Elevated Growth Temperatures Research Articles

Thermal stabilization of putative karyoskeletal protein-enriched fractions from Saccharomyces cerevisiae.

Elevated growth temperature (heat shock) promoted the structural stability of karyoskeletal protein-enriched fractions isolated from Saccharomyces cerevisiae. Similar stabilization could be induced by brief incubation of nuclei at 37 degrees C in vitro. These results are similar to those reported for higher eucaryotes and have practical implications for investigation of the karyoskeleton in S. cerevisiae.

Thermal stabilization of putative karyoskeletal protein-enriched fractions from Saccharomyces cerevisiae.

Elevated growth temperature (heat shock) promoted the structural stability of karyoskeletal protein-enriched fractions isolated from Saccharomyces cerevisiae. Similar stabilization could be induced by brief incubation of nuclei at 37 degrees C in vitro. These results are similar to those reported for higher eucaryotes and have practical implications for investigation of the karyoskeleton in S. cerevisiae.

PLASMIDS OF CLOSTRIDIUM BOTULINUM TYPE A, B, E, F, AND OTHER CLOSTRIDIAL SPECIES

ABSTRACTPlasmids from 46 strains of Clostridium botulinum types A, B, E, and F were isolated using a modification of the Minton and Morris procedure (1981). When these strains were examined, 54% contained one or more plasmids ranging in molecular weight from ∼ 69 to 3.4 megadaltons (MDa). Attempted curing of plasmids for two C. botulinum type A strains, 73A and Hall, using acridine orange and elevated growth temperatures (42 to 45°C) produced no changes in plasmid profiles or toxicity as determined by agarose gel electrophoresis and immunodiffusion tests, respectively. However, exposure of type E C. botulinum strain BL764 to 1000 Krad gamma irradiation resulted in loss of one plasmid (8.4 MDa) but toxicity was retained.

Plasmid curing in bacteria

Plasmids are extrachromosomal pieces of double-stranded circular DNA which have the capability to replicate independently of the host chromosome, yet coexist with it [1]. To date, many species of bacteria isolated from diverse habitats are known to contain plasmid DNA [2,3]. Some plasmids are stable and can be maintained through successive generations by being partioned to each daughter cell during cell division. This allows each cell to receive at least one plasmid copy. In recent years, plasmids have been observed in a wide variety of bacteria. In part, this is due to the development of new procedures that allow the detection, isolation, and molecular characterization of plasmid DNA. When working with some plasmid-containing bacteria, it is often desirable to obtain a plasmid-cured derivative. This allows a direct comparison to be made between the plasmid-containing and plasmid-cured cells. Some plasmids undergo spontaneous segregation and deletion. However, the majority are extremely stable, and require the use of curing agents or other procedures (elevated growth temperature, thymine starvation), to increase the frequency of spontaneous segregation [4]. The usefulness of curing agents is unpredictable in many bacterial strains, as there are no standard protocols applicable to all plasmids. However, there are some procedures that have provided good results with certain species. The aims of this manuscript are to summarize and review plasmid curing agents and procedures. In view of the importance of plasmids in specifying antibiotic and metal-resistance; metabolic properties; pathogenicity; host specificity and nodulation ( Rhizobium spp.); conjugal properties, and replication-maintenance properties [2], reproducible procedures for obtaining plasmid-cured derivatives are necessary.

Plasmid curing in bacteria

Plasmids are extrachromosomal pieces of double-stranded circular DNA which have the capability to replicate independently of the host chromosome, yet coexist with it [1]. To date, many species of bacteria isolated from diverse habitats are known to contain plasmid DNA [2,3]. Some plasmids are stable and can be maintained through successive generations by being partioned to each daughter cell during cell division. This allows each cell to receive at least one plasmid copy. In recent years, plasmids have been observed in a wide variety of bacteria. In part, this is due to the development of new procedures that allow the detection, isolation, and molecular characterization of plasmid DNA. When working with some plasmid-containing bacteria, it is often desirable to obtain a plasmid-cured derivative. This allows a direct comparison to be made between the plasmid-containing and plasmid-cured cells. Some plasmids undergo spontaneous segregation and deletion. However, the majority are extremely stable, and require the use of curing agents or other procedures (elevated growth temperature, thymine starvation), to increase the frequency of spontaneous segregation [4]. The usefulness of curing agents is unpredictable in many bacterial strains, as there are no standard protocols applicable to all plasmids. However, there are some procedures that have provided good results with certain species. The aims of this manuscript are to summarize and review plasmid curing agents and procedures. In view of the importance of plasmids in specifying antibiotic and metal-resistance; metabolic properties; pathogenicity; host specificity and nodulation (Rhizobium spp.); conjugal properties, and replication-maintenance properties [2], reproducible procedures for obtaining plasmid-cured derivatives are necessary.

Reflection high-energy electron diffraction intensity oscillation study of Ga desorption from molecular beam epitaxially grown AlxGa1−xAs

Reflection high-energy electron diffraction (RHEED) intensity oscillations are used as an in situ probe to study Ga desorption during molecular beam epitaxial growth of AlxGa1−xAs. A simple thermodynamic model is developed which, in the limit of low As4 : group III beam flux ratios, accurately predicts the measured changes in AlxGa1−xAs growth rate and composition due to Ga desorption at elevated growth temperatures. The desorbing Ga flux is observed to be less in thin epitaxial layers of GaAs growing on top of AlGaAs than in bulk GaAs, giving rise to a growth rate transient at heterointerfaces.

Effect of growth temperature and salt concentration on the fatty acid composition ofFlavobacterium halmephilumCCM2831

In this study the fatty acid composition of Flavobacterium halmephilum CCM2831 grown at different temperatures and salt concentrations is reported. At elevated growth temperatures the amount of cellular saturated long-chain acids (C18:0 and C20:0) and the branched-chain acid br-C17:0 increased, and the concentrations of both cyclic acids and the branched chain acid br-C15:0 decreased. Increasing the salt concentration in the medium resulted in a gradual increase in cellular cyclopropanoic acids and a concomitant decrease in the monounsaturated fatty acid content.

Effect of growth temperature and salt concentration on the fatty acid composition of Flavobacterium halmephilum CCM2831

In this study the fatty acid composition of Flavobacterium halmephilum CCM2831 grown at different temperatures and salt concentrations is reported. At elevated growth temperatures the amount of cellular saturated long-chain acids (C18:0 and C20:0) and the branched-chain acid br-C17:0 increased, and the concentrations of both cyclic acids and the branched chain acid br-C15:0 decreased. Increasing the salt concentration in the medium resulted in a gradual increase in cellular cyclopropanoic acids and a concomitant decrease in the monounsaturated fatty acid content.

Maintenance of plasmid pSC101 in Escherichia coli requires the host primase.

The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures. Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322. No segregation of the primase-encoding RP4 derivative was observed.

Electron microscopy of the secondary structure in partially denatured rRNAs of Escherichia coli and Bacillus stearothermophilus.

Partially denatured 16S and 23S rRNAs from the thermophile Bacillus stearothermophilus show characteristic loop patterns when observed by electron microscopy. The patterns are very similar to those seen in rRNAs from Escherichia coli. At least 2 of 4 most stable interactions in 16S rRNA and 8 of 12 interactions in 23S rRNA are in common for the two species. These interactions correspond well to features of secondary structure in models inferred for rRNA from phylogenetic sequence comparisons and chemical modification studies. However, two additional large loops, enclosing large portions of the 23S rRNA, have been detected in B. stearothermophilus for the first time, and even though other loops are similar, their relative frequencies vary in the two species. Much of the variation is consistent with relative delta G degree values for putative base-paired stems at the base of different loops; but the 5'-terminal loops in 23S rRNA, for example, are unaccountably far less stable in B. stearothermophilus. Also, in general, structural features are not differentially stabilized in B. stearothermophilus; the relative stability of secondary structure in its ribosomes at elevated growth temperatures must involve interactions with ribosomal proteins or other cellular components.

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature.

The effect of elevated growth temperature on nif mRNA synthesis, nif protein synthesis, and nitrogenase activity was investigated in wild-type and NifL- strains of Klebsiella pneumoniae. Nitrogenase activity is not affected at 41 degrees C; however, nitrogenase is not synthesized at that temperature. Transcription of three nif operons studied is repressed at 41 degrees C. We show that the nifL protein is not required for repression by high temperature and propose that repression of nif at 41 degrees C results from a reversible inactivation of the nifA gene product.

Influence of temperature upon dislocation mobility and elastic limit of single crystal HgI 2

The practical importance of studying mechanical properties and dislocation structure of HgI 2 is reviewed briefly. Specifically, the performance of single crystal HgI 2 radiation detectors is evidently sensitive to crystalline imperfections, including dislocations. The dislocation structure, in turn, may be altered during crystal growth and detector fabrication; the nature of such alterations depends upon the mechanical properties of the crystal and the stresses to which the crystal is subjected. This work examines the influence of temperature upon dislocation mobility and plasticity in vapor grown single crystals of mercuric iodide. Dislocation mobility is determined by measuring the lengths of the longest arms of dislocation etch pit rosettes on (001) surfaces following microhardness indentation and chemical etch. Measurements were made at temperatures ranging from room temperature to the phase transition temperature of 127°C. Dislocation mobility was found to increase with temperature, with the effect accelerating as the 127°C phase transition is approached. Increasing temperature was also found to lower the critical resolved shear stress for plastic deformation or slip on (001) planes. In these contexts, the vapor grown crystals are clearly “softer” at their elevated growth temperatures. The results are discussed in terms of a dislocation model involving “soft” and “hard” glide dislocations.

ESR studies on the lipid bilayers of separated outer and cytoplasmic membranes of a moderately halophilic bacterium.

Electron spin resonance (ESR) studies were performed on the isolated outer and cytoplasmic membranes from cells of a moderately halophilic bacterium Pseudomonas halosaccharolytica ATCC 29423, which were grown in 2 M NaCl medium at different temperatures. The cytoplasmic membrane contained more acidic phospholipids (50% or more; including most of the diphosphatidylglycerol) than the outer membrane (40%) in 25 and 30 °C grown cells, while in both membranes of 35 °C grown cells the relative content of acidic phospholipids was almost the same. With elevated growth temperature C19-cyclopropanoic fatty acids in both membranes increased.The physical properties of lipid bilayers of both membranes were studied by a spin-label method using N-oxyl spin-labelled stearic acids. Isolated membranes suspended in a medium with or without 2 M NaCl showed no appreciable differences in ESR spectra on changing the sodium chloride concentration in the medium. The lipid bilayers of both membranes gave almost uniform rotational correlation times for the first 12 carbon atoms of the hydrocarbon chains starting from the polar surface of the bilayers. The difference between rotational correlation times of either membrane and those of whole cells and liposomes of extractable lipids of this bacterium was that the rotational correlation times of both membranes gave similar absolute values regardless of change in the growth temperature and varied similarly with the temperature of ESR measurements. The surface lipid layers up to C12 had a phase transition temperature near the growth temperature of the bacterium. The rotational activation energies were 5–10 kcal/mol (1 cal = 4.1868 J) for the liquid phase and 10–25 kcal/mol for the solid phase. In general, the order parameters of both membranes decreased gradually with increasing temperature of ESR measurements, except for rapid decreases and discontinuous drops near the growth temperature with the spin-probe labelled at C12 of stearic acid (12NS). Such discontinuous decreases in order parameter of 12NS were not observed for the whole cell membrane nor for liposomes of total extractable lipids of this bacterium.

Improved mobility in OM-VPE-grown Ga 1− x In x As

The use of a tirmethylindium-trimethylarsenic adduct, which is synthesised in the gas phase, as the In source, and AsH3 as the primary As source is reported for the growth of high-quality Ga1−xInxAs (x<0.20). The improved epitaxial layers have higher mobilities and lower background doping than those grown with trimethylarsenic as the sole As source. The effects of elevated growth temperatures are also reported.

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.

Lipid and protein composition of membranes of Bacillus megaterium variants in the temperature range 5 to 70 degrees C.

Membranes were prepared from four temperature range variants of Bacillus megaterium: one obligate thermophile, one facultative thermophile, one mesophile, and one facultative psychrophile, covering the temperature interval between 5 and 70 degrees C. The following changes in membrane composition were apparent with increasing growth temperatures: (i) the relative amount of iso fatty acids increased and that of anteiso acids decreased, the ratio of iso acids to anteiso acids being 0.34 at 5 degrees C and 3.95 at 70 degrees C, and the pair iso/anteiso acids thus seemed to parallel the pair saturated/unsaturated acids in their ability to regulate membrane fluidity; (ii) the relative/unsaturated acids in their ability to regulate membrane fluidity; (ii) the relative amount of long-chain acids (C16 to C18) increased fivefold over that of short-chain acids (C14 and C15) between 5 and 70 degrees C; (iii) the relative amount of phosphatidylethanolamine increased, and this phospholipid accordingly dominated in the thermophilic strains, whereas diphosphatidylglycerol was predominant in the two other strains; and (iv) the ratio of micromoles of phospholipid to milligrams of membrane protein increased three-fold between 5 and 70 degrees C. Moreover, a quantitative variation in membrane proteins was evident between the different strains. Briefly, membrane phospholipids with higher melting points and packing densities appeared to be synthesized at elevated growth temperatures.

Occurrence of a High Temperature Sensitivity of Chloroplast Ribosome Formation in Several Higher Plants

A specific high temperature-induced deficiency of chloroplast ribosome formation, as indicated by the absence of chloroplast rRNA, has been observed in the leaves of light- or dark-grown seedlings of Avena sativa L., Hordeum vulgare L., and Triticum aestivum L. at certain temperatures between 28 and 34 C. While the growth of the leaves (size, morphology, total amino nitrogen content) was little affected by the elevated temperature, chlorophyll accumulation was strongly inhibited, amounting to only 2 to 20% of its content in 22 C-grown leaves which were used as a reference for normal development. The carotenoid contents were also lower but still reached at least 15 to 20% of the corresponding measurements at 22 C. Ribulose-1,5-bisphosphate carboxylase was absent at the higher temperature while NADP-glyceralde-hydephosphate dehydrogenase reached high activities. For the peroxisomal marker enzyme hydroxypyruvate reductase, 30 to 70% of the activity present in 22 C-grown leaves was found in extracts from high temperature-grown leaves. Fumarase reached 1.5- to 4-fold higher activities at the elevated growth temperature than at 22 C. Leaves of Pisum sativum L. were completely chlorotic and deficient of 70S ribosomes at 33 C but simultaneously suffered from a severe general inhibition of their growth. In Zea mays L., a formation of chlorotic leaves was not observed at elevated temperatures.

Production of Megacins C and Cx: Presumptive Evidence of Extrachromosomal Control

Exposure of growing cultures of Bacillus megaterium C4A(-) to ethidium bromide or an elevated growth temperature was found to eliminate megacin C production. Ethidium bromide resulted in a cure rate of up to 13%. Growth at 43 degrees C gave a cure rate of up to 99%. Megacin C production was lost spontaneously at a rate of 4% or less. There was a greater rate of spontaneous loss of megacin Cx production by B. megaterium 337, up to 14%. Growth at 43 degrees C resulted in a cure rate of up to 24% in this organism. Reversion to the Meg(+) state by cured clones has never been demonstrated. These observations support the hypothesis that production of megacins C and Cx is plasmid mediated. Meg(-) clones adsorbed more megacin than either parent strain and were more susceptible to megacin action.

Formation of chloroplast pigments and sterols in rye leaves deficient in plastid ribosomes

1. In rye (Secale cereale L.) leaves the formation of plastidic ribosomes is sensitive to elevated growth temperatures. Parallel to the loss of 70S ribosomes, in leaves growing at 32° chlorophyll accumulation was also prevented. Except for the tips of the first leaves which still contained some 70S ribosomes, the leaves were chlorotic. The amount of chlorophyll formed at 32° depended on the light intensity and decreased with higher intensities. After return to normal temperature (22°) chlorotic parts of the first leaves greened to a varying extent while those parts of most 2. or 3. leaves which had been formed in light at 32° remained permanently bleached until they died. Those parts of 2. and 3. leaves which were newly formed at 22° became normally green again. - 2. Formation and distribution of total and individual carotenoids were compared after development at 22° and 32°. In dark-grown leaves the higher growth temperature had no marked influence on the quantity or composition of carotenoids. At 22° the content of total carotenoids was 5fold and that of β-carotene 25fold increased by light. At 32° these light-induced increases were much lower. Only 41% of the total carotenoids and 18% of the β-carotene formed at 22° in light were found at 32°. Of the carotenoids present at 32°, 76% were located in the light green tips of the leaves. In plastids isolated from completely chlorotic leaf parts, carotenoids were still present and were even the predominant pigments. - 3. The contents of total sterols, the fractions of free sterols, sterol glycosides and esters, and the composition of individual sterols were compared in rye leaves grown at 22° and at 32°, in light or darkness. Light had little effect on the total sterol contents per leaf. However, more than 2fold higher sterol contents were observed in leaves grown at 32°, as compared to those from 22°. The amounts of most sterol fractions and individual sterols were similarly increased at the higher temperature but the sterol glycosides being relatively more increased than the total sterols.

A colicin-tolerant mutant of Escherichia coli with reduced levels of cyclic AMP and the regulation of lambdoid phage lysogeny.

Wild-type phages λ and ϕ80 plate very differently on the colicin-tolerant mutant 483 (originally designated as ER437 by Nomura and Witten 1967) at different incubation temperatures. Phage λ gives very high levels of lysogenic colonies at all temperatures and thus reduced plating efficiencies. In marked contrast, phage ϕ80 plates with normal efficiencies at all growth temperatures, forming turbid-solid centred plaques at 30° and clear plaques at elevated growth temperatures.