Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15 degrees C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees C membranes, while 15 degrees C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15 degrees C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15 degrees C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37 degrees C, but only 50% at 15 degrees C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15 degrees C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca2+, such that these proteins are synthesized only at the higher temperature (37 degrees C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28 degrees C during nutrient-limited growth. Thus, temperature and Ca2+ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

A Highly Thermosensitive and Permeable Mutant of the Marine Yeast Cryptococcus aureus G7a Potentially Useful for Single-Cell Protein Production and its Nutritive Components

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.     

Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells.

Proliferation of mammalian cells can be controlled by low cultivation temperature. However, depending on cell type and expression system, varying effects of a temperature shift on heterologous protein production have been reported. Here, we characterize growth behavior and productivity of the Chinese hamster ovary (CHO) cell line XM111-10 engineered to synthesize the model-product-secreted alkaline phosphatase (SEAP). Shift of cultivation temperature from 37 degrees C to 30 degrees C caused a growth arrest mainly in the G1 phase of the cell cycle concomitant with an up to 1.7-fold increase of specific productivity. A low temperature cultivation provided 3.4 times higher overall product yield compared to a standard cultivation at 37 degrees C. The cellular and molecular mechanisms underlying the effects of low temperature on growth and productivity of mammalian cells are poorly understood. Separation of total protein extracts by two-dimensional gel electrophoresis showed altered expression levels of CHO-K1 proteins after decrease in cultivation temperature to 30 degrees C. These changes in the proteome suggest that mammalian cells respond actively to low temperature by synthesizing specific cold-inducible proteins. In addition, we provide the first evidence that the cold response of mammalian cells includes changes in postranslational protein modifications. Two CHO proteins were found to be phosphorylated at tyrosine residues following downshift of cultivation temperature to 30 degrees C. Elucidating cellular events during cold exposure is necessary for further optimization of host-cell lines and expression systems and can provide new strategies for metabolic engineering.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.     

Ecology, inhibitory activity, and morphogenesis of a marine antagonistic bacterium belonging to the Roseobacter clade

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30 degrees C but died rapidly at 37 degrees C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37 degrees C in less than 2 days and at 25 degrees C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.     

Immunochemistry of Yersinia enterocolitica O3 grown at different temperatures.

Comparative agglutinations of homogeneous stable suspensions prepared with Yersinia enterocolitica growth at 37 degrees C and at 25 degrees C were performed with anti-sera prepared in rabbits with the bacteria grown at both these temperatures. Sera prepared with live Y. enterocolitica grown at 37 degrees C agglutinated both suspensions at a much lower titre than the sera prepared with formaldehyde-treated bacteria is grown at 25 degrees C. All the sera in which strongly precipitating antibodies were induced reacted, in agar-gel, against native and heated proteins. The small amounts of antipolysaccharides induced in all the sera reacted only in the ring test against the bacterial polysaccharides. The absorption of the sera prepared with live Y. enterocolitica grown at 37 degrees C, with antigens synthesized at 25 degrees C did not remove all the homologous antibodies; apparently, some determinants are specific for the bacteria grown at 37 degrees C. Morphological changes of the small rods to elongated bacilli and filamentous forms were observed in most cultures of the Y. enterocolitica grown at 37 degrees C; these changes coincided with a low yield of proteins and point to an inhibitory effect of the 37 degrees C temperature.     

Temperature-dependent variation in the extent of methylation of ribosomal proteins L7 and L12 in Escherichia coli.

The amount of epsilon-N-monomethyllysine in ribosomal proteins L7 and L12 in Escherichia coli is dependent upon the cell growth temperature. At 37 degrees C or above, very small amounts were detected. Dramatic increase in the content of epsilon-N-monomethyllysine in these proteins was observed when the growth temperature was lowered.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Effects of growth temperature on protoplast membrane properties in Bacillus megaterium.

Changes in the protoplast membrane of the KM strain of Bacillus megaterium were assessed after growth at 20, 30, or 37 degrees, C. Although the overall membrane concentrations of lipids and proteins were virtually unchanged, increased culture temperature resulted in cells with membranes that contained relatively more unbranched and long-chain fatty acids and more acidic phospholipids, as well as different proportions and numbers of individual proteins. Electrophoretic analysis revealed 23, 31, or 29 protein bands, respectively, in membranes from cells grown at the three temperatures. Protoplasts from cells grown at higher temperatures were considerably less susceptible to lysis by shearing forces. As judged by passive leakage at 30 degrees C, intact cells from cultures grown at 37 degrees C were the least permeable to erythritol. Relatively low ambient concentrations of Ca2+ or Mg2+ protected protoplasts from osmotic lysis but even much higher concentrations left erythritol leakage virtually unaffected. Thus, growth temperature affected not only membrane lipis but also membrane proteins and these changes resulted in membranes with altered mechanical properties and permeabilities.     

Temperature and the formation of radiation-induced chromosome aberrations. I. The effect of irradiation temperature

Irradiation temperature, changed from 37 degrees C to 4 degrees C, acts as a dose-modifying factor with regard to the dose-yield relationship for dicentric chromosome aberrations in human lymphocytes irradiated with 150 kV X-rays. The temperature dependence of the aberration yield observed at constant dose is S-shaped, with a sharp rise near 15 degrees C from a lower plateau below 12 degrees C to a higher plateau beyond 17 degrees C. The aberration yield is determined by the irradiation temperature, irrespective of fast temperature changes from 4 degrees C to 37 degrees C or from 37 degrees C to 4 degrees C, applied at various delay times before and after irradiation. It is concluded that irradiation temperature influences the formation of chromatin lesions rather than their interaction.     

High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.