal damage

X-irradiation of Chinese hamster cells at temperatures above 37°C results in enhanced killing response. The magnitude of this thermal effect increases with increasing temperature and varies inversely with dose rate during the exposure of the cells to the combined effects of elevated temperature and ionizing radiation. Postirradiation incubation at an elevated temperature is also effective in enhancing the response but not preirradiation hyperthermia. Split-dose experiments demonstrate that hyperthermia also inhibits the repair of sublethal damage for temperatures up to ～41°C. Above 41°C, lethal damage expression is enhanced as well. Fluctuations in the age-response structure of cells x-irradiated at 42°C are reduced, a result consistent with a reduced capacity for sublethal damage when cells are hyperthermic during irradiation.     

Changes in cell morphology of Listeria monocytogenes and Shewanella putrefaciens resulting from the action of protamine.

Protamine, which is an antibacterial basic peptide, was shown to alter the cell morphology of Listeria monocytogenes and Shewanella putrefaciens. Atomic force microscopy revealed that protamine smoothed the surface of cells, formed holes in the cell envelope, and caused fusion of S. putrefaciens cells. Immunoelectron microscopy of protamine-treated cells of both L. monocytogenes and S. putrefaciens showed great damage to the cell wall and condensation of the cytoplasm. Respiration of the cells was decreased due to treatment with sublethal concentrations of protamine, probably due to leakage or loss of cell envelope potential. It was concluded that protamine disrupted the outer surface structure and condensed the cytoplasm of sensitive cells and, in sublethal concentrations, altered membrane structures, thereby eliminating respiration.     

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane.     

Towards a unifying theory of late stochastic effects of ionizing radiation

The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10mW/cm(2)), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41°C, while apoptosis was induced only when the medium temperature achieved 40°C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line.     

Abnormal cell division caused by inclusion bodies in E. coli; increased resistance against external stress

Inclusion body formation occurs naturally in prokaryotic cells, but is particularly common when heterologous foreign proteins are overexpressed in bacterial systems. The plant disease virus protein CMV 3a (cucumber mosaic virus movement protein) and the 56 kDa Orientia tsutsugamushi (OT56) protein (an outer membrane protein), which causes tsutsugamushi disease, were expressed in Escherichia coli, and found to form inclusion bodies. Confocal laser scanning microscopy revealed that these inclusion bodies are localized at the cellular poles within E. coli. Cells expressing inclusion bodies appeared to be interconnected, and divided abnormally. The clustered cells exhibited biofilm-like characteristics in that the interior cells of the community were protected by the antibiotic resistance of the outer cells. We compared the number of colony-forming units in inclusion body-forming versus non-forming E. coli to demonstrate the effects of lysozyme, sonication or antibiotic treatment. E. coli clustering provided significantly improved protection against cell disruption/lysis by physical and biochemical stress. This is the first report that shows that abnormal cell division caused by inclusion body formation can cause cellular clustering, resulting in improved resistance to stress in vitro.     

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.     

Seasonal variation in survival of Escherichia coli exposed in situ in membrane diffusion chambers containing filtered and nonfiltered estuarine water.

Human fecal Escherichia coli isolates were exposed over a seasonal cycle to estuarine water in diffusion chambers filled with double-filtered (0.45 and 0.2 microns) and nonfiltered water. Laboratory manipulations of E. coli cultures before estuarine exposure were reduced to minimize sublethal stress, and nonselective or resuscitative enumeration techniques were employed to maximize recovery of stressed cells. E. coli was capable of extended survival during in situ exposure to estuarine water, provided eucaryotes were excluded from diffusion chambers. Survival was directly related to temperature in absence of the eucaryote component of the natural microbiota. Although it was not possible to prevent eventual bacterial contamination in double-filtered water, there was no direct evidence that such contamination affected E. coli survival. Conversely, E. coli disappearance was most pronounced at warmer temperatures in the presence of the natural microbiota, and decline coincided with increasing eucaryote densities. In contrast, the decline of E. coli during winter was similar in both filtered and nonfiltered seawater.     

Destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.

High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.     

Seasonal variation in survival of Escherichia coli exposed in situ in membrane diffusion chambers containing filtered and nonfiltered estuarine water

Human fecal Escherichia coli isolates were exposed over a seasonal cycle to estuarine water in diffusion chambers filled with double-filtered (0.45 and 0.2 microns) and nonfiltered water. Laboratory manipulations of E. coli cultures before estuarine exposure were reduced to minimize sublethal stress, and nonselective or resuscitative enumeration techniques were employed to maximize recovery of stressed cells. E. coli was capable of extended survival during in situ exposure to estuarine water, provided eucaryotes were excluded from diffusion chambers. Survival was directly related to temperature in absence of the eucaryote component of the natural microbiota. Although it was not possible to prevent eventual bacterial contamination in double-filtered water, there was no direct evidence that such contamination affected E. coli survival. Conversely, E. coli disappearance was most pronounced at warmer temperatures in the presence of the natural microbiota, and decline coincided with increasing eucaryote densities. In contrast, the decline of E. coli during winter was similar in both filtered and nonfiltered seawater.     

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt’s lymphoma (Raji) cells

Abstract

We investigated the effects of 1.8?MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt’s lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8?GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350?W/kg in a CO₂ incubator. The duration of the exposure was 24?h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW?+?Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.     

Effect of membrane fatty acid substitution and temperature on repair of sublethal damage in mammalian cells

Repair of sublethal radiation damage (SLD) has been investigated as a function of temperature in mouse fibroblast LM cells with different membrane lipid composition. Rigidification or fluidization of the cellular membranes was accomplished by incorporation of myristic acid and arachidonic acid, respectively, in the phospholipids of the membranes. The SLD repair after radiation was essentially the same for the cells with the more rigid (saturated fatty acid) membranes and the cells with the more fluid (polyunsaturated fatty acid) membranes. This observation was made for repair at 37 degrees C as well as for repair at hypothermic temperatures. Incorporation of polyunsaturated fatty acid protected the cells against hypothermic death. These experiments demonstrate that although membranes are likely targets for cell killing by low temperature treatments, membrane lipids are probably not involved in the repair of sublethal radiation damage. It must be concluded that neither the degree of polyunsaturation of the lipids nor the degree of fluidity of the membrane is important for radiation-induced killing of mammalian cells.     

Effects of extremely low frequency (50 Hz) magnetic field on morphological and biochemical properties of human keratinocytes

We investigated the effects on human keratinocytes (HaCaT) of exposure to a sinusoidal magnetic field of 2 mT (50 Hz). These cells are a good model for studying interaction of nonionising radiation, because they are not shielded from fields in vivo and also because they are resistant to both mechanical and thermal stimuli. We performed scanning microscopy which showed modification in shape and morphology in exposed cells. This modification is related to differential actin distribution as revealed by phalloidin fluorescence analysis. Moreover, the exposed cells show increased clonogenic capacity, as well as increased cellular growth as showed by clonogenicity assays and growth curves. Indirect immunofluorescence analysis using a fluorescent antibody against involucrin and beta4 integrin, which are respectively differentiation and adhesion markers, revealed an increase of involucrin expression and segregation of beta4 integrin in the cell membrane in cells exposed to 50 Hz; a higher percentage of the exposed cells shows a modified pattern of adhesion and differentiation markers. We also present evidence that exposure of HaCaT cells can interfere with protein kinase activity. Our observations confirm the hypothesis that electromagnetic fields at 50 Hz may modify cell membrane morphology and interfere with initiation of the signal cascade pathway and cellular adhesion.     

On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria provides a protective barrier for natural occurring inhibitors. Pressure mediated OM permeabilisation therefore contributes to the elimination of Escherichia coli and Salmonella by pressure preservation processes. Pressure mediated inactivation, sublethal injury, and membrane permeabilisation of E. coli were determined using two strains differing in their barotolerance. Pressure treatment of E. coli TMW 2.427 at 300, 500 and 600 MPa for 40 min resulted in a 0, 1, and greater 6 log decrease of viable cell counts, respectively. The kinetics of OM and cytoplasmic membrane permeabilisation after pressure treatment were determined by staining of pressure treated cells with the fluorescent dyes propidium iodide (PI) and 1-N-phenylnaphtylamine (NPN), respectively. A slight increase of PI fluorescence was observed at conditions resulting in a greater 6 log decrease of viable cell counts only. In contrast, increased NPN fluorescence indicating OM permeabilisation was observed prior to cell death and sublethal injury. An on-line assay for determination of pressure mediated OM damage based on NPN fluorescence was established to distinguish between reversible and irreversible OM damage. Generally, the same degree of outer membrane damage was observed by either on line or off line determinations. However, whereas reversible membrane damage occurred fast and in thermodynamic equilibrium with pressure conditions, irreversible outer membrane damage was a time dependent process.