Initial studies on aBacillus subtilis mutant lacking the dnaK-homologue protein

A sequence of 412bp, spanning the terminal half of thegrpE and the proximal portion of thednaK-homologues inBacillus subtilis, was amplified with PCR technology. This fragment was cloned into pJH101, anEscherichia coli plasmid, and transformed intoB. subtilis strain YB886. Several chloramphenicol-resistant colonies were obtained from this transformation. The integration of the plasmid into theB. subtilis chromosome was verified by restriction endonuclease analysis and Southern hybridization. Strain BUL101, a chloramphenicol-resistant transformant, lacked the DnaK-homologue as demonstrated by two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. BUL101 grew at slower rates than parental cells at both 37°C and 48°C, produced abnormal cell shapes at 48°C, and was unable to grow at 51°C. The 412bp fragment did not exhibit detectable promoter activity.     

The Architectonics of Colonies of Bacillus subtilis 2335

Colonies grown from vegetative Bacillus subtilis 2335 cells had a standard structure, with bacillar cells occupying the whole colony volume. At the same time, the colonies of this bacterium grown from germinated spores had an abnormal structure characterized by the location of cells in a surface layer 100–200 m thick at the colony boundary with the air. The glycocalyx of the colonies grown from spores was characterized by a wetting angle _e of 120°–160°, whereas that of the colonies grown from vegetative cells had an angle _eas low as 5°–30°. It is suggested that spores and vegetative cells follow different strategies of substrate colonization and that the architectonics of bacterial colonies is determined by the physicochemical properties of the glycocalyx.     

Thermal Resistance ofBacillus cereus spores in custard preparations

Summary The thermal resistance ofBacillus cereus spores expressed in terms ofD value (an expression of time in minutes required for the heat destruction of one log cycle of spores) in custard preparations decreased from 90°C to 100°C. With an increase in pH of custard preparations from 6.2 to 7.2, theD value ofB. cereus spores increased from 3.1 to 3.7 min at 90°C, from 2.5 to 3.0 min at 95°C and from 1.7 to 2.2 min at 100°C. However, theZ value (the change in temperature necessary to cause a tenfold change in theD value) remained unaffected by the change in pH. A decrease in the number of heatedB. cereus spores was observed with the increase in the concentration of the antimicrobial agent sodium benzoate and the antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

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Résistance thermique des spores deBacillus cereus dans les préparations de crème

Résumé La résistance thermique de spores deBacilluscereus, exprimée par la grandeur D (l'expression du temps en minutes requis pour la destuction thermique d'une décade logarithmique de spores) dans les préparations de crème, décroît de 90 °C à 100 °C. Avec une augmentation de pH des préparations de crème de 6.2 à 7.2, la valeur D pour les spores deB. cereus augmente de 3.1 à 3.7 min. à 90 °C, de 2.5 à 3.0 min. à 95 °C et de 1.7 à 2.2 min. à 100 °C. Toutefois, la grandeur Z (le changement de température requis pour engendrer un changement d'un facteur 10 dans la valeur D) n'est pas affectée par ce changement de pH. Une diminution du nombre de spores deB. cereus chauffé est observée avec l'augmentation de la concentration du benzoate de soude, un agent antimicrobien, ainsi que de l'anisole hydroxy-butylé et du toluène hydroxybutylé, deux agents antioxidants.

     

Recoverability of heat-injured Bacillus spores by lysozyme and EDTA or alkaline thioglycollate

The D_95°C value of Bacillus thuringiensis spores plated in the presence of lysozyme increased from 3.0 min to 3.6 min by post-treatment of heat-injured spores with 50mm EDTA. In the case of Bacillus alvei and Bacillus polymyxa spores D-values decreased from 4.9 to 4.3 min and from 4.7 to 4.1 min respectively. Post-treatment of heat-injured spores treated with alkaline thioglycollate increased D_95°C values of Bacillus alvei from 4.2 to 5.3 min, B. thuringiensis 3.6 to 4.7 min, and Bacillus polymyxa from 4.2 to 5.0 min when spores were plated in the presence of lysozyme. Electron micrographs of heat-injured B. alvei spores treated with sodium thioglycollate indicated that the coat layers of the treated spores were granulated and less intact than the control spores.     

Sporulation in distilled water

Spores are formed when vegetative cells of sporing aerobes are shaken with distilled water at 37°. These spores are derived from the small number of cells which survive lysis. The sporulation process involves increase and concentration of solid material in the cell, and is achieved at the expense of the products of lysis of 80 to 90 per cent of the resuspended cells.     

Procurement of spore‐free Bacillus anthracis for molecular typing outside of BSL3 environment

Aims: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint^® analysis and pulse field gel electrophoresis (PFGE). Methods and Results: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45°C under normal air, enhanced CO₂, microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80°C for 30 min and processed for and analysed by either Riboprinter^® or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. Conclusions: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter^® and PFGE. Significance and Impact of the Study: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore‐formers.     

Chlorophyll a Fluorescence Analysis in Response to Excitation Irradiance in Bean Plants (Phaseolus vulgaris L. and Vigna unguiculata L. Walp) Submitted to High Temperature Stress

Bean plants Phaseolus vulgaris L. (cv. Carioca and Negro Huasteco) and Vigna unguiculata L. Walp (cv. Epace-10) were grown in a growth chamber with a photosynthetic photon flux density of 200 mol m^–2 s^–1 at leaf level and air temperature of 25+1 °C. Fully expanded, first pair leaves of 12-d-old plants were submitted for 90 min to high temperature (25, 30, 35, 40, 45, and 48 °C). Chlorophyll a fluorescence parameters (ETR, q_P, q_N, and F₀) were investigated using a modulated fluorimeter at 25 °C during recovery considered here as 48 h after stress induction period. An accentuated decrease in q_P and an increase in q_N at 48 °C in Carioca and Negro Huasteco was not observed in Epace-10. In response to excitation irradiance a great potential for ETR was found in Negro Huasteco at 25 °C, also demonstrated by net photosynthetic rate. At 48 °C ETR was high for Epace-10 while it was equal to zero for Carioca and Negro Huasteco. Tolerance to high temperature observed in Epace-10 provided important information about the adaptative characteristics of Vigna cultivars to warm climates.     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

Heat resistance of Clostridium sordellii spores

The thermal destruction kinetics of Clostridium sordellii spores was studied in this research. Decimal reduction times (D values) for C. sordellii ATCC 9714 spores ranged between 175.60 min for D₈₀ (the D value for spore suspensions treated at 80 °C) and 11.22 min for D₉₅. The thermal resistance (Z) and temperature coefficient (Q₁₀) values of spores were calculated to be as high as 12.59 °C and 6.23, respectively. At 95 °C, the relative thermal death rate and relative thermal death time of C. sordellii ATCC 9714 spores were found to be 0.0085/min and 118 min, respectively, indicating that the death rate of spores was 118 times lower at 95 °C than at 121.1 °C. Heat treatments at up to 85 °C for 120 min failed to cause a 100-fold destruction in spore populations of C. sordellii ATCC 9714. By contrast, spore counts were reduced by 2log₁₀ cycles within 73 min and 23 min at 90 °C and 95 °C, respectively. This is the first published report of thermal inactivation of C. sordellii spores; however, further studies are needed to confirm these results in real food samples.     

Survival kinetics of Salmonella enterica serotype senftenberg (S. senftenberg) after heat and acid stress

The survival kinetics of two clinical isolates of Salmonella senftenberg were studied after heat and acid stress. The strains survived better at 53 and 55 °C after heat shock of 30 min at 50 °C or overnight heat adaptation at 45 °C. An increase in the decimal reduction time, D, of heat-shocked [10.2 min (53 °C) and 9.37 min (55 °C)] and heat-adapted [8.12 min (53 °C) and 7.8 min (55 °C)] cells was observed compared with the non-stressed cells [6.87 min (53 °C), 6.56 min 55 °C)]. A significant difference was also observed in the survival of acid-adapted to acid non-adapted S. senftenberg bacteria.     

A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase

Summary [C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25°, but which is deficient in cytochromes b and aa ₃ when grown at 37° (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37°, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37°-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rates of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37°-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.     

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

The effect of hypothermic treatment on the repair or expression of X-ray damage measured in split dose experiments on L5178Y-S cells

Summary The nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25° C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37° C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37° C followed by 4 h holding at 25° C prior to the second half of a split dose, or 5 h holding at 25° C without a 37° C incubation during the interval between doses. Longer incubations at 37° C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37° C was observed only during the first 30 min; thereafter,³H-dThdR incorporation washigher than in unirradiated controls. Theexcess synthesis effect was removed by shifting irradiated cells to 25° C holding. The inhibition observed at 25° C was reversed by shifting to 37° C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37° C, and by 3 h at 25° C; DNA double-strand break (DSB) repair plateaus at 80% (37° C) and 60% (25° C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.A preliminary report of this work was presented at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA 1987, USA     

Activation ofStreptomyces viridochromogenes spores by detergents

Constitutively dormant spores ofStreptomyces viridochromogenes germinate rapidly following treatment with 1.0% of the detergents Tween 80, sodium dodecyl sulfate (SDS), or sodium heptadodecyl sulfate. Six other detergents did not activate the spores. Activation by SDS was studied further. The spores were not activated following treatment with 0.09% or less of SDS for 60 min at 37°C. Activation was complete within 1 to 2 min of treatment with 1.0% SDS. the SDS-activated spores became deactivated during incubation in buffer. Deactivation was slow at 4°C and complete after incubation for 12 h at 25°C or 6 h at 37°C. The endogenous respiratory rate of the spores was increased 3-fold by SDS activation.     

Enzymes and germination of spores of the yeast Saccharomyces cerevisiae

Enzymes in spores of the yeast Saccharomyces cerevisiae were determined and compared with those in vegetative cells. The activities of phosphatase, oxidase and enzymes in the glycolytic bypass were high (about 3–10 fold) in spores, whereas those of dipeptidase, protease, DNase, RNase and TCA cycle enzymes were low in spores, being only 50% of the activities in vegetative cells. Enzymes in the glycolysis, glutathione and acetate metabolic pathways remained unchanged before and after sporulation. Germination of the yeast spores was repressed in the presence of d-aspartate, d-glutamate, d,l-methionine, d,l-cysteine, l-isoleucine, l-histidine and l-threonine, or bivalent metal ions such as Ni²⁺, Co²⁺ and Zn²⁺.