Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8^T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h⁻¹) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min⁻¹ g⁻¹). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).     

Effect of low winter temperatures on milk production of dairy cows grazed on farms in a warm temperate climate (Australia)

Two-day milk production figures for dairymen at Armidale (31° S Lat.) and Goulburn (35° S Lat.), Australia, were compared with minimum daily temperatures. Comparisons were made between production levels on days with temperatures above and below 0°C, and between days with varying intensities of cold: differences in production were minor. The infrequent, more prolonged periods of cold weather (cold being days on which the minimum temperature was below 0°C) were also associated with only very slight reductions in milk output; and the most pronounced decline in production was not attributable to low temperatures. Although short-term fluctuations in output were not related to low temperature events or low temperatures in combination with rain, thermal stress on plant growth during winter contributed to a seasonal downturn in production and below-average annual milk yields in areas with low winter temperatures.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Effect of different carbon sources on membrane permeability,membrane fluidity,and fatty acid composition of a psychrotrophic Acinetobacter sp. HH1-1 during growth at low temperatures and after cold shock

The effect of different carbon sources on the ability of a psychrotrophic Acinetobacter sp., strain HH1-1, to grow at low temperatures and respond to cold shock was investigated by monitoring cell membrane permeability, membrane fluidity and fatty acid composition. Cells were grown in batch cultures with acetate, Tween 80 or olive oil as the sole source of carbon and incubated at 25, 5^°C or subjected to a 25 to 5 ^°C decrease in growth temperature (cold shock). Cell membrane changes were observed following cold shock for all carbon sources. Cells became leaky and membranes less fluid immediately after cold shock. The fatty acid composition of cells also varied significantly with carbon source. A higher content of oleic acid (cis-9-octadecenoic acid – 18:1) was observed in cells grown in the presence of Tween 80 and olive oil compared to cells grown in the presence of acetate. Increased content of palmitoleic acid (cis-9-hexadecenoic acid – 16:1) observed during growth at 5^°C and following cold shock indicated that this fatty acid may be important for growth at low temperatures. Acetate-grown cells responded more quickly to cold shock than did Tween 80 or olive oil-grown cells by restoring membrane fluidity and by taking K+ back into the cells. In addition, acetate-grown cells modified the content of fatty acid cis-9-hexadecenoic acid at 2h post cold shock as opposed to 24h post cold shock in cells grown in the presence of Tween 80 or olive oil. This research indicated that cells are most affected by rapid decreases in growth temperature and growth at low temperatures when cells utilized olive oil as the sole source of carbon.     

MICROCLIMATIC EFFECTS ON WATER RELATIONS,LEAF TEMPERATURES,AND THE DISTRIBUTION OF HERACLEUM LANATUM AT HIGH ELEVATIONS

The small-scale distribution of an understory herb, Heracleum lanatum, was evaluated in terms of leaf temperature and water relations limitations due to a large leaf size (630 cm²). Diurnal variations in transpiration (4 to 60 mg m⁻² s⁻¹) were influenced by fluctuations in solar irradiance, wind speed, leaf temperature and stomatal conductance. Computer simulations indicated that leaf temperatures in a forest clearing would be > 12 C above air temperature, with maximum transpiration rates of 140 mg m⁻² s⁻¹, and daily water loss to be over 200% greater than values at natural understory locations. Simulations of nocturnal temperature relations indicated ~100 W m ⁻² less incident longwave irradiance in the forest clearing as compared to the understory (560 vs. 660 W m⁻² at 400 hr). This difference led to predicted leaf temperatures being as low as 6 C below air temperature in the forest clearing while measured leaf temperatures in the forest understory were within 1.5 C of air temperature throughout the night. Furthermore, minimum air temperatures were at or below 6 C on 36% of the nights during the summer growth period indicating that in open areas leaves of H. lanatum would frequently be below 0 C and subject to possible freeze damage. Heracleum lanatum may be more abundant in the shaded understory of the subalpine forest because exposure in open environments would result in high leaf temperatures and increased transpirational water loss during the day, as well as low leaf temperatures with the possibility of freeze damage at night.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of polyunsaturated fatty acids on the growth and differentiation of the cellular slime mould, Dictyostelium discoideum

Cells of Dictyostelium discoideum grown on media containing polyunsaturated fatty acid (PUFA) exhibit impaired differentiation when placed on a solid surface in the absence of all nutrients. Differentiation is inhibited at all temperatures, and although the inhibition is somewhat less pronounced at low temperatures, there is neither a lowering of the optimum temperature of aggregation nor a reversal of inhibition at low temperatures. Furthermore, although the in vitro aggregation of vegetative cells and the reaggregation of dispersed aggregation-phase cells are markedly temperature dependent, PUFA supplementation does not markedly influence this dependence. These data are not consistent with the hypothesis that impaired differentiation is due to increased plasma membrane fluidity. PUFA had no adverse effect on cell growth at temperatures at or below the optimum growth temperature, 22 °C. At 25 °C, however, there was considerable inhibition and at 27 °C growth was completely eliminated in the presence of PUFA.     

Is digestive capacity limiting growth at low temperatures in roach?

In roach Rutilus rutilus growth ceases below a temperature threshold of 12° C. This cessation of growth is accompanied by a reduction in feeding. Do roach decrease feeding in the cold because of reduced energy demand, caused by the decelerating effect of low temperature on metabolism and growth, or is feeding directly limited by low temperatures, leading to reduced growth rates? It was found that at low temperatures the intake and digestion of food may be limited by reduced activities of digestive enzymes. Trypsin, amylase and γ‐glutamyl transferase showed a negative compensation with respect to temperature, resulting in very low activities at acclimation temperatures of ≤12° C. Trypsin activity, falling from 400·5 ± 131·2 U g^?1 fresh mass of the gut at 27° C to 12·5 U g^?1 fresh mass at 4° C, displayed the strongest linear correlation with growth rates, suggesting that trypsin activities may set a limit to growth in the low temperature range. If protein digestion is limiting at low temperatures, this should be reflected in reduced concentrations of amino acid in the white muscle. The size of the total amino acid pool was not affected by temperature acclimation and ranged between 19·2 ± 6·2 and 25·2 ± 3·6 µmol g^?1 fresh mass of the white muscle. A decrease, however, was found of several amino acids, mainly of threonine and glutamine, in the low temperature range. Low concentrations of the essential amino acid threonine (0·14 ± 0·03 µmol g^?1 fresh mass at 12° C and 0·12 ± 0·05 µmol g^?1 fresh mass at 4° C) were probably due to nutritional or digestional limitations and may therefore have resulted from reduced trypsin activity in the cold. The non‐essential amino acid glutamine, however, can be endogenously synthesized and its low level observed at 4° C (0·16 ± 0·09 µmol g^?1 fresh mass) was not necessarily a result of low trypsin activities. It is more likely that low temperatures impair glutamine synthesis. The possibility that glutamine concentrations may be down regulated under conditions when anabolic processes are not advantageous is discussed.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Biomass production in mass cultures of marine phytoplankton at varying temperatures

Natural populations of marine phytoplankton obtained from a large outdoor pond were grown on waste water-sea water mixtures in laboratory continuous cultures in the temperature range 5–33 °C. Virtually all of the influent inorganic nitrogen (14.0 mg l^?1) was assimilated at every temperature tested. There was, however, a distinct change in dominant species with temperature: below 19.8 °C Phaeodactylum tricornutum was dominant, at 27 °C Nilzschia sp. was the main species, and as the temperature increased above 27 °C a blue-green alga, Oscillatoria sp., became increasingly dominant. There is some indication that the excellent growth of P. tricornutum below 10 °C was related to a dramatic increase in the nutrient content per cell as the temperature decreased. Thus at low temperatures reduced division rates are compensated for by increased nutrient uptake rates. It follows that there is a transfer of phytoplankton protein from numerous small cells at intermediate temperatures to large cells that are reduced in numbers at lower temperatures but which represent the same total organic matter. The effect of this phenomenon on annual food chain efficiencies in both controlled and natural marine ecosystems is unknown.     

The combined relationship of temperature and molybdenum concentration to nitrogen fixation byAnabaena cylindrica

The joint effects of growth temperature, incubation temperature, and molybdenum concentration on the nitrogen fixation rate ofAnabaena cylindrica were determined using the acetylene-reduction technique. The nitrogen-fixation response to increased molybdenum concentration varied among three growth temperatures (15°, 23°, and 30° C). The pattern of rate change was similar within a growth temperature but increased overall in magnitude with the three incubation temperatures (also 15°, 23°, and 30° C). The maximum rate of nitrogen fixation occurred at 30°C regardless of previous growth temperature. The minimum molybdenum concentration necessary to yield substantial acetylene reduction varied with growth temperature: at 15°C, 15g 1^–1 was effective; at 23°C, less than 5g 1^–1 was effective; and at 30°C, 50g 1^–1 was effective. At all three growth temperatures, increases in molybdenum concentration above the minimum effective concentration produced increases in acetylene reduction. However, at higher molybdenum concentrations inhibition of nitrogen fixation occurred.     

Growth and productivity of the psychrophilic marine diatoms Thalassiosira antarctica Comber and Nitzschia frigida Grunow in batch cultures at temperatures below the freezing point of sea water

Summary The diatoms Nitzschia frigida and Thalassiosira antarctica grow exponentially even at temperatures between-4 and -6°C and a salinity between 73 and 100 Under these conditions the light saturation of growth is reached in continuous light at a scalar quantum irradiance of between 7 mol·m^–2·s^–1 and 10 mol · m^–2 · s^–1. The increase in salinity retards growth more than a decrease in temperature. For N. frigida the limit of growth is at -8°C (S = 145%.). At increasing quantum irradiance, the chlorophyll content per unit cell volume decreases, whereas there is a significant increase in the carbon content of the exponentially growing cells. In addition, there is hardly any change in the protein content. The results show that both species of diatom can survive in ice without forming resting spores and even grow at extremely low temperatures.     

The adjustment of photosynthetically grown cells of Rhodospirillum rubrum to aerobic light conditions

Summary The growth of photosynthetically precultured cells of Rhodospirillum rubrum under aerobic condition in light is investigated. Special emphasis is given to the question of whether the photosynthetic electron transport chain is influenced under these conditions. Light-induced absorbance changes under anaerobic conditions show that although in whole cells a variation can be noted, the reactions of isolated membranes decrease only very slowly and parallel to each other. The photophosphorylation activity remains constant on a bacteriochlorophyll basis. On a cell mass basis this activity decreases parallel to the decreasing bacteriochlorophyll content. Light-dependent NAD⁺ reduction by ascorbate-DCPI remains constant on a bacteriochlorophyll basis, whereas succinate supported NAD⁺ reduction in light increases. On a cell mass basis the activity of succinate supported NAD⁺ reduction stays nearly constant, thus showing similar responses to the presence of oxygen in light as the NADH oxidase system. NADH oxidase activity increases on a bacteriochlorophyll basis and does not change on a cell mass basis. Parallel to the NADH-oxidase system, oxygen uptake in the dark by whole cells does not change after aerobiosis in light. Light inhibits respiration even after several generations of growth in the presence of oxygen; however, the inhibition decreases slowly. Light inhibition of respiration can be totally overcome by the addition of the uncoupler CCCP. These results indicate that light-dependent electron transport is not influenced significantly by the presence of oxygen. Although the respiratory system is formed, cells preferentially grow photosynthetically. Respiration takes over when the amount of bacteriochlorophyll reaches very low values.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - BChl bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DCPI Na-2,6-dichlorophenol-indophenol - NAD(P)⁺ nictotinamide-adenine-di; nucleotide (phosphate) - NADPH reduced NAD(P)⁺ - TMPD N,N,N-N-tetra; methyl-p-phenyldiamine     

Effect of temperature variations on growth,reproduction, amino acid synthesis,fat and sugar content in ulva fasciata delile plants

This work deals with the effect of temperature variations on growth, reproduction, amino acid synthesis, fat and sugar content in Ulva fasciata in habiting the littoral region of the Mediterranean shore bordering Alexandria.Higher temperatures above 30°C have lethal effects on some parts of the algal fronds while the other parts are rather resistant. The latter are scattered groups of cells, which usually regenerate new plants when carried even to distant parts due to detrimental action of waves and currents. They serve a good means for vegetative propagation and spread better than zooids, which probably perish under such severe and violent conditions.The rate of growth of Ulva indicated by the increase in dry weight and total nitrogen, increases at rising temperature till the latter becomes limiting.Higher temperatures favour the growth and development of sporophytic phases, known by their deep green colouration, while temperatures below 20°C favour the production of gametophytes, identified by their yellowish green colour. This might explain why most of the algal crops of Ulva fasciata produced from parental stock (3 crops per year i.e. annually), are sporophytes.Moderate temperatures were favourable for growth, reproduction and the various metabolic processes. Amino acids and sugar contents increase with the rise of temperature, reaching their maximum around 25°C, while fat content increases at still lower temperatures.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Microcalorimetric investigations of the metabolism of yeasts

Summary Microcalorimetric experiments on growth ofSaccharomyces under oxygen and nitrogen pressure between 0 and 10 kp/cm² are described. Within this range there are no alterations of the metabolism by the pressureper se but increased cell volume and a pronounced number of cells are observed. With nitrogen the enthalpy change amounts to a value of 130 cal/g glucose invariable with pressure. For oxygen a maximum heat evolution of 650 cal/g glucose is found in stirred cultures at the minimum pressure of almost 0 kp/cm². With rising O₂ pressure one observes a strong repression of heat flux which drops to a minimum value at 2 kp/cm². This repression is overcome by substrate concentrations less than 2 mg/ml. In unstirred cultures exposed to oxygen pressure the growth is determined by the geometrical and temporal distribution of cells and oxygen in the vessels. The calorimetric data are discussed in view of the mean volume and the dry weight of the cells.Herrn Prof. Dr. W. Stein zum 60. Geburtstag gewidmet.     

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K_m ATP or a change in the kinetics of Mg⁺⁺ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.     

Phloem Transport and the Regulation of Growth of Sorghum bicolor (Moench) at Low Temperature

Leaf expansion in Sorghum bicolor (Moench) was severely retarded by low night temperatures (5 C). However, this was not reflected in the early measurements of relative growth rate, indicating that the response was not associated with a deterioration of the photosynthetic system. For plants grown at 30/25 C (day/night) and subsequently held at an ambient temperature of 30 C, phloem transport, as measured either by the movement of ¹⁴C-photosynthate through a zone of controlled temperature or by accumulation of dry matter distal to this zone, was inhibited by temperatures below 10 C. The speed of movement of ³²P through the temperature controlled zone was more sensitive to temperature with reductions apparent below 20 C. Although there was some recovery in the movement of ³²P following 3 days equilibration at low temperature (1 to 10 C), the new values (approximately 100 centimeters per hour) were still only about one-third of those obtained in the high temperature controls. For plants held at an ambient temperature of 21 C, which is well below the optimum for growth, translocation was only inhibited by temperatures below 5 C. Although low temperature may reduce the carrying capacity of the phloem of S. bicolor, this is unlikely to be an important factor in regulating the growth of the plants at low temperatures.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.